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LSU Doctoral Dissertations Graduate School
2013
Group 3 late Embryogenesis abundant proteinsfrom embryos of Artemia francisana : molecularcharacteristics, expression and functionLeaf Chandra BoswellLouisiana State University and Agricultural and Mechanical College
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Recommended CitationBoswell, Leaf Chandra, "Group 3 late Embryogenesis abundant proteins from embryos of Artemia francisana : molecularcharacteristics, expression and function" (2013). LSU Doctoral Dissertations. 1527.https://digitalcommons.lsu.edu/gradschool_dissertations/1527
GROUP 3 LATE EMBRYOGENESIS ABUNDANT PROTEINS FROM
EMBRYOS OF ARTEMIA FRANCISCANA: MOLECULAR
CHARACTERISTICS, EXPRESSION AND FUNCTION
A Dissertation
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
The Department of Biological Sciences
by
Leaf Chandra Boswell
B.S. Biological Sciences, Louisiana State University, 2005
December 2013
ii
For my family, for their continuous love and support
in all aspects of my life.
iii
ACKNOWLEDGMENTS
First I would like to thank my mentor, Dr. Steven Hand for his guidance in
performing research and in the presentation of data. I would also like to thank Dr.
Michael Menze (Eastern Illinoise University) for helping me learn the fundamentals of
lab work and for helpful discussions in many areas of this project. Assistance with DNA
sequencing was provided by Dr. Scott W. Herke, manager of the Genomics Facility in the
Department of Biological Sciences at LSU. The Mass Spectrometry Facility in the
Department of Chemistry at LSU is also acknowledged for sample analysis. I would like
to thank Dr. Ted Gauthier for helpful advice regarding circular dichroism experiments. I
also thank Dr. Simon Chang for graciously providing PFK used in the protein
stabilization studies. Staff of the LSU Socolofsky Microscopy Center, especially Dr.
Holly Hale-Donze, is acknowledged for assisting with the imaging studies. Thanks are
extended to Dr. Hal Holloway and the LSU School of Veterinary Medicine Histology
Laboratory for paraffin embedding, sectioning and related processing. This study was
supported by National Science Foundation grant IOS-0920254.
iv
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ............................................................................................... iii
LIST OF TABLES ...............................................................................................................v
LIST OF FIGURES ........................................................................................................... vi
ABSTRACT ..................................................................................................................... viii
CHAPTER 1 INTRODUCTION .......................................................................................10
1.1 Research Aims of This Dissertation ................................................................17
CHAPTER 2 QUANTIFICATION OF CELLULAR PROTEIN EXPRESSION
AND MOLECULAR FEATURES OF GROUP 3 LEA PROTEINS
FROM EMBRYOS OF ARTEMIA FRANCISCANA .............................................19
2.1 Introduction ......................................................................................................19
2.2 Methods............................................................................................................22
2.3 Results ..............................................................................................................29
2.4 Discussion ........................................................................................................38
CHAPTER 3 GROUP 3 LEA PROTEINS FROM EMBRYOS OF ARTEMIA
FRANCISCANA: STRUCTURAL PROPERTIES AND PROTECTIVE
ABILITIES DURING DESICCATION ................................................................43
3.1 Introduction ......................................................................................................43
3.2 Methods............................................................................................................46
3.3 Results ..............................................................................................................51
3.4 Discussion ........................................................................................................61
CHAPTER 4 INTRACELLULAR LOCALIZATION OF GROUP 3 LEA
PROTEINS IN EMBRYOS OF ARTEMIA FRANCISCANA ................................66
4.1 Introduction ......................................................................................................66
4.2 Methods............................................................................................................69
4.3 Results ..............................................................................................................72
4.4 Discussion ........................................................................................................77
CHAPTER 5 SUMMARY AND FUTURE DIRECTIONS ..............................................81
LITERATURE CITED ......................................................................................................87
VITA ................................................................................................................................101
v
LIST OF TABLES
Table 2.1 Mass spectrometry confirms that the two molecular mass forms of
AfrLEA2 share sequence similarity with the bona fide purified protein ...............31
vi
LIST OF FIGURES
Figure 1.1 mRNA expression profile for the gene Afrlea3m from A. franciscana
embryos ..................................................................................................................14
Figure 2.1 Western blot analyses of purified recombinant proteins and various
extracts from Artemia franciscana .........................................................................30
Figure 2.2 PCR products amplified with cDNA prepared from diapause embryos
of A. franciscana ....................................................................................................32
Figure 2.3 Sequence comparisons for the four cDNA sequences amplified with
primers designed against Afrlea3m ........................................................................33
Figure 2.4 Quantification of AfrLEA2 protein in extracts of A. franciscana by
Western blot analysis .............................................................................................34
Figure 2.5 AfrLEA2 concentrations from diapause through 8 h of pre-emergence
development ...........................................................................................................35
Figure 2.6 Quantification of mitochondrial LEA proteins by Western blot analysis
in heat-treated extracts of A. franciscana ..............................................................36
Figure 2.7 Protein concentrations of AfrLEA3m_43, AfrLEA3m, and
AfrLEA3m_29 for diapause, 0-8 h of pre-emergence development, and
24 h nauplius larvae ...............................................................................................37
Figure 3.1 CD spectra of recombinant AfrLEA2 in the hydrated state, in the
presence of SDS and TFE, and after desiccation ...................................................52
Figure 3.2 Structural composition of recombinant AfrLEA2 as calculated from the
respective CD data with algorithms described in the Methods section .................52
Figure 3.3 CD spectra of recombinant AfrLEA3m in the hydrated state, in the
presence of SDS and TFE, and after desiccation ...................................................53
Figure 3.4 Structural composition of recombinant AfrLEA3m as calculated from
the respective CD data with algorithms described in the Methods section ...........53
Figure 3.5 CD analysis of BSA in the hydrated state, in the presence of SDS and
TFE, and after desiccation .....................................................................................55
Figure 3.6 Structural composition of BSA as calculated from the respective CD
data with algorithms described in the Methods section .........................................55
vii
Figure 3.7 Residual LDH activity after desiccation for one week without additives
(control) or in the presence of different protectants ...............................................57
Figure 3.8 Residual LDH activity after desiccation for one week without additives
(control) or in the presence of different protectants ...............................................57
Figure 3.9 Residual PFK activity after desiccation for 24 h without additives
(control) or in the presence of different protectants ...............................................58
Figure 3.10 Residual PFK activity after desiccation for 24 h without additives
(control) or in the presence of different protectants ...............................................58
Figure 3.11 Residual CS activity after two 24 h bouts of drying without additives
(control) or in the presence of different protectants ...............................................60
Figure 3.12 Residual CS activity after two 24 h bouts of drying without additives
(control) or in the presence of different protectants ...............................................60
Figure 4.1 Embryos of A. franciscana visualized at 0 h of pre-emergence
development by differential interference contrast (DIC) microscopy ...................73
Figure 4.2 Subcellular localization of AfrLEA2 in embryos of A. franciscana
visualized at 0 h of pre-emergence development ...................................................74
Figure 4.3 Subcellular localization of AfrLEA2 in embryos of A. franciscana
visualized after 6 h of pre-emergence development ..............................................75
Figure 4.4 Subcellular localization of AfrLEA3m (and its closely related variants
recognized by AfrLEA3m antibody) in embryos of A. franciscana
visualized at 0 h of pre-emergence development ...................................................76
viii
ABSTRACT
Late Embryogenesis Abundant (LEA) proteins are highly hydrophilic,
intrinsically disordered proteins whose expression has been correlated with desiccation
tolerance in anhydrobiotic organisms. Embryos of the brine shrimp, A. franciscana,
contain high titers of group 3 LEA proteins during desiccation-tolerant stages such as
diapause and pre-emergence development. Here I report the sequencing of three novel
variants of AfrLEA3m mRNA (Afrlea3m_47, Afrlea3m_43 and Afrlea3m_29), whose
deduced protein sequences are predicted to localize to the mitochondrion. These mRNAs
are very similar to Afrlea3m, but each has a stretch of sequence that is absent in at least
one of the others. In addition Afrlea3m_43 has five single nucleotide changes scattered
across its sequence, and Afrlea3m_47 and Afrlea3m_43 have three single nucleotide
differences in the section of sequence shared only by these two variants.
Protein expression for AfrLEA2, AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29
is highest in diapause embryos and decreases throughout development to their lowest
levels in desiccation-sensitive nauplius larvae. This pattern of protein expression is in
agreement with previously reported mRNA expression for AfrLEA2 and AfrLEA3m and
supports a role for LEA proteins in desiccation tolerance of embryos. When adjustment
is made for mitochondria matrix volume, the effective concentrations of cytoplasmic
versus mitochondrial group 3 LEA proteins are similar in vivo, and the values provide
guidance for the design of in vitro functional studies with these proteins. Investigations
of protein secondary structure show AfrLEA2 and AfrLEA3m to be intrinsically
disordered in solution and that they gain structure during desiccation and in the presence
ix
of the solvents TFE and SDS. I also show that during drying recombinant AfrLEA2 and
AfrLEA3m confer protection to three desiccation-sensitive enzymes (lactate
dehydrogenase, phosphofructokinase and citrate synthase). The degree of protective
ability was found to depend on the target enzyme chosen. The strongest degree of
stabilization was observed when a given LEA protein was used in the presence of the
stabilizing sugar trehalose, which is naturally accumulated by A. franciscana embryos.
Finally, AfrLEA2 is shown by immunohistochemistry to reside in the cytoplasm and
nucleus of embryonic cells of A. franciscana, and the AfrLEA3m proteins are localized
to the mitochondrion. The presence of LEA proteins in multiple subcellular
compartments suggests a requirement to protect biological structures in many areas of a
cell in order for an organism to survive desiccation stress.
10
CHAPTER 1
INTRODUCTION
Late Embryogenesis Abundant (LEA) proteins are historically predicted to
function in desiccation tolerance, mainly based on the observation that their expression
correlates to desiccation tolerant stages (Tunnacliffe and Wise 2007; Hand et al. 2011).
This proposed role in desiccation tolerance is directly supported by studies such as those
by Gal et al. (2004), and Battista et al. (2001), which show reduced LEA protein
expression, in a nematode and a bacterium respectively, decreases tolerance of the
organism to water stress. Specific roles suggested for LEA proteins include stabilization
of sugar glasses (vitrified, noncrystalline structure in cells promoted by sugars like
trehalose) (Wolkers et al. 2001; Hoekstra 2005; Shimizu et al. 2010), protein stabilization
via protein-protein interaction or ‘molecular shield’ activity (Tompa and Kovacs 2010;
Chakrabortee et al. 2012), membrane stabilization (Tunnacliffe and Wise 2007; Tolleter
et al. 2010), and formation of structural networks (Wise and Tunnacliffe 2004). Of these
predicted functions, the ability of LEA proteins to protect proteins from aggregation and
preserve their activity, stabilize membranes, and strengthen sugar glasses during water
stress are best defined (see current reviews Tunnacliffe and Wise 2007; Tunnacliffe et al.
2010; Hand et al. 2011; Hincha and Thalhammer 2012). Other functions such as ion
sequestration and hydration buffers have been suggested (for review see Tunnacliffe and
Wise 2007), but the biological significance of such functions have been challenged (Hand
et al. 2011). The primary objective of research presented in this dissertation is to better
11
the current understanding for the role of LEA proteins in desiccation tolerance, and to
provide further classification of five Group 3 LEA proteins from embryos of A.
franciscana.
LEA proteins, as named by Galau et al. (1986), were originally identified in
embryos of wheat and cotton over 30 years ago (Cuming and Lane 1979; Dure et al.
1981; Galau and Dure 1981). Since their initial discovery LEA proteins have been
documented not only in plants (Cuming 1999; Hoekstra et al. 2001; Tunnacliffe and Wise
2007; Shih et al. 2008), but also bacteria (Stacy and Aalen 1998; Dure 2001; Battista et
al. 2001), cyanobacteria (Close and Lammers 1993), a slime mold (Eichinger et al. 2005),
fungi (Mtwisha et al. 1998; Sales et al. 2000; Katinka et al. 2001; Abba et al. 2006), and
more recently animals, such as nematodes (Solomon et al. 2000; Browne et al. 2002; Gal
et al. 2003, 2004; Browne et al. 2004; Tyson et al. 2007; Haegeman et al. 2009), rotifers
(Tunnacliffe et al. 2005; Denekamp et al. 2009; Denekamp et al. 2010), embryos of brine
shrimp (Hand et al. 2007; Sharon et al. 2009; Menze et al. 2009; Chen et al. 2009;
Warner et al. 2010; Warner et al. 2012; Marunde et al. 2013), springtails (Clark et al.
2007; Bahrndorff et al. 2009), and a chironomid insect larvae (Kikawada et al. 2006).
The name, Late Embryogenesis Abundant, stems from the observation that LEA proteins
accumulate late in the maturation process of plant seeds (Galau et al. 1986). In addition
many plant LEA proteins accumulate in response to abscisic acid (ABA) and water stress
(Cuming 1999; Bartels 2005). The expression of non-plant LEA proteins typically
correlate to desiccation tolerant stages (for reviews, see Tunnacliffe and Wise 2007;
Hand et al. 2011).
12
Original classification of LEA proteins by Dure et al. (1989) defined three groups
(group 1, 2, and 3) based on common amino acid domains. Dure’s original classification
has been followed by various proposed naming schemes, many of which are
contradictory, including an alternative naming scheme proposed by Dure himself, which
labels each group based on a cottonseed prototype (Dure 1993). Most LEA proteins fall
within group 1 (D-19, PFAM LEA_5), group 2 (D-11, PFAM Dehydrin) and group 3 (D-
7, PFAM LEA_4), but other minor groups have been proposed including group 4 (D-113,
PFAM LEA_1), group 5 (D-29, PFAM LEA_4), group 6 (D-34 PFAM SMP), Lea5 (D-
73, PFAM LEA_3), and Lea14 (D-95, PFAM LEA_2) (Bray 1993; Galau et al. 1993;
Bray 1994; Tunnacliffe et al. 2010). However, currently there is only consensus on
groups 1, 2, and 3. Wise (2003) re-examined LEA protein classification with newly-
developed bioinformatics tools, which led to redistribution of group 4 and 5 members to
groups 2 and 3, thus eliminating groups 4 and 5, and there is argument that group 6,
Lea5, and Lea14 should not be considered part of the LEA protein family (Tunnacliffe
and Wise 2007).
The majority of LEA proteins classified to the three major groups have a biased
amino acid composition resulting in high hydrophilicity and a consequential lack of
secondary structure in solution (Tunnacliffe and Wise 2007). The high hydrophilicity of
LEA proteins also contributes to solubility at elevated temperatures; a characteristic
widely used during purification. LEA protein similarities, beyond hydrophilicity, are
often disparate between major groups. For example LEA proteins often vary in size and
net charge even within a defined group (Tunnacliffe and Wise 2007). According to Dure
et al. (1989) group 1 LEA proteins are characterized by the presence of a hydrophilic 20-
13
amino acid motif, and group 3 proteins by the presence of multiple repeats of an 11-
amino acid motif, although often with low homology (e.g. Grelet et al. 2005; Hand et al.
2007). Group 2 proteins, often referred to as dehydrins, are characterized by the presence
of at least two out of three sequence motifs named Y, S and K by Close (1997).
Anhydrobiotic embryos of A. franciscana, a model species for studying
desiccation tolerance, possess a multitude of group 1 and group 3 LEA proteins (Hand et
al. 2007; Menze et al. 2009; Sharon et al. 2009; Chen et al. 2009; Warner et al. 2010; Wu
et al. 2011; Warner et al. 2012; Marunde et al. 2013). LEA proteins expressed by A.
franciscana have been localized to both the cytoplasm (Hand et al. 2007) and, for the first
time in animals, the mitochondrion (Menze et al. 2009). Previously the only known
mitochondrial LEA protein had been documented from seeds of the pea plant, Pisum
sativum (Grelet et al. 2005). In Chapter 2 of this dissertation, I characterize three novel
LEA proteins from A. franciscana that are located in the mitochondrion. Chapter 2 also
investigates the protein expression levels for a cytosolic AfrLEA protein (AfrLEA2), and
three mitochondrial AfrLEA proteins (AfrLEA3m, AfrLEA3m_29, and AfrLEA3m_47).
mRNA expression data, previously published for AfrLEA2 (Hand et al. 2007) and
AfrLEA3m (Menze et al. 2009), indicate that the mRNA encoding each of these two
proteins are upregulated in desiccation-tolerant developmental stages when compared to
desiccation-intolerant nauplius larvae (for example see Fig 1.1). Partially discordant
mRNA/protein expression has been reported for LEA proteins expressed by a nematode
(Goyal et al. 2005a), emphasizing the importance of characterizing expression patterns
for both mRNA and protein. In addition to general protein expression levels of four
14
Figure 1.1 mRNA expression profile for the gene Afrlea3m from A. franciscana embryos.
LEA mRNA is maintained 9-11 fold higher in the two desiccation-tolerant embryonic
stages when compared to the desiccation-intolerant nauplius larva. Double asterisks
indicate that the paired means are statistically different (t-test, p < 0.05) (redrawn from
Menze et al. 2009).
group 3 LEA proteins across several developmental time points, Chapter 2 provides
physiological concentrations for one cytosolic and three mitochondrial LEA proteins.
Cellular titers, such as the ones provided, are currently lacking for LEA proteins.
Many LEA proteins are predicted to adopt a primarily α-helical structure (Dure et
al. 1989). However, in solution a number of LEA proteins with predicted α-helical
structure are found to be predominantly unstructured (Tunnacliffe and Wise 2007;
Tunnacliffe et al. 2010; Hand et al. 2011; Hincha and Thalhammer 2012). Attempts to
15
crystallize LEA proteins have been unsuccessful (e.g. McCubbin et al. 1985), which has
been attributed to their intrinsic disorder and high degree of hydration (Tunnacliffe and
Wise 2007). Structural information for a LEA protein in the hydrated state was first
presented by McCubbin et al. (1985) indicating that Em, a group 1 LEA protein isolated
from wheat embryos, is largely unstructured in solution. Ultimately, a small group 3
LEA protein expressed in pollen of Typha latifolia was found to gain structure upon
desiccation (Wolkers et al. 2001). Investigation into the structure of LEA proteins is
important as it may help shed light on LEA protein function. Chapter 3 of this
dissertation uses circular dichroism (CD) spectroscopy in order to deduce the secondary
structure of two recombinant LEA proteins in solution, in the presence of two solvents
[sodium dodecyl sulfate (SDS) and trifluoroethanol (TFE)] and in the dry state.
The gain of structure upon desiccation has bolstered the prediction that LEA
proteins may in fact function in the dry state. Accordingly, numerous LEA proteins have
been shown to protect proteins and/or membranes upon desiccation (for recent reviews
see Tunnacliffe and Wise 2007; Tunnacliffe et al. 2010; Hand et al. 2011; Hincha and
Thalhammer 2012). The ability of a LEA protein to provide protection during water
stress through an anti-aggregation effect was first shown by Goyal et al. (2005b), for two
LEA proteins from Aphelenchus avenae. These proteins were able to prevent
aggregation and protect the activity of both CS and LDH during desiccation and
subsequent rehydration. However, unlike classical molecular chaperones, the same two
proteins afforded no protection to these two enzymes during heat stress. Therefore,
Goyal et al. (2005b) propose that LEA proteins are acting as a novel form of molecular
chaperone, for which they coin the term “molecular shield”. In this context LEA proteins
16
would prevent the aggregation of desiccation sensitive molecules by serving as a physical
barrier among sensitive molecules. The ability of two recombinant group 3 LEA proteins
from A. franciscana embryos to protect multiple enzymes of both cytosolic and
mitochondrial origin is investigated in Chapter 3. The use of LEA proteins and enzymes
from different cellular compartments allows for a novel mix-and-match approach to
distinguish whether a mitochondrial LEA protein preferentially protects mitochondrial
enzymes, while a cytosolic LEA protein preferentially protects cytosolic enzymes, or if
the protective effect of LEA proteins is universal.
Protection of subcellular components, such as the mitochondrion, is undoubtedly
necessary if a cell is to survive desiccation (Liu et al. 2005; Hand and Hagedorn 2008;
Menze and Hand 2009). This need for protection of membrane bound organelles is
supported by subcellular targeting of LEA proteins, which has been documented in many
species, both plant and animal alike (Tunnacliffe and Wise 2007; Hand et al. 2011).
Known subcellular locations for plant LEA proteins include the cytoplasm, nucleus,
chloroplast, endoplasmic reticulum, vacuoles, peroxisomes, and the plasma membrane
(Tunnacliffe and Wise 2007). Mitochondrial localization was first reported in plants for a
LEA protein (PsLEAm) from seeds of Pisum sativum (Grelet et al. 2005), and soon
thereafter in the brine shrimp A. franciscana (Menze et al. 2009). In addition to the
mitochondrion, animal LEA proteins have also been localized to the endoplasmic
reticulum, the Golgi, and secreted into the extracellular space (Tripathi et al. 2012).
Chapter 4 uses immunohistochemistry to confirm the predicted subcellular localization of
AfrLEA2 and AfrLEA3m in embryos of the brine shrimp A. franciscana.
17
1.1 Research Aims of This Dissertation
The overall objective of this dissertation is to improve our current understanding
for the role of LEA proteins in desiccation tolerance through molecular characterization,
expression data, and functional studies of group 3 LEA proteins from embryos of A.
franciscana. In Chapter 2, I sequence and characterize three novel group 3 LEA proteins
from embryos of A. franciscana, which we name AfrLEA3m_29, AfrLEA3m_43, and
AfrLEA3m_47. Through sequencing, I illustrate these three LEA proteins to share
sequence similarities with AfrLEA3m, the original mitochondrial LEA protein reported
from A. franciscana. I performed Western blot analysis on protein extracts from several
developmental time points in order to determine if protein expression corresponded to
mRNA expression. Lastly, using Western blot analysis, I created standard curves with
known amounts of recombinant AfrLEA2 and AfrLEA3m in order to calculate
endogenous protein levels for AfrLEA2, AfrLEA3m, AfrLEA3m_29 and AfrLEA3m_43.
I also provide evidence that endogenous AfrLEA2 exist primarily as a dimer in vivo.
The focus of Chapter 3 is the secondary structure of recombinant AfrLEA2 and
AfrLEA3m, as well as the ability of these two LEA proteins to protect the activity of
desiccation sensitive proteins during drying and subsequent rehydration. I use circular
dichroism spectroscopy to resolve the secondary structure of AfrLEA2 and AfrLEA3m
both in solution and after desiccation, as well as to test the effects of SDS, TFE on LEA
protein structure. I then evaluate the protective ability of AfrLEA2 and AfrLEA3m by
using a novel mix-and-match technique. Through use of this mix-and-match technique I
investigate whether LEA proteins provide universal protection to multiple enzymes or
18
perhaps a cytosolic LEA protein preferentially protects cytosolic enzymes, while a
mitochondrial LEA protein preferentially protects mitochondrial enzymes.
Chapter 4 focuses on the intracellular localization of AfrLEA2, and the four
mitochondrial LEA proteins, AfrLEA3m, AfrLEA3m_29, AfrLEAm_43 and
AfrLEA3m_47. All four of the aforementioned mitochondrial LEA proteins are
recognized by the antibody raised against AfrLEA3m. Therefore, the localization study
investigates the four mitochondrial LEA proteins as a group. Bioinformatics software
predict AfrLEA2 to reside in the cytosol, and AfrLEA3m to translocate to the
mitochondrion. Previous studies have confirmed that AfrLEA3m translocates to the
mitochondrion in mammalian cells (Menze et al. 2009; Li et al. 2012). However, the
intracellular locations predicted for AfrLEA2 and AfrLEA3m have not been
experimentally confirmed in embryos of A. franciscana. I use immunohistochemistry to
confirm the predicted intracellular location for AfrLEA2 and the mitochondrial LEA
proteins recognized by antibody produced against AfrLEA3m.
19
CHAPTER 2
QUANTIFICATION OF CELLULAR PROTEIN EXPRESSION AND
MOLECULAR FEATURES OF GROUP 3 LEA PROTEINS FROM
EMBRYOS OF ARTEMIA FRANCISCANA 2.1 Introduction
When considering the ability to survive water stress, the most extreme examples
are anhydrobiotic organisms, which can survive extended periods of almost complete
desiccation (Keilin 1959; Crowe and Clegg 1973; Crowe and Madin 1974; Crowe and
Clegg 1978; Clegg 2005; Watanabe 2006; Cornette and Kikawada 2011; Welnicz et al.
2011). In nature, anhydrobiotic organisms such as nematodes and tardigrades routinely
experience dehydration down to 2% tissue water (Crowe and Madin 1974; Alpert 2006),
and the brine shrimp embryo can survive even lower residual water content under
aggressive experimental drying in the laboratory (Clegg et al. 1978; Hengherr et al.
2011b, a). As research on this topic progresses, it is becoming clear that desiccation
tolerance relies on a number of different mechanisms and requires the stabilization of
individual organelles in addition to cytosolic components (Pouchkina-Stantcheva et al.
2007; Tunnacliffe and Wise 2007; Hand and Hagedorn 2008; Atkin and Macherel 2009;
Hand et al. 2011; Tripathi et al. 2012). The accumulation of low molecular weight
organic solutes, such as trehalose, is often seen in desiccation tolerant organisms. These
organic solutes aid in macromolecular protection at low water contents (Yancey et al.
1982; Yancey 2005). In addition to organic solutes, several types of protective
macromolecules are correlated with desiccation tolerance, including Late Embryogenesis
Abundant (LEA) proteins and small stress proteins like Artemia P26, Hsp 21 and Hsp 22
20
(Clegg et al. 1994; Liang et al. 1997a; Liang et al. 1997b; Willsie and Clegg 2001; Clegg
2005; Qiu and Macrae 2008a; Qiu and MacRae 2008b). Desiccation-tolerant embryos of
A. franciscana possess a multitude of LEA proteins (Hand et al. 2007; Menze et al. 2009;
Sharon et al. 2009; Chen et al. 2009; Warner et al. 2010; Wu et al. 2011; Warner et al.
2012). In the present study I sequence the mRNA of three novel AfrLEA3m variants,
and quantify protein expression for AfrLEA2, AfrLEA3m, AfrLEA3m_43, and
AfrLEA3m_29 during diapause and development in A. franciscana. I also report
evidence that cytoplasmic-targeted AfrLEA2 exists primarily as a homodimer in vivo.
Western blot analysis of mitochondria isolated from 0 h post-diapause embryos reveals
four distinct bands when probed with antiserum against AfrLEA3m that have been shown
by mass spectrometry to correspond to the three novel mRNA sequenced in this study
and AfrLEA3m (Boswell et al., 2013).
To date all of the animal LEA proteins described have been assigned to group 3
(for classification scheme see Wise 2003), with the exception of group 1 LEA proteins
discovered in A. franciscana (Sharon et al. 2009; Warner et al. 2010; Wu et al. 2011;
Marunde et al. 2013). Group 3 LEA proteins are predicted to have high alpha-helix
content, but have been found experimentally to be unfolded when fully hydrated in
aqueous solution (Goyal et al. 2003). Interestingly, Goyal et al. (2003) found that a
group 3 LEA protein from an anhydrobiotic nematode adopted a α-helical structure upon
desiccation, with a possible coiled-coil formation. Group 3 LEA proteins are
characterized as being highly hydrophilic, intrinsically unstructured proteins with an
overrepresentation of charged and acidic amino acid residues (Tunnacliffe and Wise
2007; Battaglia et al. 2008).
21
Various functions have been proposed for LEA proteins based on their natively
unfolded structure and the correlation of gene expression to desiccation tolerance.
Predicted physiological roles for LEA proteins include stabilization of sugar glasses
(vitrified, noncrystalline structure in cells promoted by sugars like trehalose) (Wolkers et
al. 2001; Hoekstra 2005; Shimizu et al. 2010), protein stabilization via protein-protein
interaction or ‘molecular shield’ activity (Tompa and Kovacs 2010; Chakrabortee et al.
2012), membrane stabilization (Tunnacliffe and Wise 2007; Tolleter et al. 2010), ion
sequestration (Grelet et al. 2005), and formation of structural networks (Wise and
Tunnacliffe 2004). Such networks of LEA proteins have been hypothesized to increase
cellular resistance to physical stresses imposed by desiccation (Goyal et al. 2003).
Experimentally, LEA proteins prevent protein aggregation, protect enzyme function, and
maintain membrane integrity during water stress (for reviews see Tunnacliffe and Wise
2007; Hand et al. 2011; Hincha and Thalhammer 2012). However, the exact mechanisms
for these protective abilities continue to be explored. Few studies attempt to rigorously
estimate the effective cellular concentrations of LEA proteins (e.g., see excellent results
for cotton seeds Roberts et al. 1993). As a consequence, some functional roles projected
from in vitro experiments may not be applicable in vivo because the concentrations used
for in vitro characterization of LEA proteins are often arbitrary and may be unrealistic.
In the present study, the titer of cytoplasmic-localized LEA protein (AfrLEA2) was 0.79
± 0.21 to 1.85 ± 0.15 mg/g cellular water across development, and the combined
mitochondrial-targeted LEA proteins (AfrLEA3m, AfrLEA3m_29, AfrLEA3m_43) was
roughly 1.2-2.2 mg/ml matrix volume for post-diapause embryos. Such estimates suggest
that the effective concentrations of cytoplasmic versus mitochondrial group 3 LEA
22
proteins are similar in vivo and provide guidance for the design of in vitro functional
studies with these proteins.
2.2 Methods
Cloning, expression and antibody production for recombinant AfrLEA2 and AfrLEA3m
The original nucleic acid sequences for Afrlea2 (GenBank accession no.
EU477187) and Afrlea3m (GenBank accession no. FJ592175) cloned from A. franciscana
embryos (Hand et al. 2007; Menze et al. 2009) were amplified from our existing A.
franciscana cDNA library. Each gene was ligated into pET-30a (an expression vector
with a T7 lac promoter; Novagen, Rockland, MA) and then Rosetta™ 2(DE3) Singles™
Competent Cells (Novagen) were transformed with the genes according to the
manufacturer’s instructions. AfrLEA2 was expressed with an N-terminal 6X-His tag,
and AfrLEA3m was expressed with a C-terminal 6X-His tag so as not to interfere with
the mitochondrial localization sequence found at the N-terminus. Expression of
recombinant AfrLEA protein was induced by the addition of 1 mM IPTG for 2-3 h, and
confirmed by SDS PAGE and protein staining with Coomassie Blue. Bacterial cells were
pelleted by centrifugation (5,000 x g, 15 min) at 4ºC and chemically lysed using
Bugbuster® Protein Extraction Reagent (Novagen) in the presence of a protease inhibitor
cocktail, P8849 (Sigma-Aldrich, St Louis, MO). After removal of cellular debris by
centrifugation, the cell lysate was subjected to affinity chromatography on a HisTrap™
FF crude column (GE Healthcare, Waukesha, WI; 1 ml or 5 ml size, depending on
experimental requirements). Affinity purification binding buffer contained 20 mM
sodium phosphate, 0.5 M NaCl, and 20 mM imidazol, pH 7.5. A step elution was
performed using an elution buffer containing 20 mM sodium phosphate, 0.5 M NaCl, and
23
0.5 M imidazol, pH 7.5. Flow rate was set at the maximum rate recommended by the
manufacturer (1 ml/min for 1 ml column, or 5 ml/min for 5 ml column). Fractions
containing recombinant protein were heat treated at 95ºC for 20 min followed by
centrifugation (20,000 x g, 30 min) to separate the soluble fraction. The soluble fraction
was dialyzed overnight against the starting buffer for anion exchange (20 mM
triethanolamine, 10 mM NaCl, pH 7.0). The sample was then applied to an anion
exchange column (HiTrap™ Q FF; GE Healthcare). The elution buffer contained 20 mM
triethanolamine, and 1 M NaCl, pH 7.0. The fractions containing pure recombinant
protein, as assessed by SDS-PAGE and protein staining, were exchanged into LEA
storage buffer (20 mM HEPES, 50 mM NaCl, pH 7.5) and concentrated using Amicon®
Ultra Centrifugal filters (Ultracel®-10K; Millipore, Billencia, MA). Antibodies were
raised in chickens against recombinant AfrLEA2 and AfrLEA3m by Aves Labs, Inc.
(Tigard, OR).
Preparation of cDNA and sequencing of additional Afrlea3m-related genes
In extracts of mitochondria isolated from A. franciscana, four protein bands were
identified with the AfrLEA3m antibody produced above (see Results). Consequently, we
suspected that multiple mRNA species might be detected with cDNA prepared from A.
franciscana embryos. Total RNA was isolated from diapause embryos using an RNeasy
Midi kit (Qiagen, Valencia, CA), and then a DyNAmo cDNA synthesis kit (New England
Biolabs, Ipswich, MA) was used for reverse transcription according to manufacturer’s
instructions. Primers for Afrlea3m amplified four products, which were cloned with a
pENTR™/D-TOPO® Cloning Kit (Invitrogen, Carlsbad, CA) as described in the
manufacturer instructions. One Shot® TOP10 Chemically Competent E. coli
24
(Invitrogen) were transformed with these genes. Direct colony PCR was performed to
screen for transformed colonies. Colonies were identified that contained each of the four
inserts, and a QIAprep 96 Turbo Miniprep Kit (Qiagen) was used to purify plasmid DNA
from each. Sequencing was conducted with BigDye terminator chemistry and an ABI
PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Molecular mass determination by SDS-PAGE
The molecular mass of recombinant and endogenous LEA proteins were
determined by SDS-PAGE as described by Hames (1998). Briefly, the log of molecular
mass for biotinylated protein standards (Cell Signaling Technology, Danvers, MA) was
plotted against relative migration distance (Rf) for the proteins after separation by SDS-
PAGE. Rf was calculated as the migration distance of a protein divided by the migration
distance of the dye front. The Rf values for LEA proteins were used to interpolate their
molecular masses from the standard curves. The reported masses are the result of six
separate measurements on three independent gels. AfrLEA2 samples were analyzed on
7% gels for optimal determination of size.
Mass Spectrometry
In-gel trypsin digestion of proteins separated by SDS-PAGE was performed as
described by Shevchenko et al. (2006). Briefly, bands of interest were excised from a
Coomassie-stained SDS gel and destained in 100 µl of 100 mM ammonium
bicarbonate/acetonitrile (1:1, vol/vol) for about 30 min. After destaining, 500 µl of 100%
acetonitrile was added to the destain mixture to dehydrate the gel pieces. The gel pieces
were then transferred to 50 µl of a trypsin cocktail (13 ng protease/µl of a solution
25
containing 10 mM ammonium bicarbonate and 10% (vol/vol) acetonitrile) and incubated
for 90 min to saturate the gel pieces with trypsin. Sequencing grade trypsin (cat. #
V5111) was obtained from Promega (Fitchburg, WI). Next 10-20 µl of 100 mM
ammonium bicarbonate was added to the trypsin cocktail, and gel pieces were incubated
overnight at 37ºC for complete protein digestion. Peptide products were extracted by
adding 100 µl of extraction solution (5% formic acid/acetonitrile (1:2, vol/vol)) to the
trypsin cocktail and incubated on a shaker for 15 min at 37ºC. The liquid fraction
containing the peptide digestion products was collected and dried in a vacuum centrifuge.
Samples were submitted to the Mass Spectrometry Facility in the Department of
Chemistry (Louisiana State University) and analyzed by LC-MS-MS on a QSTAR XL
quadrupole time-of-flight mass spectrometer (Applied Biosystems). The MS/MS data for
each protein digest was submitted for a database search using Mascot from Matrix
Sciences (Boston, MA).
Preparation of protein extracts from diapause and post-diapause embryos
Diapause embryos were collected from the surface of the Great Salt Lake (Ogden,
UT) in fall 2011. Diapause embryos were maintained at ambient temperature in 1.25 M
NaCl containing 200 units/ml nystatin, 50 mg/ml kanamycin, and 50 mg/ml penicillin-
streptomycin; were protected from light. Prior to use diapause embryos were rinsed and
incubated in 35 ppt artificial seawater (Instant Ocean; Aquarium Systems, Mentor, OH)
at room temperature with shaking (110 rpm) for 4 days (Reynolds and Hand 2004) to
allow hatching of any embryos that had broken diapause. Hatched nauplius larvae were
removed and intact diapause embryos were used for experiments. Post-diapause embryos
of A. franciscana were obtained in the dry state from Great Salt Lake Artemia (Ogden,
26
UT; grade: laboratory reference standard) and stored at -20ºC. Prior to use these
dehydrated embryos were hydrated overnight in ice-cold 0.25 M NaCl. Embryos for the
0 h time point were processed immediately after hydration at 0ºC. Other embryos were
transferred to fresh 0.25 M NaCl at 23ºC and incubated with shaking (110 rpm) to
promote pre-emergence development, and embryos were sampled at or 2, 4, 6, and 8 h.
Prior to homogenization embryos were filtered and then blotted between two sheets of
Whatman no. 41 filter paper to remove interstitial water. Blotting was performed
according to Clegg (1974). To obtain nauplius larvae, hydrated embryos were incubated
in 35 ppt artificial seawater for 24 h at 23ºC with shaking (110 rpm). Nauplius larvae
were separated from unhatched embryos and shells, and then filtered and blotted.
For quantification of AfrLEA2 by Western blot (see below), 100 mg of embryos
or 24 h nauplii were transferred directly into 1.9 ml of Laemmli sample buffer [62.5 mM
Tris-HCl (pH 6.8), 2 % SDS, 10 % glycerol, 5 % 2-mercaptoethanol (Laemmli 1970)]
and homogenized in a ground glass homogenizer for 5-7 min. The homogenate was then
heated at 95ºC for 5 min and centrifuged (10,000 x g, 10 min) to remove the insoluble
debris like shell and chitin fragments. In order to quantify the mitochondrial AfrLEA
proteins, it was necessary to first enrich these protein in extracts, because due to their
mitochondrial location these proteins comprise a smaller percentage of total cellular
protein compared to the cytosolic AfrLEA2. Accordingly, 200 mg of embryos (or 24 h
nauplii) were instead homogenized into the non-denaturing LEA storage buffer described
above. The homogenate was heated at 95ºC for 20 min and centrifuged (20,000 x g, 30
min, 4ºC) to sediment the heat-insoluble fraction. The soluble supernatant, which
contains only heat-stable macromolecules like LEA proteins, was retained and combined
27
3:1 with 4X Laemmli sample buffer. Protein concentration was obtained for all samples
using a Lowry assay as described by Peterson (1977).
Western Blot analysis
For A. franciscana samples 10 µg of total protein was loaded per lane on SDS
acrylamide gels (4 % stacking gel, 11 % resolving gel) and electrophoresed for 80 min at
125 V in a Bio-Rad mini-Protean 3 cell. Proteins were then transferred to a nitrocellulose
membrane (Bio-Rad, Hercules, CA) in a transfer buffer containing 192 mM glycine, 20%
methanol, 0.025% SDS, and 25 mM Tris; acceptable transfer was confirmed by staining
the membranes with Ponceau S. Membranes were then blocked in a 5 % fat free dry milk
solution for 1 h. Incubation with primary antibody raised against recombinant AfrLEA
protein (Aves Labs Inc) was performed overnight at 4ºC, and then blots were washed for
a total of 1 h with four changes of TBS-T (0.1% Tween 20, 20 mM Tris-HCl, 500 mM
NaCl, pH 7.6). Next the membranes were incubated for 1 h at room temperature with the
HRP-linked secondary antibody (goat anti-chicken; Aves Labs Inc) and washed as above.
Protein bands were visualized with LumiGLO chemiluminescent substrate (Cell
Signaling Technology).
Quantity One Basic 4.6.9 software (Bio-Rad Laboratories) was used to quantify
band intensities. Global background subtraction was applied to each image analyzed.
These intensities were converted to ng AfrLEA protein per band by comparing intensity
values to a standard curve generated with known amounts of pure recombinant AfrLEA
protein. Experimental samples for AfrLEA2 were normalized to -tubulin as a loading
control before any further calculations were performed. Then the values for ng
protein/band were converted to mg protein/ml embryo water based on water content data
28
previously published (Glasheen and Hand 1989). Mitochondrial AfrLEA proteins were
quantified similarly except they were not normalized to a housekeeper protein due to the
heat treatment step required prior to Western blotting. Values for mitochondrial AfrLEA
proteins were reported as µg/g wet tissue. Finally, to facilitate comparison to the
effective in vivo concentration of AfrLEA2, mitochondrial AfrLEA proteins were also
expressed as mg protein/ml matrix volume. This calculation is based upon an estimated
mitochondrial volume of 5% of the total cell volume for post-diapause embryos of A.
franciscana (Rees et al. 1989), and an estimate that matrix volume is about 50% of total
mitochondrial volume in the semi-condensed/condensed states (Hackenbrock 1968;
Scalettar et al. 1991).
Detection of glycoproteins in polyacrylamide gels
In-gel detection of glycoproteins was performed using the Periodic Acid Schiff
(PAS) method. Schiff’s fuchsin-sulfite reagent was obtained from Sigma-Aldrich and
detection was performed as described by the supplier. Briefly, gels were incubated in
fixative solution (40% ethanol and 7% acetic acid) for 1.5 h with four changes of solution
and then left in the fixative overnight. Afterwards, the fixative solution was again
refreshed twice, each followed by a 30 min incubation. Oxidation of glycoprotein bands
was accomplished by immersing gels in a solution containing 1% periodic acid and 3%
acetic acid for 60 min. Gels were then washed ten times (10 min each) to remove traces
of periodic acid before incubation in Schiff’s Reagent for 60 min in the dark. Stained
gels were washed in a solution with 0.58% potassium disulfite and 3% acetic acid to
remove any background.
29
2.3 Results
Properties of AfrLEA2 in embryos of A. franciscana
As previously reported, AfrLEA2 is a group 3 LEA protein (38.9 kDa) that is
predicted to reside in the cytoplasmic compartment (Hand et al. 2007). This predicted
location was confirmed in HepG2 cells transfected with the GFP-tagged protein (Li et al,
2012). Recombinant AfrLEA2 has a total molecular mass of 43.1 kDa (38.9 kDa plus 4.2
kDa for a 6X-His tag and associated sequence) but migrates on SDS gels at a calculated
mass of 49.3 ± 0.9 (mean ± SD; n = 3) (Fig. 2.1). This higher apparent molecular mass
with SDS-PAGE may be explained by the observation that some intrinsically disordered
proteins have a reduced binding for SDS and therefore frequently exhibit decreased
migration on SDS gels (Tompa 2002). Based on the migration of recombinant AfrLEA2,
we expect endogenous AfrLEA2 to migrate at about 44 kDa. Surprisingly, when A.
franciscana extracts are electrophoresed on an 11% polyacrylamide gel and probed with
anti-AfrLEA2 polyclonal antibody, a protein band is detected around 75 kDa (data not
shown). Upon closer examination with a 7% polyacrylamide gel to provide better
resolution of larger proteins, three bands are discernible, with calculated apparent
molecular masses of 82.0 ± 0.4, 75.3 ± 1.0, and 72.7 ± 0.5 kDa (means ± SD; n = 3) (Fig
2.1). For most developmental stages of A. franciscana where AfrLEA2 is expressed, the
82 kDa form is predominant. Based on the molecular mass and further evidence below,
we suggest that the 82 kDa protein is a dimer of the 38.9 kDa AfrLEA2, and the smaller
30
Figure 2.1 Western blot analyses of purified recombinant proteins and various extracts
from Artemia franciscana. Lane one was loaded with molecular mass standards, lane
two with 1 µg of recombinant AfrLEA2 protein, and lane three with 10 µg protein of an
extract prepared from diapause embryos. With extracts prepared from diapause embryos,
anti-AfrLEA2 antibody reveals three bands on a 7% polyacrylamide gel with apparent
molecular masses of 82.0 ± 0.4, 75.3 ± 1.0, and 72.7 ± 0.5 kDa. Arrows indicate dimers
and trimers formed by recombinant AfrLEA2.
bands may be degradation products or even processed AfrLEA2 (cf. Goyal et al. 2005a;
Kikawada et al. 2006).
Oligomers of LEA proteins resistant to SDS dissociation have been previously
documented by PAGE (Goyal et al. 2003), and we detected apparent dimers and trimers
of purified recombinant AfrLEA2 by SDS-PAGE with anti-AfrLEA2 antibodies (Fig 2.1;
97.6 kDa and 137.5 kDa), which supports the ability of the protein to form oligomers.
31
Table 2.1 Mass spectrometry confirms that the two molecular mass forms of AfrLEA2
share sequence similarity with the bona fide purified protein. Mascot ions scores are -
10*Log(P), where P is the probability that the observed match is a random event.
Individual ions scores, or combined scores where multiple peptides are identified from a
single protein, greater than 60 indicate identity or extensive homology (p<0.05).
Apparent multimers of recombinant AfrLEA2 are also visualized using an anti-6X-His
antibody (data not shown), which suggests the multimers are not non-specific products
that cross-react with AfrLEA2 antibody. While only small numbers of cytoplasmic
proteins are typically glycosylated, in-gel staining with PAS reagent did not indicate
AfrLEA2 to be glycosylated, based on the absence of PAS-positive bands of appropriate
size in embryo extracts enriched for LEA proteins by heat purification. In-gel trypsin
digests were performed on gel slices from the regions where the dimer and monomer
migrate, and the peptides were analyzed by mass spectrometry (LC-MS-MS). One or
more peptide fragments from both areas were found to share sequence identity to bona
fide AfrLEA2 based on robust scores (Table 2.1). Finally, with Afrlea2-specific
primers, PCR amplification was performed on cDNA prepared from diapause embryos.
Only a single 1.1 kb product was generated, which is the expected size of mRNA
encoding for AfrLEA2 (364 amino acids) (Fig. 2.2). The combined evidence indicates
that the predominant 82 kDa protein visualized on Western blots with AfrLEA2 antibody
is a dimer of the 38.9 kDa AfrLEA2.
AfrLEA2 monomer AfrLEA2 dimer Score Peptide Score Peptide
47 K.SISDAAYFTGK.G 83 K.GIGETVKADADVVEGMASTGYEK.L
56 K.INAIQTPEEMDHER.L
101 K.GIGETVKADADVVEGMASTGYEK.L
32
Figure 2.2 PCR products amplified with cDNA prepared from diapause embryos of A.
franciscana. Lane one was loaded with a DNA ladder (kb) and lanes two and three with
products from PCR reactions as indicated. Primers designed against Afrlea2 yielded a
single product of approximately 1.1 kb (Afrlea2 lane). Primers designed against Afrea3m
amplified four products that migrate at 1.2, 1.0, 0.9 and 0.7 kb (Afrlea3m lane)
Multiple independently-encoded variants of AfrLEA3m
With Afrlea3m primers and cDNA template prepared from diapause embryos,
PCR amplification generated four products (Fig. 2.2). In order to gain more insight into
33
Figure 2.3 Sequence comparisons for the four cDNA sequences amplified with primers
designed against Afrlea3m. The green boxed regions (nucleotide 1 – 84) indicate the
mitochondrial leader sequence that is shared by all four cDNAs. Each of the other four
boxed regions (yellow, red, light blue, dark blue) indicate stretches of virtually-identical
sequence that are present in some of the cDNAs but absent in at least one.
AfrLEA3m_47: yellow box = nucleotide 466 – 801, red box = nt 989 – 1036, dark blue
box = nt 1037 – 1084 and light blue box = nt 1085 – 1183. AfrLEA3m_43: yellow box =
nt 466 – 801 and light blue box = nt 989 – 1087. AfrLEA3m: red box = nt 653 – 700,
dark blue box = nt 701 – 748 and light blue box = nt 749 – 847. AfrLEA3m_29: red box
= nt 653 – 700. None of the mRNA variants contain sequence that was not shared by at
least one other isoform. Solid black lines indicate virtually-identical sequence shared by
all four cDNAs
the nature of these mRNAs, each of the four bands amplified from cDNA was cloned and
sequenced. The protein encoded by the 924 bp cDNA is identical to AfrLEA3m that was
previously reported (Menze et al. 2009). Like AfrLEA3m, all three deduced proteins
(AfrLEA3m_47, AfrLEA3m_43 and AfrLEA3m_29; suffixes indicate masses deduced
from cDNA sequence) possess mitochondrial targeting sequences and thus are predicted
to localize to the mitochondrion with high probability (Target P, MitoProt II, and
Predator). The deduced protein sequences for the three new isoforms are highly
hydrophilic as determined by Kite and Doolittle hydropathy plots (data not shown). The
sequences for all four cDNAs are very similar, but each has a stretch of sequence that is
absent in at least one of the others (Fig. 2.3). In addition Afrlea3m_43 has five single
nucleotide changes scattered across its sequence that do not match the other three
cDNAs(data not shown), and Afrlea3m_47 and Afrlea3m_43 have three single nucleotide
differences in the section of sequence shared by these two variants (Fig. 2.3, yellow bar).
34
Thus we conclude that these mRNA species are independently encoded, but closely
related variants, of Afrlea3m.
Protein expression of AfrLEA2 during development
By comparing band intensities to a standard curve created using recombinant
AfrLEA2 (Fig. 2.4), we were able to estimate the quantity of AfrLEA2 (subforms
combined) present in each sample. These values were then converted to mg LEA protein
per ml embryo water. Because AfrLEA2 is a cytoplasmic-localized protein, this
concentration unit provides a meaningful estimate of its effective titer in vivo. AfrLEA2
is most abundant in diapause and decreases throughout development to undetectable
levels in 24 h nauplius larvae (Fig. 2.4A and Fig 2.5). This pattern is in agreement with
the mRNA expression profile reported for Afrlea2 (Hand et al. 2007) and further supports
Figure 2.4 Quantification of AfrLEA2 protein in extracts of A. franciscana by Western
blot analysis. A) Expression levels for AfrLEA2 are shown at various stages of the life
cycle [diapause; pre-emergence development (hours 0, 2, 4, 6, 8,); and nauplius larvae
(24 h)]. AfrLEA2 is most abundant in diapause and decreases throughout development to
undetectable levels in nauplius larvae. α-tubulin is included as a loading control for each
time point. B) Concentration dependency of recombinant AfrLEA2 as measured with
anti-AfrLEA2 antibody. C) Standard curve for recombinant AfrLEA2 (R2 = .986).
35
Figure 2.5 AfrLEA2 concentrations from diapause through 8 h of pre-emergence
development. All values were normalized to α-tubulin. Asterisks (*) indicate that the
means are statistically different from diapause values (1-way-ANOVA, Tukey, P < 0.05).
Conversion of AfrLEA2 concentrations to ‘per ml embryo water’ is based on water
content data previously published (Glasheen and Hand 1989)
The role for LEA proteins in desiccation tolerance in A. franciscana, a physiological
feature that disappears beginning at the larval stage. Based on these results there are 1.85
± 0.15 mg (mean ± SD; n = 3) of AfrLEA2 per ml embryo water in diapause embryos
(Fig. 2.5), or 5.05 µg of AfrLEA2 per mg total embryo protein.
Quantification of protein expression for AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29
during development
As described above, a standard curve (Fig. 2.6) was used to determine the
concentrations of AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29 present in each
sample. The low amount of expressed AfrLEA3m_47 made it problematic to quantify.
36
Figure 2.6 Quantification of mitochondrial LEA proteins by Western blot analysis in
heat-treated extracts of A. franciscana. A) Expression levels for mitochondrial LEA
proteins are shown at various stages of the life cycle [diapause, pre-emergence
development (hours 0, 2, 4, 6, 8); and nauplius larvae (24 h)]. AfrLEA3m isoforms are
most abundant in diapause and decrease throughout development to the lowest levels
observed, which are found in nauplius larvae. Equal amounts of total protein in extracts
were loaded for each time point. B) Concentration dependency of recombinant
AfrLEA3m as measured with anti-AfrLEA3m antibody. C) Standard curve for
recombinant AfrLEA3m. (R2 = .99)
Because these three proteins are localized to the mitochondrial compartment, the
concentrations of each mitochondrial AfrLEA were initially expressed as µg protein per g
wet tissue (Fig. 2.7). There were significant decreases in content for each of the three
proteins from diapause through pre-emergence development (0 – 8 h), and then even
further decreases occurred in 24 h nauplius larvae (Fig. 2.7). These trends in protein
expression mirror the mRNA expression data reported for Afrlea3m (Menze et al. 2009).
Finally, to estimate an effective in vivo concentration, the amount of combined
mitochondrial-targeted LEA proteins (AfrLEA3m, AfrLEA3m_29, and, AfrLEA3m_43)
was also expressed as mg protein/ml mitochondrial matrix volume. For post-diapause
37
Figure 2.7 Protein concentrations of AfrLEA3m_43, AfrLEA3m, and AfrLEA3m_29 for
diapause, 0-8 h of pre-emergence development, and 24 h nauplius larvae. For each LEA
protein, the asterisks (*) indicate that the means for pre-emergence and larval stages are
statistically different from their respective diapause value (1-way-ANOVA, Tukey, P <
0.05). AfrLEA3m_47 was too faint to reliably quantify across development.
embryos, this value is approximately 1.2 mg protein/ml matrix volume. Considering that
the mitochondrial density in cells is comparable between diapause and post-diapause
embryos (Reynolds and Hand, 2004), the value would be approximately 2.2 mg/ml
during diapause. Interestingly, such estimates suggest that the effective concentrations
of cytoplasmic versus mitochondrial group 3 LEA proteins are comparable in vivo and
provide guidance for the design of in vitro functional studies with these proteins.
38
2.4 Discussion
In the present study we have characterized the protein expression levels for four
group 3 LEA proteins throughout A. franciscana development. The four mitochondrial
LEA mRNA studied here share similar sequence identity, but contain multiple single
base pair differences, so we predict that these mRNA are encoded by separated genes.
We have previously reported mRNA expression for AfrLEA2 (Hand et al. 2007) and
AfrLEA3m (Menze et al. 2009) to be highest in desiccation tolerant stages (diapause and
post-diapause embryos) when compared to desiccation-sensitive nauplius larvae. The
protein expression patterns reported in this study are in agreement with the mRNA
expression and provide further evidence that LEA proteins play a role in desiccation
tolerance.
Finally, we have also experimentally measured physiologically-relevant quantities
of a cytoplasmic (AfrLEA2) and three mitochondrial (AfrLEA3m_43, AfrLEA3m, and
AfrLEA3m_29) LEA proteins across development in A. franciscana. Values of this type
are useful to more accurately evaluate whether concentration-dependent properties
identified for LEA proteins in vitro are relevant for in vivo settings.
Our results provide evidence that endogenous AfrLEA2 exists primarily as a
dimer in A. franciscana embryos. The presence of SDS-resistant oligomers have been
previously reported for LEA proteins and other hydrophilic proteins (Goyal et al. 2003;
Maskin et al. 2007; Bahrndorff et al. 2009), but this is the first report to our knowledge of
a LEA protein existing primarily as a dimer in vivo. In addition to molecular mass, key
evidence for the 82 kDa dimer includes the amplification with Afrlea2-specific primers of
only a single PCR product, and that this product is of the correct size for a mRNA
39
encoding the 38.9 kDa monomer. Further, a few bases upstream of the Afrlea2 gene is
sequence for a translational stop codon. Mass spectrometry of the dimer supports
sequence similarity to the monomer. At high concentrations, our purified recombinant
AfrLEA2 will form dimers and trimers in vitro that are resistant to SDS dissociation. The
smaller bands (75.3 and 72.7 kDa) recognized by the anti-AfrLEA2 antibody could be
processed forms of the AfrLEA2 dimer, as reported by Goyal et al. (2005a) for a Group 3
LEA protein (AavLEA1) from the nematode Aphelenchus avenae. These authors provide
evidence for non-random cleavage that could increase the specific activity of the LEA
protein, whereby two shorter proteins are more effective molecular shields than one
larger one. Alternatively, intrinsically disordered proteins, such as LEA proteins, are
susceptible to random degradation due to their unstructured nature (Receveur-Brechot et
al. 2006; Uversky and Dunker 2010).
Unlike the results for Afrlea2, four distinct bands were amplified from cDNA of
diapause embryos with primers designed for Afrlea3m. While general architectural
features of the coding sequences display similarities that include sequence identity at
their N-termini (Fig. 3), the multiple single-nucleotide differences distributed across the
sequences preclude splice variants as an explanation and suggest these four mRNAs are
products of separate, independent genes. A similar case is seen in rotifers, where two
LEA mRNAs are very similar but arise from two individual genes on different
chromosomes (Pouchkina-Stantcheva et al. 2007). Also, very similar variants of Group 1
LEA proteins have been documented in A. franciscana and attributed to independent
genes (Sharon et al. 2009; Warner et al. 2010; Warner et al. 2012; Marunde et al. 2013).
40
As research into the function of LEA proteins continues it is important to consider
their endogenous cellular titer, and how LEA protein concentration relates to proposed in
vivo functions. Considering that an individual LEA protein family can represent up to
3.86% of total cytosolic protein in plant seeds (Roberts et al. 1993), and that most
organisms express a multitude of LEAs, it is becoming apparent that LEA proteins can
embody a large proportion of total cellular protein. We have shown that AfrLEA2, one
of the multiple cytosolic LEA proteins expressed in A. franciscana, has a cellular
concentration of 1.85 mg protein/ml cell water, and three of the known mitochondrial
LEA proteins from A. franciscana have a combined concentration of 2.2 mg protein/ml
mitochondrial matrix volume. These values can be used to re-evaluate previous
predictions for LEA protein function, as well as to guide the design of future
experiments. For example, Tolleter et al. (2007) predict that LEAM, a mitochondrial
protein expressed in pea seeds, provides protection to the inner mitochondrial membrane
during desiccation. According to their calculations, LEAM would need to represent
about 0.6% of total matrix protein in order to provide protection to about one-third of the
inner membrane surface (an estimate of the protein-free area). With total matrix protein
in the range of 400 mg/ml, this predicted amount of 2.4 mg/ml LEAM is not
unreasonable based on our estimation that three mitochondrial LEA proteins from A.
franciscana embryos are present at a combined concentration of 2.2 mg/ml.
Another important consideration is the design and interpretation of in vitro studies
used to attribute functional characteristics to various LEA proteins. Knowledge of the
endogenous titers of LEA proteins is fundamental because it can be used as a starting
point for estimating mass ratios of LEA protein to target molecule in vivo. The ability of
41
LEA proteins to stabilize sugar glasses (Wolkers et al. 2001), model membranes (Tolleter
et al. 2010) and proteins (Goyal et al. 2005b) have been investigated in vitro at mass
ratios of LEA protein to target as high as 2:1, 1:3 and 40:1, respectively. Although there
may be situations where the use of endogenous levels of either LEA proteins or target
molecules are not practical (due to cost or availability), it is important to take the cellular
titers of LEA proteins into consideration when interpreting results, especially when
translating functional characteristics of LEA proteins from in vitro to in vivo conditions.
Furthermore, an open question exists as to why the presence of LEA proteins without
protective sugars is sufficient for desiccation tolerance in some anhydrobiotic species,
while in other tolerant animals, high concentrations of glass-forming sugars (e.g.,
trehalose) are preferentially accumulated during drying together with LEA proteins (cf.
Hand et al. 2011). Perhaps the absolute cellular titer of LEA proteins expressible in a
given cell type/organism governs the apparent need for trehalose.
Several reports now indicate that a multitude of LEA proteins can be expressed in
a given anhydrobiotic organism, which brings into question why it is necessary for one
organism to express so many different LEA variants. Differential subcellular targeting
may be one reason for the presence of so many LEA proteins in a single organism. To
date, plant LEA proteins have been found in the cytoplasm, mitochondrion, nucleus,
chloroplast, endoplasmic reticulum, vacuole, peroxisome, and plasma membrane
(Tunnacliffe and Wise 2007). Animal LEA proteins have also been found commonly in
the cytoplasm (for review see Hand et al. 2011) as well as multiple subcellular locations
including the mitochondrion (Grelet et al. 2005; Menze et al. 2009; Warner et al. 2010;
Warner et al. 2012), nucleus (Warner et al. 2012), endoplasmic reticulum, golgi, and
42
extracellular space (Tripathi et al. 2012). In addition to differential subcellular targeting,
LEA proteins have been shown to stabilize different classes of macromolecules during
water stress. For example, some LEA proteins provide protection to target proteins and
do not protect lipid membranes, while others stabilize lipid membranes and afford no
protection to proteins (Pouchkina-Stantcheva et al. 2007). Considered together it is
possible that differential subcellular targeting and the ability of individual LEA proteins
to stabilize different types of macromolecules may explain the necessity for multiple
LEAs within a single organism.
In conclusion, our results for differential ontogenetic expression of LEA proteins
support their involvement in desiccation tolerance in A. franciscana, and we provide
physiological protein concentrations for LEA proteins in two different cellular
compartments, the cytoplasm and the mitochondrion. Appreciating the cellular titers of
LEA proteins in different organisms is important to further our understanding of how
these proteins function, and can be used as a guide to design future in vitro experiments.
This data contributes to our growing understanding of the multitude of LEA proteins
expressed in A. franciscana (Hand et al. 2007; Menze et al. 2009; Sharon et al. 2009;
Chen et al. 2009; Warner et al. 2010; Wu et al. 2011; Warner et al. 2012), which arguably
should be considered an animal extremophile (Clegg 2011).
43
CHAPTER 3
GROUP 3 LEA PROTEINS FROM EMBRYOS OF ARTEMIA
FRANCISCANA: STRUCTURAL PROPERTIES AND PROTECTIVE
ABILITIES DURING DESICCATION
3.1 Introduction
Group 3 Late Embryogenesis Abundant (LEA) proteins are a family of proteins
accumulated by organisms, both plant and animal alike, in relation to water stress
(Tunnacliffe and Wise 2007; Tunnacliffe et al. 2010; Hand et al. 2011). Major features
of LEA proteins include high hydrophilicity and low sequence complexity (Cuming
1999; Tunnacliffe and Wise 2007; Hand et al. 2011). One well characterized function
attributed to LEA proteins is their ability to protect the activity of desiccation-sensitive
enzymes against multiple types of water stress (for recent reviews see Tunnacliffe and
Wise 2007; Tunnacliffe et al. 2010; Hand et al. 2011; Hincha and Thalhammer 2012).
Most LEA proteins are predicted to adopt a predominantly α-helical structure in the dried
state. However, investigations into LEA secondary structure reveal that the majority of
LEA proteins are predominantly disordered in solution (Wolkers et al. 2001; Goyal et al.
2003; Shih et al. 2004; Pouchkina-Stantcheva et al. 2007; Tolleter et al. 2007;
Thalhammer et al. 2010; Popova et al. 2011; Hundertmark et al. 2012; Shih et al. 2012).
Gain of structure by LEA proteins during dehydration has led to the hypothesis that LEA
proteins may function specifically in the dry state (e.g. Li and He 2009). Alternatively,
there is also evidence that LEA proteins might function as unstructured proteins in the
hydrated state. Several studies have shown that LEA proteins are able to reduce the
aggregation of polyglutamine (polyQ) or amyloid β-peptide when co-expressed in
44
mammalian cells (Chakrabortee et al. 2007; Liu et al. 2011; Chakrabortee et al. 2012a).
Marunde et al. (2013) showed that a group 1 LEA protein can improve cell viability and
mitochondrial function at very modest levels of water stress, which are unlikely to
promote substantial coiling of LEA proteins. Regardless of whether LEA proteins
function in both hydrated and dry states, structural characterization is an important step in
a comprehensive assessment of individual LEA proteins. In this chapter I investigate the
secondary structure of two group 3 LEA proteins from embryos of A. franciscana
(AfrLEA2 and AfrLEA3m) dried and in solution, as well as their capacity to adopt
secondary structure after the addition of sodium dodecyl sulfate (SDS), and
trifluoroethanol (TFE). In addition to structural studies, I tested the ability of
recombinant AfrLEA2 and AfrLEA3m, both alone and in concert with trehalose, to
afford protection to three different target enzymes during desiccation and subsequent
rehydration.
It is well documented that group 2 LEA proteins have the capability to protect
proteins against freezing (for review see Tunnacliffe and Wise 2007), and protection
during freezing has also been reported for group 3 LEA proteins, although not as
extensively as for group 2 (Honjoh et al. 2000; Goyal et al. 2005). In addition to
protection against water stress imposed by freezing, LEA proteins from groups 1, 2, and
3 can afford protection to enzymes during desiccation (Sanchez-Ballesta et al. 2004;
Grelet et al. 2005; Reyes et al. 2005; Goyal et al. 2005). The ability of LEA proteins to
protect the activity of desiccation-sensitive enzymes from the deleterious effects of
dehydration can, at least partially, be attributed to an ability to prevent enzyme
aggregation. AavLEA1, from the nematode A. avenae, prevents the desiccation induced
45
aggregation of both CS and LDH, thereby protecting the activity of these two enzymes
(Goyal et al. 2005). Goyal et al. (2005) propose that the unordered flexible structure of
LEA proteins allows them to function as a physical barrier between aggregation prone
molecules, a function which they term “molecular shield” activity. Although protection
against protein aggregation is imperative if an organism is to survive desiccation,
prevention of protein denaturation must also be considered (Tompa and Kovacs 2010).
Compared to classic chaperones, direct interactions between LEA proteins and target
molecules are not as well characterized, but evidence for loose interaction has been
reported. Cor15am from Arabidopsis thaliana is capable of direct association with LDH
in vitro as shown by crosslinking experiments (Nakayama et al. 2007). In vivo
experiments were also performed and although no stable interactions were detected,
Cor15am was found to consistently co-purify with the large and small subunits of
Rubisco after crosslinking. Chakrabortee et al. (2012b) provide further evidence for
loose interaction between AavLEA1 tagged with mCherry and a polyQ protein using
quantitative Förster resonance energy transfer.
In addition to protective macromolecules such as LEA proteins, anhydrobiotic
organisms typically accumulate organic solutes such as trehalose, which aid in
macromolecular protection during water stress (Yancey et al. 1982; Yancey 2005).
Trehalose constitutes as much as 20% dry weight of A. franciscana embryos (Crowe et
al. 1987). LEA proteins and trehalose in combination are capable of providing a
synergistic protection to target molecules (Goyal et al. 2005). It is pertinent to note here
that trehalose is not an absolute requirement for desiccation tolerance because it is not
accumulated by bdelloid rotifers (Lapinski and Tunnacliffe 2003; Caprioli et al. 2004) or
46
various tardigrades (Hengherr et al. 2008); however, trehalose undoubtedly plays a role in
the organisms in which it is accumulated. The importance of trehalose is exemplified in
one organism which accumulates the sugar by the observation that A. avenae is not able
to survive desiccation unless ample time is provided for the conversion of glycogen to
trehalose, as occurs during slow drying (Madin and Crowe 1975; Crowe et al. 1977).
3.2 Methods
Recombinant LEA Proteins from A. franciscana
Preparation and purification of recombinant AfrLEA2 and AfrLEA3m were
accomplished according to the procedures described in Boswell et al. (2013). Briefly, the
original nucleic acid sequences were amplified from our existing cDNA library from A.
franciscana, ligated into expression vectors, and then competent bacterial cells were
transformed with the genes. AfrLEA2 was expressed with an N-terminal 6X-His tag, and
AfrLEA3m was expressed with a C-terminal 6X-His tag so as not to interfere with the
mitochondrial localization sequence found at the N-terminus. Bacterial cells were lysed
in the presence of protease inhibitors, cellular debris were removed by centrifugation, and
the resulting supernatant subjected to affinity chromatography on a HisTrap™ FF crude
column (GE Healthcare, Waukesha, WI). Fractions containing recombinant protein were
heat treated and centrifuged to separate the soluble fraction. The protein samples were
then applied to an anion exchange column (HiTrap™ Q FF; GE Healthcare). After
elution, the fractions containing pure recombinant protein were exchanged into LEA
storage buffer and concentrated using Amicon® Ultra Centrifugal filters (Ultracel®-10K;
Millipore, Billencia, MA).
47
Recombinant AfrLEA2 has a total molecular mass of 43.1 kDa (38.9 kDa plus 4.2
kDa for a 6X-His tag and associated vector sequence). AfrLEA3m is a mitochondrial
LEA protein with a deduced molecular mass of 34.1 kDa, which includes the 3.2 kDa
mitochondrial targeting sequence.
Far-UV Circular Dichroism Spectroscopy
CD spectra were recorded on a Jasco J-815 spectropolarimeter (Jasco Analytical
Instruments, Easton, MD). The pathlength was 0.1 cm and measurements were taken for
wavelengths from 190 – 250 nm. Spectra were measured at a protein concentration of
0.14 mg/ml for recombinant AfrLEA2, 0.164 mg/ml for recombinant AfrLEA3m and
0.164 for bovine serum albumin (BSA). The buffer (blank) spectrum was subtracted
from each sample spectrum. After blank subtraction each spectrum was converted to
mean residue ellipticity and smoothed using the Savitzky-Golay method (Savitzky and
Golay 1964) with a convolution width of nine. Buffer subtraction, conversion to mean
residue ellipticity, and smoothing was performed using the Spectra Manager Software
(Jasco Analytical Instruments). For measurements of dried proteins, 50 µl of protein
solution (at the respective concentrations above) were dried on one side of a demountable
cuvette overnight in a drybox containing the desiccant Drierite (W.A. Hammond Drierite
Co. Ltd., Xenia, OH). A pathlength of 0.01 cm was used for conversion of dry data to
mean residue ellipticity.
Secondary structure analyses were performed with the DICHROWEB Web server
(Whitmore and Wallace 2004, 2008) using the following algorithms: CONTINLL
(Provencher and Glockner 1981; van Stokkum et al. 1990), and SELCON3 (Sreerama et
al. 1999; Sreerama and Woody 2000). Reference dataset 7 was used for all analyses
48
because this dataset is optimized for 190 – 240 nm wavelengths and contains denatured
proteins (Sreerama and Woody 2000). Protein disorder was predicted using the
PONDR® VL-XT predictor (Molecular Kinetics Inc., Indianapolis, IN) (Romero et al.
1997; Li et al. 1999; Romero et al. 2001).
Desiccation and Activity Assay of Lactate Dehydrogenase (LDH)
LDH from rabbit muscle was obtained from Sigma Aldrich (St Louis, MO;
product code L2500). Before use, LDH was exchanged into LEA storage buffer (20 mM
Hepes, 50 mM NaCl, pH 7.5) using Amicon® Ultra Centrifugal filters (Ultracel®-10K;
Millipore, Billencia, MA). Then 10 µl droplets of 50 µg/ml LDH, with or without
protectants, were dried in 1.5 ml microcentrifuge tubes at room temperature in a drybox
containing Drierite for one week. Samples were re-hydrated with 20 µl of LEA storage
buffer (diluted two-fold) for 1 h on ice. Control assays of LDH activity were performed
prior to desiccation by adding 5 µl of LDH sample (50 µg/ml) to a final reaction volume
of 1.0 ml, which contained 0.2 M Tris-HCl buffer (pH 7.3), 660 µM NADH and 3 mM
sodium pyruvate. LDH activity after desiccation was measured as described for controls,
except that 10 µl of LDH sample were added to account for the two-fold dilution of the
enzyme during rehydration. Change in A340 was recorded for 1.5 min, and LDH activity
was reported as a percentage of the rate measured for non-dried controls. Each sample
was compared to control values that contained the same mixture of protectants in order to
account for an observed increase in LDH activity in the presence of higher concentrations
of protectant protein. It is appropriate to note that the use of LDH in droplets at ≤ 20
µg/ml yields artifactual results because of non-specific binding of LDH protein to vial
surfaces and time-dependent inactivation that can readily be detected with control
49
samples. Reported values are the average of three separate drying trials each with three
nested replicates (n = 9).
Desiccation and Activity Assay Phosphofructokinase (PFK)
The PFK used in this study was a purified recombinant form of the rabbit muscle
enzyme and was a generous gift from Dr. Simon Chang. Prior to use, PFK was
exchanged into a 100 mM sodium phosphate buffer (pH 8.0) containing 1 mM EDTA
and 5 mM dithiothreitol (DTT) using Amicon® Ultra Centrifugal filters. Then 10 µl
droplets of 150 µg/ml PFK, with or without protectants, were dried in 1.5 ml
microcentrifuge tubes at room temperature in a drybox containing Drierite for 24 h.
Samples were then rehydrated with 20 µl of 50 mM sodium phosphate buffer (pH 8.0)
containing 0.5 mM EDTA and 2.5 mM DTT for 1 h on ice.
Activity was assayed essentially as described by Bock and Frieden (1974).
Briefly, reactions (1 ml assay volume) were performed at 25ºC in a 42 mM Tris-acetate
buffer (pH 8.0) containing 51 mM KCl, 5.1 mM NH4Cl with final concentrations of 0.16
mM NADH, 2 mM ATP, 2 mM fructose-6-phosphate, 2 mM magnesium acetate, 2.5
units glycerol-3-phosphate dehydrogenase, 25 units triosephosphate isomerase, and 2
units aldolase. Prior to use, all accessory enzymes (Sigma Aldrich) were exchanged into
a 100 mM Tris-acetate buffer (pH 8.0, at 6ºC) with 0.1 mM EDTA. PFK activity was
measured for control samples prior to desiccation by adding 5 µl of PFK sample (150
µg/ml) to a total reaction volume of 1 ml reaction mixture described above; for dried
samples 10 µl of PFK was added to account for the two-fold dilution of the enzyme
during rehydration. Change in A340 was recorded for 2 min, and PFK activity was
reported as a percentage of the rate measured for non-dried controls. Each sample was
50
compared to control values that contained the same mixture of protectants in order to
account for an observed increase in PFK activity in the presence of higher concentrations
of protectant protein. Reported values are the average of three separate drying trials each
with three nested replicates (n = 9).
Desiccation and Activity Assay of Citrate Synthase (CS)
CS from porcine heart was obtained from Sigma Aldrich (product code: C3260).
Before use, CS was exchanged into 1 M Tris-HCl (pH 8.1) using Amicon® Ultra
Centrifugal filters. Then 10 µl droplets of 50 µg/ml CS, with or without protectants, were
dried at room temperature in a drybox containing Drierite for 24 h. After one round of
drying, each sample was resuspended with 10 µl H2O and dried a second time for 24 h.
Double dried samples were re-hydrated with 20 µl of 0.5 M Tris-HCl (pH 8.1) for 1 h on
ice. Control CS activity assays were performed prior to desiccation by adding 5 µl of CS
sample (50 µg/ml) to a final reaction volume of 1 ml, which contained 0.1 M Tris-HCl
buffer (pH 8.1), 0.1 mM 5,5’-dithiobis-(2-nitrobenzoic acid) and 0.2 mM acetyl-CoA.
The reaction was initiated by the addition of 0.5 mM oxaloacetate. CS activity after
desiccation was measured as described for controls, except that 10 µl of CS sample were
added to a final reaction volume of 1 ml in order to account for the two-fold dilution of
the enzyme during rehydration. Change in A340 was recorded for 2.0 min, and CS
activity was reported as a percentage of the rate measured for non-dried controls. Each
sample was compared to control values that contained the same mixture of protectants in
order to account for an observed increase in CS activity in the presence of higher
concentrations of protectant protein. Reported values are the average of three separate
drying trials each with three nested replicates (n = 9).
51
3.3 Results
Secondary structure of LEA proteins
Intrinsic disorder predicted by PONDR was 39.84% for AfrLEA2 and 37.13% for
AfrLEA3m. These predictions of disorder as well as secondary structure predictions for
AfrLEA2 and AfrLEA3m reported previously (Hand et al. 2007; Menze et al. 2009) were
tested using CD spectroscopy. In solution the CD spectrum of AfrLEA2 exhibits features
typical of a predominantly disordered, random coiled protein with a minimal ellipticity at
around 200 nm (Fig. 3.1). The presence of either 70% TFE or 2% SDS promoted
AfrLEA2 to adopt substantial α-helical structure as indicated by spectra containing a
double minimum near 208 and 222 nm and a strong positive band at 191 nm (Fig 3.1).
Secondary structure estimates confirm the apparent gain of α-helix content by AfrLEA2
in the presence of 2% SDS and 70 % TFE, with an increase from 4% α-helix content in
solution, to 24% and 41% respectively (Fig. 3.2). Dry AfrLEA2 gains structure and
exhibits a spectrum similar to that induced by the presence of TFE, indicating that
AfrLEA2 adopts a predominantly α-helical conformation upon desiccation (Fig. 3.1). In
fact desiccation of AfrLEA2 causes an increase in α-helix content from 4% in solution to
46% dry as determined from the CD spectra (Fig. 3.2).
Similar to what was observed for AfrLEA2, AfrLEA3m was found to be
predominantly disordered in solution with a propensity to adopt a more α-helical
structure in 2% SDS or 70% TFE solutions (Fig. 3.3); α-helix content increases from 2%
52
Figure 3.1 CD spectra of recombinant AfrLEA2 in the hydrated state, in the presence of
SDS and TFE, and after desiccation.
Figure 3.2 Structural composition of recombinant AfrLEA2 as calculated from the
respective CD data with algorithms described in the Methods section.
53
Figure 3.3 CD spectra of recombinant AfrLEA3m in the hydrated state, in the presence of
SDS and TFE, and after desiccation.
Figure 3.4 Structural composition of recombinant AfrLEA3m as calculated from the
respective CD data with algorithms described in the Methods section.
54
for the hydrated protein to 41% and 36 % respectively (Fig. 3.4). Drying of AfrLEA3m
does not seem to induce as large of a structural shift in the CD spectra as that seen for
AfrLEA2 (Fig. 3.3), but the secondary structure estimation indicates that α-helix content
does increase notably from 2% in solution to 18% dry (Fig. 3.4). AfrLEA3m possesses a
greater percentage of β-sheet in the dry state compared to AfrLEA2, which could explain
the lower α-helix of AfrLEA3m. Finally, it is appropriate to note that both recombinant
proteins contain approximately 10% sequence that is atypical of mature LEA protein.
AfrLEA2 contains a 4.2 kDa segment that represents the 6X-His tag and associated
vector sequence, while the AfrLEA3m protein includes a hydrophobic targeting sequence
of 3.2 kDa.
CD spectroscopy measurements were also gathered for the globular protein BSA.
BSA spectra served two purposes: first as a control protein to check the accuracy of both
CD measurements and deconvolution software, and second as a control protein to which
we could compare the structural changes of AfrLEA2 and AfrLEA3m. The spectrum of
BSA in solution was that of a predominantly α-helical protein (Fig. 3.5). Takeda et al.
(1987) estimated the secondary structure of BSA to contain 66% α-helix, 3% β-sheet and
31% random coil. Secondary structure estimates from my CD data are similar -- 56% α-
helix, 6% β-sheet, and 26% random coil. The presence of either 2% SDS or 70% TFE
did not cause a substantial shift in the BSA spectrum (Fig. 3.5). However, drying did
shift the CD spectra of BSA (Fig. 3.5), but towards a pattern that indicates a greater
percentage of random coil (26% in solution to 52% when dry; Fig. 3.6). This spectra
change is consistent with denaturation of the globular protein in the dried state.
55
Figure 3.5 CD analysis of BSA in the hydrated state, in the presence of SDS and TFE,
and after desiccation.
Figure 3.6 Structural composition of BSA as calculated from the respective CD data with
algorithms described in the Methods section.
56
Protection of enzyme activity by LEA proteins during desiccation
Drying studies with target enzyme-LEA protein combinations were performed to
test the protective abilities of the two recombinant AfrLEA proteins against dehydration-
induced damage. To evaluate whether a cytoplasmic LEA protein preferentially protects
cytoplasmic enzymes, while a mitochondrial LEA protein preferentially protects
mitochondria-localized enzymes, we used a mix-and-match design by choosing target
enzymes that reside in each of these two cellular compartments. Cytoplasmic enzymes
chosen were LDH and PFK, and the mitochondrial enzyme was CS.
After desiccation and storage in the dry state for one week, LDH when rehydrated
exhibited a residual activity of 31 ± 8% (mean ± SD) compared to non-dried control
activity (Fig. 3.7 and 3.8). The residual activity of LDH increased to 66 ± 6% (mean ±
SD) when dried in the presence of 100 mM trehalose. The protection afforded LDH by
the two LEA proteins was similar, but not statistically different from the protection seen
with BSA (1-way-ANOVA, Tukey, P > 0.05), both in the presence and absence of 100
mM trehalose (Fig. 3.7 and 3.8).
The second cytoplasmic enzyme tested was PFK. This enzyme is considered one
of the most dehydration-sensitive enzymes known (Crowe et al. 1987). It is completely
and irreversibly inactivated during freeze drying (Carpenter et al. 1987; Carpenter and
Crowe 1989) and during air drying to less ≤ 3% initial sample water (superfused with
CaSO4-dried nitrogen at 33-35oC; Carpenter and Crowe 1988), perhaps due to the
formation of inactive dimers (Crowe et al. 1992). After drying at room temperature for
24 h, PFK displayed a residual activity of 18 ± 3% (mean ± SD), which was only
57
Figure 3.7 Residual LDH activity after desiccation for one week without additives
(control) or in the presence of different protectants. The protective ability of AfrLEA2
both with and without 100 mM trehalose is compared to equivalent BSA solutions and to
100 mM trehalose alone. Values are reported as percent of the initial LDH activity
measured prior to desiccation (mean ± SD, n = 9).
Figure 3.8 Residual LDH activity after desiccation for one week without additives
(control) or in the presence of different protectants. The protective ability of AfrLEA3m
both with and without 100 mM trehalose is compared to equivalent BSA solutions and to
100 mM trehalose alone. Values are reported as percent of the initial LDH activity
measured prior to desiccation (mean ± SD, n = 9).
58
Figure 3.9 Residual PFK activity after desiccation for 24 h without additives (control) or
in the presence of different protectants. The protective ability of AfrLEA2 both with and
without 100 mM trehalose is compared to equivalent BSA solutions and to 100 mM
trehalose alone. Values are reported as percent of the initial PFK activity measured prior
to desiccation (mean ± SD, n = 9).
Figure 3.10 Residual PFK activity after desiccation for 24 h without additives (control) or
in the presence of different protectants. The protective ability of AfrLEA3m both with
and without 100 mM trehalose is compared to equivalent BSA solutions and to 100 mM
trehalose alone. Values are reported as percent of the initial PFK activity measured prior
to desiccation (mean ± SD, n = 9).
59
increased to 24 ± 5% in the presence of 100 mM trehalose (Fig. 3.9 and 3.10). AfrLEA2
and AfrLEA3m again performed similarly, but both LEA proteins protected PFK far
better than BSA with or without trehalose. In fact, BSA alone did not afford any
protection to PFK, while a remarkable 98 ± 4% of control (non-dried) activity was
preserved when the enzyme was dried in the presence of 400 µg AfrLEA2 plus 100 mM
trehalose (Fig. 3.9), and 103 ± 8% of control activity was preserved when PFK was dried
in the presence of 400 µg AfrLEA3m plus 100 mM trehalose (Fig. 3.10). To our
knowledge this is the first time the protective ability of LEA proteins has been tested with
a target protein possessing such high sensitivity to desiccation.
Lastly I tested the mitochondrial enzyme CS, which was double dried prior to
each assay for residual activity. CS retained 20 ± 3% of control activity after the final
rehydration (Fig. 3.11 and 3.12). Trehalose provided a high level of protection to CS, as
evidenced by a residual activity of 76 ± 5%. As seen with the target enzyme LDH, the
protective abilities of the two LEA proteins plus 100 mM trehalose were similar to one
another, but not statistically different from the protection observed with BSA plus
trehalose (1-way-ANOVA, Tukey, P > 0.05). However, the presence of 400 µg
AfrLEA3m alone protected 69 ± 4 % of the control CS activity (Fig. 3.12), which is
significantly more protection than was provided by the same concentration of either
AfrLEA2 or BSA (1-way-ANOVA, Tukey, P < 0.05; Fig. 3.11).
60
Figure 3.11 Residual CS activity after two 24 h bouts of drying without additives
(control) or in the presence of different protectants. The protective ability of AfrLEA2
both with and without 100 mM trehalose is compared to equivalent BSA solutions and to
100 mM trehalose alone. Values are reported as percent of the initial CS activity
measured prior to desiccation (mean ± SD, n = 9).
Figure 3.12 Residual CS activity after two 24 h bouts of drying without additives
(control) or in the presence of different protectants. The protective ability of AfrLEA3m
both with and without 100 mM trehalose is compared to equivalent BSA solutions and to
100 mM trehalose alone. Values are reported as percent of the initial CS activity
measured prior to desiccation (mean ± SD, n = 9).
61
3.4 Discussion
Despite the high content of α-helix predicted by bioinformatics software (Hand et
al. 2007; Menze et al. 2009), both recombinant AfrLEA2 and AfrLEA3m are
predominantly disordered in solution. This intrinsic disorder in the hydrated state is a
common theme for LEA proteins and is attributed to their highly hydrophilic nature
(Tunnacliffe and Wise 2007). Both AfrLEA2 and AfrLEA3m adopt a higher percentage
of α-helical structure in the presence of the solvents SDS and TFE, and also are found to
gain structure in the dried state. The ability of LEA proteins to gain structure after drying
was first documented by Wolkers et al. (2001). We also show that both AfrLEA2 and
AfrLEA3m can protect sensitive enzymes from the damages imposed by desiccation,
thereby preserving enzyme activity that is typically lost after drying.
The ability of AfrLEA2 and AfrLEA3m to gain structure during drying is in
agreement with many recent studies on LEA proteins (for review see Tunnacliffe et al.
2010; Hand et al. 2011; Hincha and Thalhammer 2012; Tunnacliffe and Wise 2007). A
predominant lack of secondary structure in solution places LEA proteins within a large
class of proteins most commonly called intrinsically disordered proteins or IDPs (for
review see Uversky and Dunker 2010). Buffer solutions containing 70% TFE were used
in order to probe the conformational propensities of recombinant AfrLEA2 and
AfrLEA3m. Both recombinant LEA proteins were found to gain α-helical structure in
the presence of 70% TFE, while the secondary structure of the control protein BSA
remained effectively unchanged. Although the exact mechanism through which this
desolvating agent promotes secondary structure is debated, α-helix induction is thought to
be due largely to desolvation of the polypeptide backbone (Kentsis and Sosnick 1998).
62
Therefore, it can be argued that the promotion of α-helical structure in AfrLEA2 and
AfrLEA3m by TFE is pertinent to secondary structure gained during desiccation. SDS
was also found to also induce α-helical structure in both AfrLEA2 and AfrLEA3m,
although not as effectively as TFE.
The spectra for AfrLEA2 and AfrLEA3m in the dried state indicate that both
proteins gain structure during desiccation. AfrLEA2 adopts a substantial α-helical
structure while AfrLEA3m gains both α-helix and turns. In contrast, BSA becomes more
disordered during drying. This observed loss of structure suggests denaturation, which
would not be unusual for a globular protein like BSA. Of the three conditions evaluated
by CD in the present study (TFE, SDS, dried), the α-helix content of 59% predicted for
AfrLEA2 by bioinformatics software best matches the content observed for the dried
state (45.6%). In addition, intrinsic disorder predicted by PONDR for AfrLEA2
(39.84%) also best matches secondary structure calculated for the dry state (35.7%
random coil). This outcome is consistent with the general case that predictive algorithms,
when applied to LEA protein, often predict the structure of the dried molecule (Tolleter et
al. 2007; Thalhammer et al. 2010; Popova et al. 2011). Similar to what was seen for
AfrLEA2, intrinsic disorder predicted for AfrLEA3m (37.13%) best matches secondary
structure percent calculated for the dry state (39.05%). However, in contrast to
AfrLEA2, AfrLEA3m is predicted to contain 73% α-helix, but based on CD spectroscopy
only adopts 18% α-helix when desiccated. Secondary structure of a group 3 LEA protein
from pollen was shown to be dependent on both the speed of drying and the presence of
sucrose (Wolkers et al. 2001). Rapid drying, as opposed to the slow drying performed for
AfrLEA2 and AfrLEA3m, promoted a higher proportion of α-helical structure; the
63
presence of sucrose caused the LEA protein to adopt an almost entirely α-helical structure
(Wolkers et al. 2001). How such factors might alter the final dried structures of
AfrLEA2 and AfrLEA3m should be tested.
As discussed earlier, gain of structure upon desiccation has led to the prediction
that LEA proteins may function preferentially in the dry state. However, the ability of
LEA proteins to prevent protein aggregation has been shown in solution, also indicating
functionality in the hydrated state (e.g. Chakrabortee et al. 2007). It is likely that both
scenarios are possible. An individual LEA protein could function as a molecular shield
in solution, and the same LEA protein could gain structure as water is removed to further
protect the cell in the dry state by interacting with membranes, stabilizing sugar glasses,
and forming filamentous networks (for extended reviews, see Tunnacliffe and Wise 2007;
Hand et al. 2011).
Experiments evaluating the capacity of AfrLEA2 and AfrLEA3m to protect
desiccation-sensitive, target enzymes from damage during drying showed that this ability
depends on the target protein chosen. For LDH, neither AfrLEA2 nor AfrLEA3m were
able to afford better protection than that provided by BSA, which is in apparent contrast
with reports for other LEA proteins in the literature. However, it should be noted that
AfrLEA2 and AfrLEA3m did afford a high degree of protection to LDH similar to that
seen with other LEA proteins (e.g. Goyal et al. 2005); the difference is that BSA
stabilized LDH in my study far more than previously reported. Small differences in the
final water content of dried samples could explain this inconsistency. Reyes et al. (2005)
reported that in the presence of BSA, LDH exhibited 75% residual activity after being
dried to 2% water content, but activity dropped below 40% at a water content < 2%.
64
Another aspect that has differed substantially among studies is the concentration of LDH
in the test mixture. In the present study LDH was dried at an initial concentration of 50
µg/ml because preliminary observations showed that at lower concentrations the enzyme
lost activity in a time-dependent fashion simply stored on ice for 1 h during rehydration
(data not shown). In comparison, multiple groups have dried or frozen LDH at
concentrations lower than 10 µg/ml (Miller et al. 1998; Honjoh et al. 2000; Sanchez-
Ballesta et al. 2004; Reyes et al. 2005; Goyal et al. 2005; Nakayama et al. 2007). The use
of such low concentrations of LDH could result in artifactual results due to non-specific
adsorption of LDH to vial surfaces.
In the present study AfrLEA2 and AfrLEA3m, when combined with trehalose, are
able to protect nearly 100% of control PFK activity when rehydrated 24 h after
desiccation, and this protection is far greater than that seen with BSA plus trehalose.
Furthermore, the combined effect of LEA protein plus trehalose seems to be synergistic
compared to either of the agents alone. The stabilization of PFK with trehalose alone is
virtually identical to that reported by Carpenter et al. (1987) under similar slow drying
conditions as used herein. Results for the two cytosolic enzymes tested (LDH, PFK) do
not indicate that a cytosolic LEA protein is able to provide better protection to cytosolic
enzymes than is a mitochondrial LEA protein.
Compared to PFK, CS shows significant resistant to desiccation damage and
therefore was subjected to two 24 h bouts of drying in order to cause more damage to the
enzyme. This double drying technique has been utilized previously with CS (for e.g. see
Pouchkina-Stantcheva et al. 2007). The protective abilities of the two LEA proteins
when combined with trehalose are similar to each other and also to BSA plus trehalose.
65
However, AfrLEA3m alone, at the highest concentration tested, provided significantly
more protection to CS than did AfrLEA2 or BSA. A somewhat similar situation of
preferential protection has been reported for LEA protein-lipid membrane interaction,
where a mitochondrial-targeted LEA protein preferentially protected liposomes with a
lipid composition that mimicked the endogenous composition of the inner mitochondrial
membrane, as compared to liposomes with a more generic composition (Tolleter et al.
2010). Previously, a group 3 LEA protein in the absence trehalose was reported to
protect almost 100% of CS activity after two rounds of desiccation (Goyal et al. 2005).
The apparent discrepancy between this previous study and the values presented here
could be due to differences in drying protocols between experiments. Although Goyal et
al. (2005) perform two rounds of desiccation, each round consisted of vacuum drying for
1 h as opposed to our method of air drying for 24 h.
In conclusion AfrLEA2 and AfrLEA3m are intrinsically disordered proteins that
gain a considerable amount of structure upon desiccation. In addition both proteins are
able to protect desiccation-sensitive enzymes from the deleterious effects of desiccation
and subsequent rehydration. These findings serve to not only further define the
molecular characteristics and possible functions of AfrLEA2 and AfrLEA3m, but also
add to the pool of evidence that supports a role for LEA proteins in desiccation tolerance.
66
CHAPTER 4
INTRACELLULAR LOCALIZATION OF GROUP 3 LEA PROTEINS
IN EMBRYOS OF ARTEMIA FRANCISCANA
4.1 Introduction
Anhydrobiotic organisms, such as embryos of A. franciscana, are capable of
surviving almost complete loss of cellular water (Crowe and Clegg 1973; Crowe and
Madin 1974; Crowe and Clegg 1978; Crowe et al. 1992; Clegg 2005; Watanabe 2006;
Welnicz et al. 2011; Cornette and Kikawada 2011). Desiccation tolerance is
accompanied by the accumulation of one or more types of protective molecules that aid
in the survival of water stress. Included here are low molecular weight solutes such as
trehalose (Yancey et al. 1982; Yancey 2005), Late Embryogenesis Abundant (LEA)
proteins (Cuming 1999; Tunnacliffe and Wise 2007; Shih et al. 2008; Tunnacliffe et al.
2010; Hand et al. 2011), and other small stress proteins such as p26 and Artemin (Clegg
et al. 1994; Liang et al. 1997a; Liang et al. 1997b; Willsie and Clegg 2001; Clegg 2005;
Qiu and Macrae 2008a; Qiu and MacRae 2008b). Intracellular localization of protective
molecules to subcellular compartments is important during desiccation, or other forms of
stress, in order to provide complete functional protection to a cell (Hand and Hagedorn
2008; Menze and Hand 2009). In accordance with the need to protect subcellular
structures during stresses such as desiccation, LEA proteins have been identified that
localize to membrane bound organelles such as the mitochondrion in both plants (Grelet
et al. 2005) and animals (Menze et al. 2009; Warner et al. 2010). The protective sugar
67
trehalose has also been documented to reside in mitochondria of A. fransciscana embryos
(J. Reynolds, M. Menze, and S. Hand, unpublished data). In the current study I will
investigate the subcellular localization of multiple group 3 LEA proteins from embryos of
A. franciscana. The proteins to be evaluated are AfrLEA2, which is predicted to be
cytoplasmic (Hand et al. 2007), and AfrLEA3m (Menze et al. 2009), along with closely-
related isoforms AfrLEA3m_47, AfrLEA3m_43 and AfrLEA3m_29 (Boswell et al.
2013) predicted to localize to the mitochondrion. The AfrLEA3m isoforms are studied as
a group because they all are recognized by the primary antibody raised against
AfrLEA3m. Although localization studies for AfrLEA2 and AfrLEA3m have been
performed previously in mammalian cells transfected with these proteins or specific
constructs (Menze et al. 2009; Li et al. 2012), the subcellular distribution of these two
LEA proteins has never been confirmed in A. franciscana.
The intracellular localization of AfrLEA3m was first investigated experimentally
by Menze et al. (2009) who created a nucleotide construct consisting of the first 70 N-
terminal amino acids of AfrLEA3m ligated to GFP. The chimeric protein was transfected
into HepG2/C3A cells and found to co-localize with Mitotracker red, thus verifying a
mitochondrial targeting capability within the first 70 amino acids of AfrLEA3m.
Another interesting conclusion from this study is that mitochondrial import machinery, as
well as the N-terminal mitochondrial targeting sequence of AfrLEA3m, must be highly
conserved between invertebrates and mammals. Li et al. (2012) provided further
confirmation for mitochondrial localization of AfrLEA3m. In this study, full length
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AfrLEA3m expressed in HepG2 Tet-On cells was localized to mitochondria when
viewed using immunocytochemistry.
AfrLEA2 is a group 3 LEA protein that exists primarily as a homodimer in
embryos of A. franciscana (Boswell et al. 2013). The predicted cytoplasmic location of
AfrLEA2 (Hand et al. 2007) was investigated by transient transfection of a plasmid
encoding for a chimeric protein of full length AfrLEA2 plus GFP (Li et al. 2012).
Chimeric AfrLEA2-GFP was found to reside in the cytoplasm when expressed in HepG2
Tet-On cells. The chimeric AfrLEA2-GFP was also visualized in the nucleus 74 h after
transfection. Although nuclear localization is not predicted for AfrLEA2, small GFP-
tagged proteins have been documented to enter the nucleus without the presence of a
nuclear localization signal (Seibel et al. 2007). Nuclear translocation has also been
documented for p26, a small non-LEA stress protein accumulated by A. franciscana
(Clegg 2011).
In addition to the evidence for subcellular localization discussed above, two LEA
proteins from the bdelloid rotifer, Adineta ricciae, have been documented in the
endoplasmic reticulum, golgi and extracellular space (Tripathi et al. 2012). These two
proteins, ArLEA1A and ArLEA1B, are predicted by bioinformatics to contain an N-
terminal ER localization signal and a C-terminal retention signal ATEL. The presence of
the two proteins in multiple intracellular compartments and their excretion into the
extracellular space could enable widespread protection for this desiccation tolerant
organism through the expression of a limited number of protective proteins (Tripathi et
al. 2012).
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4.2 Methods
Cloning, expression and antibody production for recombinant AfrLEA2 and AfrLEA3m
Preparation and purification of recombinant AfrLEA2 and AfrLEA3m was
accomplished according to the procedures described in Boswell et al. (2013). Briefly, the
original nucleic acid sequences amplified from our existing A. franciscana cDNA library,
ligated into expression vectors, and then competent bacterial cells were transformed with
the plasmids. AfrLEA2 was expressed with an N-terminal 6X-His tag, and AfrLEA3m
was expressed with a C-terminal 6X-His tag so as not to interfere with the mitochondrial
localization sequence found at the N-terminus. Expression of recombinant LEA protein
was induced by the addition of IPTG, and confirmed by SDS PAGE. Bacterial cells were
pelleted and chemically lysed in the presence of a protease inhibitor cocktail. After
removal of cellular debris by centrifugation, the cell lysate was subjected to affinity
chromatography on a HisTrap™ FF crude column (GE Healthcare, Waukesha, WI).
Fractions containing recombinant protein were heat treated to separate the soluble
fraction, which was then dialyzed overnight against the starting buffer for anion
exchange. The sample was then applied to an anion exchange column (HiTrap™ Q FF;
GE Healthcare). The fractions containing pure recombinant protein, as assessed by SDS-
PAGE, were exchanged into LEA storage buffer (20 mM HEPES, 50 mM NaCl, pH 7.5)
and concentrated using Amicon® Ultra Centrifugal filters (Ultracel®-10K; Millipore,
Billencia, MA). Antibodies were raised in chickens against recombinant AfrLEA2 and
AfrLEA3m by Aves Labs, Inc. (Tigard, OR).
70
Preparation of A. franciscana embryos for sectioning
Post-diapause embryos of A. franciscana were obtained in the dry state from
Great Salt Lake Artemia (Ogden, UT; grade: laboratory reference standard) and stored at
-20ºC. Prior to use dehydrated embryos were hydrated overnight in ice-cold 0.25 M
NaCl. Embryos were either processed immediately following rehydration, or transferred
to fresh 0.25 M NaCl at 23ºC and incubated with shaking (110 rpm) to promote pre-
emergence development. In preparation for sectioning, embryos were dechorionated as
previously described (Kwast and Hand 1993; Reynolds and Hand 2004) by treatment
with antiformin solution (1% hypochlorate, 0.4 M sodium hydroxide, and 60 mM sodium
carbonate) for 15-20 min at room temperature, followed by three rinses with ice-cold
0.25 M NaCl. In order to deactivate the hypochlorite, embryos were incubated in ice-
cold 1% (w/v) sodium thiosulfate for 5 min and rinsed two times with ice-cold 0.25 M
NaCl followed by an incubation for 3-5 min in ice-cold 40 mM hydrochloric acid
prepared in 0.25 M NaCl, and a final three rinses with 0.25 M NaCl. Prior to further
processing it was necessary to nick the embryo wall because it is only permeable to water
and low molecular weight gasses (Clegg and Conte 1980). Briefly, embryos were
incubated in 2 M sucrose in order to reduce the internal turgor pressure of the embryo,
which prevents extrusion and damage of tissue that would otherwise occur when the cyst
wall is punctured to allow entry of fixative (Hofmann and Hand 1990; Reynolds and
Hand 2004); then embryos were nicked with a needle while being viewed under a
dissecting microscope Next embryos were transferred into 0.2 M potassium phosphate,
pH 7.8, with 4 % paraformaldehyde and 1000 mOsm sucrose for fixation. Fixed samples
were then embedded in paraffin, and 2 µm sections were prepared.
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Immunofluorescent staining
Embryo sections mounted on slides were deparaffinized by two 10 min
incubations in xylene and then gradually rehydrated with the following incubations: 10
min in 100% ethanol, 10 min in 100% ethanol repeated, 5 min in 95% ethanol, 5 min in
70% ethanol, 5 min in 50% ethanol. Finally, sections were rinsed and incubated for 5
min in phosphate buffered saline (PBS) to achieve complete rehydration. Hydrated
sections of embryos were blocked with 5% (vol/vol) normal rabbit serum (Jackson
ImmunoResearch, West Grove, PA) in PBS for 1 h. Primary antibody incubation was
performed overnight at 4ºC. Chicken anti-AfrLEA2 and chicken anti-AfrLEA3m IgY
(Aves Labs, Inc., Tigard, OR) were used at a dilution of 1:400 in 5% normal rabbit
serum, and goat anti-VDAC IgG (Santa Cruz Biotechnology, Dallas, TX) was used at a
dilution of 1:100 in normal rabbit serum. Slides were then washed six times (10 min
each) with PBS followed by incubation with the appropriate secondary antibody for 1 h.
Secondary antibodies were Alexa Fluor® 488-conjugated AffiniPure Rabbit Anti-
Chicken IgY (IgG) (H+L) (excitation, 493; emission, 519; Jackson ImmunoResearch),
and Rhodamine (TRITC)-conjugated AffiniPure Rabbit Anti-Goat IgG (H+L) (excitation,
550 nm; emission, 570 nm; Jackson ImmunoResearch) used at a dilution of 1:500 in 5%
(v/v) normal rabbit serum. After secondary antibody incubation, slides were washed six
times (10 min each) with PBS. To visualize nuclei, sections were treated with ProLong
Gold antifade reagent with DAPI (Life Technologies, Carlsbad, CA) in the case of
deconvolution microcopy. When confocal microscopy was to be used, nuclei were
stained with DRAQ5, which was added to the secondary antibody solution at a final
72
concentration of 5 µM. After incubation with secondary antibody containing DRAQ5,
cells were washed as above and treated with ProLong Gold antifade reagent.
Microscopic imaging
For deconvolution microscopy, embryo sections were viewed using a Leica DM
RXA2 upright microscope (Leica Microsystems Inc., Buffalo Grove, IL) and images
were captured with a SensiCam QE 12-bit, cooled CCD camera (PCO-TECH Inc.,
Romulus, MI). Image analysis was performed using SlideBook™ 4.1.0 software (3I
Intelligent Imaging Innovation Inc., Denver, CO). Deconvolution of images was
performed with SlideBook™ 4.1.0 software and used the no neighbors algorithm.
For confocal imaging, embryos were viewed using a Leica TCS SP2 spectral
confocal microscope with Leica LCS Software (Leica Microsystems Inc.). Three lasers,
488 nm at 50% power, 543 nm at 70% power, and 633 nm at 45% power, were used to
sequentially excite Alexa Fluor 488, TRITC, and DRAQ5. Emission passes of 500-535,
558-613, and 655-746 were used to detect the signals.
4.3 Results
Embryos of A. franciscana (Fig. 4.1) are a partial syncytium of about 4,000
nuclei. These encysted gastrula are surrounded by a complex shell that is composed of
cross-linked protein and subtended by an impermeable cuticular membrane of chitin
(Clegg and Conte 1980). Yolk platelets (lipoprotein storage organelles) are the most
abundant structure in the crowded and volume-restricted cytoplasm (Warner et al. 1972).
Mitochondria at this stage are in low abundance and only occupy ~5% of total cellular
73
Figure 4.1 Embryos of A. franciscana visualized at 0 h of pre-emergence development by
differential interference contrast (DIC) microscopy. Nuclei are stained with DAPI.
volume (Rees et al. 1989). Trehalose comprises 15% of embryo dry weight (Clegg
2005). The multilayer shell, numerous yolk platelets, and nuclei are clearly visualized
with differential interference contrast (DIC) microscopy (Fig. 4.1).
Endogenous AfrLEA2 is documented with immunohistochemistry to reside in
both the cytoplasm and nucleus of embryonic cells of A. franciscana and to be absent
from mitochondria. The nuclei, visualized with either DAPI or DRAQ5, appear to
contain AfrLEA2 when visualized at 0 h (Fig 4.2) and 6 h (Fig 4.3) of pre-emergence
development. However, the nuclear-localized AfrLEA2 did appear to increase in
embryos viewed after 6 h pre-emergence development compared to 0 h. Merged images
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Figure 4.2 Subcellular localization of AfrLEA2 in embryos of A. franciscana visualized
at 0 h of pre-emergence development. AfrLEA2 (A, D and E, green) is present in the
cytoplasm as well as the nucleus (C and E, pseudocolor blue) and does not appear to co-
localize with VDAC (B and D, red).
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Figure 4.3 Subcellular localization of AfrLEA2 in embryos of A. franciscana visualized
after 6 h of pre-emergence development. AfrLEA2 (A and C, green) is present in the
cytoplasm as well as the nucleus (B and C, blue).
show a co-localization of AfrLEA2 and nuclear staining (Fig. 4.2, E and Fig. 4.3, C) and
a lack of co-localization for AfrLEA2 and VDAC (Fig. 4.2, D). A cytoplasmic location
for AfrLEA2 is in agreement with previously reported subcellular predictions (Hand et al.
2007), and translocation to the nucleus is in agreement with the detection of a chimeric
AfrLEA2-GFP protein in the nucleus of HepG2 Tet-On cells 3 d after transfection (Li et
al. 2012).
The predicted mitochondrial location of AfrLEA3m proteins is confirmed in
embryos of A. franciscana (Fig. 4.4). The co-localization of AfrLEA3m with VDAC,
indicated by the yellow color in merged images, supports a mitochondrial location (Fig.
76
4.4, D). I note that co-localization of AfrLEA3m proteins and VDAC was not always
uniform, which could perhaps be a consequence of fluorescence from below the plane of
focus or heterogeneous amounts of AfrLEA3m across the mitochondrial population. It is
also possible that the VDAC antibody has easier access to antigen due to the proteins
location in the outer mitochondrial membrane.
Figure 4.4 Subcellular localization of AfrLEA3m (and its closely related variants
recognized by AfrLEA3m antibody) in embryos of A. franciscana visualized at 0 h of
pre-emergence development. The co-localization of AfrLEA3m proteins (A and D,
green) with VDAC (B and D, red) indicates mitochondrial targeting; areas of co-
localization appear yellow (D). Nuclei (C, pseudocolor blue) are stained with DRAQ5.
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4.4 Discussion
This study provides evidence that supports the cellular locations for AfrLEA2 and
AfrLEA3m proteins in A. franciscana embryos as predicted by bioinformatic analyses.
Although the cellular location of recombinant AfrLEA2 and AfrLEA3m have been
investigated previously when transfected into mammalian cells (Menze et al. 2009; Li et
al. 2012), the cellular localization of these two proteins, as well as of the newly identified
AfrLEA3m_47, AfrLEA3m_43, and AfrLEA3m_29 had yet to be confirmed in embryos
of A. franciscana. Multiple subcellular localizations of LEA proteins, in combination
with the capacities for individual LEA proteins to stabilize different classes of
macromolecules, may explain why a multitude of LEA proteins are expressed in a single
organism. For example, 51 different LEA proteins have been identified in the plant
Arabidopsis thaliana (Hundertmark and Hincha 2008; Bies-Etheve et al. 2008).
Predicted subcellular localization for LEA proteins from A. thaliana includes localization
to the cytosol, chloroplast, mitochondria, and entry into the secretory pathway
(Hundertmark and Hincha 2008).
Immunohistochemistry performed with AfrLEA2 antibody provides evidence for
nuclear localization of this antigen in A. franciscana embryos. This observation is in
agreement with the previously reported nuclear translocation for a chimeric AfrLEA2-
GFP protein expressed in HepG2 Tet-On cells (Li et al. 2012). AfrLEA2-GFP was found
to be cytoplasmic when visualized 24 h after transfection, but after 3 d was also detected
in the nucleus. GFP itself is known to enter the nucleus over time so care must be taken
when concluding a nuclear location based on a GFP-fusion protein (Seibel et al. 2007).
However, the visualization of AfrLEA2 in the nuclei of A. franciscana embryos supports
78
the nuclear localization observed by Li et al. (2012). Warner et al. (2012) identified five
putative LEA proteins in the nuclei of A. franciscana embryos via western blotting with
antibodies raised against group 3 LEA proteins from A. ricciae and A. avenae. However,
to our knowledge AfrLEA2 is the first rigorously-identified LEA protein to be localized
in the nucleus of animals. The accumulation of AfrLEA2 in both the cytoplasm and the
nucleus could allow a single LEA protein to protect a broader range of cellular
components. A similar example exists for a bdelloid rotifer (Tripathi et al. 2012), where
two LEA proteins were documented in the endoplasmic reticulum, golgi, and
extracellular space.
As previously stated, AfrLEA3m is predicted to localize to the mitochondrion
(Menze et al. 2009), and the newly-identified AfrLEA3m_47, AfrLEA3m_43 and
AfrLEA3m_29 are also predicted to be targeted there with the same probabilities based
on bioinformatics. AfrLEA3m_43, which has one amino acid substitution within the
predicted leader sequence, has a probability that is even higher. Immunohistochemistry
generally supports a mitochondrial localization for the proteins, although some limited
discrepancies between the AfrLEA3m proteins and VDAC were observed. As
emphasized earlier, the small cell volume (5%) occupied by mitochondria in embryos of
A. franciscana is several-fold lower than for many other cell types; for example, rat
hepatocytes are estimated to have a value of 23% (Beauvoit et al. 1994). 1994). This
feature, coupled with the limited amount of free cytoplasmic space due to numerous yolk
platelets (cf. Fig 4.1), makes resolution of the mitochondrial distribution challenging at
the level of fluorescence microscopy. Mitotracker red is the most conventional
fluorescent probe for mitochondrial imaging, but its use was unsuccessful here. Although
79
Mitotracker is retained in the mitochondrion after fixation, mitochondrial accumulation is
dependent upon an electrical potential across the inner mitochondrial membrane. Post-
diapause embryos used in this study are a partial syncytium of about 4,000 nuclei
surrounded by a complex shell impermeable to all molecules but water and low
molecular weight gasses (Clegg and Conte 1980). The shell must be nicked in order for
substances such as Mitotracker and fixative to enter A. franciscana embryos (see
Methods). Lack of Mitotracker accumulation by embryos for which the permeability
barrier has been ruptured may be due to the loss of oxidative substrates that support
electron transport and the associated membrane potential. Therefore, the addition of
substrates such as succinate to the medium during incubation with Mitotracker in the
future may improve the results with this probe.
It is probable that embryos of A. franciscana contain additional, and as yet
unidentified, LEA proteins that localize to additional subcellular compartments. For
instance, the ER and golgi of A. ricciae contain LEA proteins (Tripathi et al. 2012), and
plant LEA proteins have been found in the cytoplasm, mitochondrion, nucleus,
chloroplast, endoplasmic reticulum, vacuole, peroxisome, and plasma membrane
(Tunnacliffe and Wise 2007). Antibodies raised against two group 3 LEA proteins from
A. ricciae and Aphelenchus avenae each recognize a multitude of putative LEA proteins
in heat-soluble protein fractions prepared from isolated organelles of A. franciscana
(Warner et al. 2012). While interesting, further work is needed to verify that the proteins
recognized are in fact LEA proteins. Identification and subsequent characterization of
the full complement of LEA proteins expressed by embryos of A. franciscana awaits
release of the Artemia genome scheduled very soon.
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In conclusion, I provide evidence supporting the subcellular localization of
AfrLEA2 to both the cytoplasm and nucleus and of the AfrLEA3m proteins to the
mitochondrion. The presence of LEA proteins in multiple subcellular compartments
underscores an apparent need for protective molecules in all areas of a cell, and perhaps
even in the extracellular space, in order for an organism to survive the stresses imposed
by desiccation.
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CHAPTER 5
SUMMARY AND FUTURE DIRECTIONS
The results presented in this dissertation add to the pool of evidence that supports
a role for LEA proteins in desiccation tolerance by providing molecular characteristics,
expression data, and functional studies for group 3 LEA proteins from embryos of A.
franciscana. In chapter 2 three new Afrlea mRNA (Afrlea3m_47, Afrlea3m_43, and
Afrlea3m_29) are sequenced and characterized (Boswell et al. 2013). Deduced protein
sequences from these mRNA are very similar to the previously identified AfrLEA3m
(Menze et al. 2009) and are predicted to reside in the mitochondrion. Due to multiple
base pair differences these mRNA are predicted to arise from different genes. Protein
expression levels were investigated for four group 3 LEA proteins (AfrLEA2,
AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29) and found to be highest in diapause
embryos (a desiccation-tolerant stage) and decrease throughout pre-emergence
development to lowest levels in desiccation-intolerant nauplius larvae. Protein
expression levels for AfrLEA2, which exists primarily as an dimer in A. franciscana,
agree with mRNA expression levels reported by Hand et al. (2007), and AfrLEA2 is
present in diapause embryos at a concentration of 1.85 mg/ml embryo water. Protein
expression levels for AfrLEA3m also agree with previous mRNA reports by Menze et al.
(2009) and mitochondrial AfrLEA proteins have a combined concentration of 2.2 mg/ml
matrix volume.
Structural investigations determine AfrLEA2 and AfrLEA3m to be intrinsically
disordered proteins (Chapter 3) that gain structure after desiccation, and in the presence
of TFE or SDS. Recombinant AfrLEA2 and AfrLEA3m are also able to provide
82
protection to sensitive enzymes during desiccation. The ability of LEA proteins such as
AfrLEA2 and AfrLEA3m to gain structure in the dry state leads to a predicted dry
function (e.g. Li and He 2009), but evidence also exists that LEA proteins function in
solution (Chakrabortee et al. 2007; Liu et al. 2011; Chakrabortee et al. 2012). As
mentioned in chapter 3, it is possible that a single LEA protein could function both in the
hydrated and dry state, thereby providing maximum protection to cellular components
throughout the entire drying process.
This hypothesis that LEA proteins may show dual functionality, both in the
hydrated and dry state, can be supported by combining various pieces of literature in
attempt to present a more complete picture of LEA protein protection as a cell is
subjected to the desiccation process. LEA protein functionality in the hydrated state can
be at least partially attributed to molecular shield activity as defined by Goyal et al.
(2005). In this context LEA proteins prevent aggregation of sensitive enzymes by acting
as a physical barrier between molecules during the initial stages of dehydration.
Subsequently, as the desiccation process progresses past a water content of approximately
20% LEA proteins should begin to adopt secondary structure (Li and He 2009). At this
point LEA proteins could begin to perform functions suggested in the “dry” state. For
example, membrane interaction would require that a LEA protein first form amphipathic
α-helices, which could then insert into membranes parallel to their plane (Tolleter et al.
2007). The potential of various LEA proteins to form coiled coils during drying (Goyal
et al. 2003) provides support for the hypothesis that some LEA proteins may form
intracellular filaments during desiccation that would serve to increase the mechanical
strength of desiccated cells (Wise and Tunnacliffe 2004). This increase in mechanical
83
strength would provide protection against the physical stresses experienced by a cell
during desiccation (Wolfe et al. 1986). These hypothetical LEA protein filaments could
also work in concert with sugar glasses in a manner that has been compared to “steel-
reinforced concrete” (Goyal et al. 2003). Structure gained by LEA proteins during drying
is fully reversible (Wolkers et al. 2001), allowing return to the intrinsically disordered
state during rehydration, and prevention of protein aggregation during sub-optimal water
contents experienced until full hydration is achieved.
The possibility of dual functionality, or “moonlighting” of LEA proteins at
differing water contents is exemplified by LEA proteins such as LEAM (also called
PsLEAm) found in pea seed mitochondria (Grelet et al. 2005). LEAM is a group 3 LEA
protein that has been shown to protect both proteins and membranes from desiccation
stress (Grelet et al. 2005; Tolleter et al. 2007). There will of course be exceptions to the
dual functionality hypothesis, as is exemplified by ArLEA1B from a bdelloid rotifer
(Pouchkina-Stantcheva et al. 2007). ArLEA1B is an atypical group 3 LEA protein that is
predominantly α-helical in solution. This particular LEA protein is not able to act as a
molecular shield, as shown by an inability to protect CS from the deleterious effects of
desiccation, but does interact with membranes significantly decreasing the gel-to-liquid
crystalline phase-transition temperature (Tm) of dry liposomes.
Another structural element adopted by some LEA proteins is a solvent-exposed,
left-handed extended helix also called poly (ʟ-proline)-type (PII) conformation (Soulages
et al. 2002; Soulages et al. 2003; Mouillon et al. 2006). These groups show LEA proteins
from groups 1 and 2 to adopt increasing PII helices as a function of decreasing
temperature, but the possible implications of this observation could be more widespread.
84
Mouillon et al. (2006) suggest that the PII helix could act as an intermediate structure for
example: in between random coil and the adoption of α-helix or β-sheet during
desiccation. This group speculates that the intermediate PII helix could hold more tightly
to bound water during desiccation increasing the capability of LEA proteins to act as a
hydration buffer during desiccation. The physiological relevance of LEA proteins as a
hydration buffer has since been challenged (Hand et al. 2011); however, formation of PII
helices could still hold relevance, possibly by prolonging the molecular shield activity of
some LEA proteins to lower water contents before adoption of structure and related
function as discussed above. It is pertinent to note here that Shih et al. (2008) suggest
temperature scans performed by Goyal et al. (2003) on AavLEA1 indicate the formation
of PII helices, although this was not a conclusion of the original study. Thus, there is
evidence that LEA proteins from all three major groups are capable of forming PII
helices.
Finally, the predicted cellular location of AfrLEA2 (cytoplasm) and the four
AfrLEA proteins recognized by anti-AfrLEA3m antiserum (mitochondrial) have been
confirmed in embryos of A. franciscana (Chapter 4). In addition to the predicted
cytoplasmic location, AfrLEA2 was also located in embryo nuclei. Localization of
protective molecules such as LEA proteins to membrane bound organelles is fundamental
if an organism is to survive desiccation stress (Hand and Hagedorn 2008; Menze and
Hand 2009). In order to achieve a complete understanding of LEA protein function, the
entire complement of LEA proteins expressed in an individual organism first needs to be
documented. This possibility is on the horizon regarding A. franciscana, for which the
genome will be published shortly [Artemia Genome Day, September 2, 2013, Laboratory
85
of Aquaculture & Artemia Reference Center, Ghent University, Belgium]. As mentioned
earlier, differential cellular localization is one reason for an organism to express multiple
LEA proteins. In this case sequencing of the A. franciscana genome could allow
characterization of all AfrLEA proteins that are located to different subcellular locations.
However, differential subcellular localization alone does not fully explain the expression
of multiple LEA proteins because more than one LEA protein can be found in a single
subcellular location. Take the mitochondria from embryos of A. franciscana for
example. Currently, multiple group 1 (Warner et al. 2010; Marunde et al. 2013) and
group 3 (Menze et al. 2009; Boswell et al. 2013) LEA proteins have been identified that
localize to the mitochondrion of these desiccation-tolerant embryos. It is possible that the
presence of multiple LEA proteins in a single subcellular compartment is necessary for
the simultaneous protection of different classes of macromolecules. As the complete
array of LEA proteins in each subcellular location is discovered, we can begin to
investigate differential protection. Some LEA proteins like LEAM (Grelet et al. 2005)
may moonlight, while others like ArLEA1B (Pouchkina-Stantcheva et al. 2007) may
perform specific roles.
The mechanisms behind desiccation tolerance are not only a fundamental mystery
that we strive to comprehend for the most basic reason of increasing our ever growing
understanding of biology, but also are mechanisms that we want to be able to confer to
desiccation sensitive molecules and even entire tissue systems. In order to reach long
term goals, like stabilization of mammalian cells for example (cf., Crowe et al. 2005;
Huang and Tunnacliffe 2007; Hand and Hagedorn 2008; Li et al. 2012), we need to be
able to apply the mechanisms of desiccation tolerance learned from anhydrobiotic
86
organisms to desiccation-sensitive systems. Basic characterization of protectant
molecules, as was performed in this dissertation, is necessary in reaching goals such as
the stabilization of dry cells.
Another possible application for LEA and other intrinsically disordered proteins
lies within the medical field. Diseases like Alzheimer’s are linked to conformational
changes in certain proteins leading to their aggregation. Multiple groups have shown that
LEA proteins are capable of limiting the aggregation tendencies of proteins in vivo
(Chakrabortee et al. 2007; Liu et al. 2011; Chakrabortee et al. 2012). Therefore, it is
possible that LEA proteins could be used to prevent, or at least delay disease causing
protein aggregation.
87
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VITA
Leaf Chandra Boswell was born and raised in Covington Louisiana. As a child,
Leaf’s enthusiasm for science was enhanced by a lack of cable television and love of the
outdoors. Leaf’s father taught high school biology, chemistry, and environmental
science, which afforded her many opportunities such as attending plant identification
field trips with her father’s classes and raising a menagerie of orphaned wild animals.
After completing high school at Saint Scholastica Academy, Leaf attended Louisiana
State University and was awarded a bachelor’s degree in Biological Sciences.