CHAPTER 2

LITERATURE REVIEW

2.1 Pichia pastoris

2.1.1 History of the Pichia pastoris expression system

Pichia pastoris is a methylotrophic budding yeast that first described by

Koichi Ogata in 1969. It was initially developed as single cell protein (SCP) to be

used primarily as high protein animal feed (Ogata et al., 1969; Cereghino and Cregg,

2000). During the 1970s, Phillips Petroleum Company developed media and protocols

for growing P. pastoris on methanol in continuous culture at high cell densities (<130

g/1 dry cell weight) as shown in figure 2.1. Unfortunately, the oil crisis caused a

dramatic increased in the cost of methane and the cost of soybeans which is the major

alternative source of animal feed was decreased. These results made the economics of

SCP production from methanol were never favorable (Cereghino and Cregg, 2000).

In the early 1980’s, Salk Institute Biotechnology/Industrial Associates

(SIBIA) Inc was contracted to develop P. pastoris as an expression system for

heterologous protein production. Researchers at SIBIA isolated the gene and promoter

for alcohol oxidase, and generated vectors, strains, and corresponding protocols for

the molecular genetic manipulation of P. pastoris. The combination of the

fermentation methods developed for the SCP process and the strong regulated

expression under control of AOX1 promoter resulted in surprisingly high levels of

foreign protein expression in P. pastoris. In 1993, Phillips Petroleum sold its P.

pastoris expression system patent position to Research Corporation Technologies, the

current patent holder. In addition, Phillips Petroleum licensed Invitrogen Corporation

to sell components of the system, an arrangement that continues under Research

Corporation Technologies (Higgins and Cregg, 1998; Cereghino and Cregg, 2000).

4

Figure 2.1 The culture of P. pastoris. The left bottle shows a P. pastoris culture

grown in a flask to a density of 1 OD600 unit. The right bottle contains a sample of

the strain grown in a fermenter to a density of 130 g/1 dry cell weight (~ 500 OD600

units). (Cereghino and Cregg, 2000)

2.1.2 Methanol metabolism of Pichia pastoris

Pichia pastoris is able to utilize methanol as the sole carbon and energy

source. All methylotrophic yeast strains identified to date belong to only four genera:

Hansenula, Pichia, Candida and Torulopsis (Jungo et al, 2007). The methanol

metabolic pathway as the same in all yeasts and unique set of pathway enzymes

(Higgins and Cregg, 1998). In P. pastoris, alcohol oxidase (AOX, EC 1.1.3.13) is the

first enzyme in the methanol utilization pathway. This enzyme consists of eight

identical subunits of approximately 660 amino acids, each containing a non-

covalently bound flavin adenine dinucleotide (FAD) moiety. The subunits are

produced in the cytoplasm, transported as monomers into the peroxisomes and inside

the peroxisomes active, FAD containing octamers are formed (Cregg et al., 1988). In

cells grown on methanol, the AOX enzymes are present at high levels (up to 35% of

the total cell protein). Due to the fact that AOX has a low affinity for oxygen; the cell

therefore compensates by producing the enzyme in large amounts. (Cregg et al., 1988;

Jungo et al, 2007)

5

For the methanol utilization pathway, the oxidation of methanol to

formaldehyde and generating hydrogen peroxide in the process as shown in figure 2.2.

A by-product, hydrogen peroxide, is subsequently degraded to water and oxygen by a

second peroxisomal enzyme, catalase (Cat). The formaldehyde generated by AOX

follows one of two paths. A portion leaves the peroxisome and is further oxidized by

two cytoplasmic enzymes, formaldehyde dehydrogenase (Fld) and formate

dehydrogenase (Fdh), to generate energy for the cell. (Cereghino et al., 2005) The

remaining formaldehyde is assimilated to form cellular constituents by a cyclic

pathway that starts with the condensation of formaldehyde with xylulose 5-

monophosphate by a third peroxisomal enzyme dihydroxyacetone synthase (DHAS).

The products of this reaction, glyceraldehyde 3-phosphate and dihydroxyacetone,

leave the peroxisome and enter a cytoplasmic pathway that regenerates xylulose 5-

monophosphate and produces one net molecule of glyceraldehyde-3-phosphate for

every three turns of this cycle (Cereghino and Cregg, 2000).

Figure 2.2 The methanol pathway in Pichia pastoris. 1, alcohol oxidase; 2, catalase;

3, formaldehyde dehydrogenase; 4, formate dehydrogenase; 5, dihydroxyacetone

synthase; 6, dihydroxyacetone kinase; 7, fructose 1,6-biphosphate aldolase; 8,

fructose 1,6-bisphosphatase ( Cereghino and Cregg, 2000).

6

2.1.3 Pichia Expression system

The methylotrophic yeast Pichia pastoris has been widely used for the high-

yield expression of various proteins either by secreting into the culture supernatant or

by intracellular localization. The yeast can be genetically engineered to express

proteins for both basic research and industrial use (Leonardo et. al., 2004). Over 550

such proteins have been expressed in this yeast (Cereghino et al., 2005). P. pastoris

has many of the advantages of higher eukaryotic expression systems such as protein

processing, protein folding, and posttranslational modification, while being as easy to

manipulate as Escherichia coli or Saccharomyces cerevisiae (Mewes et al., 2002). It

is faster, easier, and less expensive to use than other eukaryotic expression systems

such as baculovirus or mammalian tissue culture, and generally gives higher

expression levels. As a yeast, it shares the advantages of molecular and genetic

manipulations with S. cerevisiae, and has the added advantage of 10 to 100-fold

higher heterologous protein expression levels. These features make P. pastoris very

useful as a protein expression system (Invitrogen Corp., 1998).

2.1.3.1 Expression vector

The foreign gene expression in Pichia pastoris requires three basic steps as

follow; the insertion of gene into an expression vector, the introduction of the

expression vector into the P. pastoris genome and the examination of potential

expression strains for the foreign gene product (Cereghino and Cregg., 2000). A

variety of P. pastoris expression vectors and host strains are available.

The plasmid vectors designed for heterologous protein expression in P.

pastoris have several common features. All expression vectors have been designed as

Escherichia coli/P. pastoris shuttle vectors (Figure 2.3), containing an origin of

replication for plasmid maintenance in E. coli and markers functional in one or both

organisms. The foreign gene expression cassette is composed of DNA sequences

containing the P. pastoris AOX1 promoter, followed by a unique restriction sites or

multiple cloning sites (MCS) for insertion of the forging coding sequence, and

followed by the transcriptional termination sequence from the P. pastoris AOX1 gene

that directs efficient 3’ processing and polyadenylation of the mRNAs. (Cereghino

and Cregg, 2000; Higgins and Cregg, 1998)

7

Figure 2.3 General diagram of P. pastoris expression vector

(Cereghino and Cregg, 2000)

Generally, the best expression results are obtained when the first ATG of the

heterologous coding sequence is inserted as close as possible to the position of the

AOX1 ATG. This position concurs with the first restriction site in most MCS

(Cereghino and Cregg, 2000). Additionally, for secretion of foreign proteins, vectors

have been constructed to contain the AOX1 promoter that encodes a secretion signal

of P. pastoris acid phosphatase gene (PHO1) or S. cerevisiae α factor prepro signal

sequence. And then, vectors with dominant drug resistance markers that allow for

enrichment of strains that receive multiple copies of foreign gene expression cassettes

during transformations have been developed (Cereghino and Cregg, 2000; Higgins

and Cregg, 1998).

The first generation of P. pastoris expression vectors, such as pHIL-D2 or

pPIC9 contained the functional histidine dehydrogenase gene (HIS4), which can be

used as a selectable marker following transformation into his4 (histidine

dehydrogenase-deficient strain) P. pastoris by the integration/transformation method

of choice. Subsequently, a gene conferring resistance to G418 (kanamycin) was added

8

to these vectors to produce the pPIC9K expression vector. This approach allows

transformation and selection for His+ transformants that are resistant to high levels of

G418 (kanamycin) and therefore can contain multiple copies of the expression vector

As the use of the HIS4 gene does not allow direct selection of multi-copy

integrants (and the gene itself is large), these constraints have led to efforts to find

alternate selection markers in order to directly select multi-copy integrants, as well as

to decrease vector size, resulting in the generation of vectors based on the ability of

the Sh ble gene product that confer resistance to the antibiotic zeocin in both bacteria

and yeast (Figure 2.4).

Figure 2.4 Vector diagram of pPICZ (Invitrogen Corp., 1998)

These zeocin-resistant vectors contain only the 5’AOX1 promoter region and

the AOX1 transcription terminator as well as the Sh ble gene from Streptoalloteichus

hindustanus. Because the ble gene serves as the selectable marker for both E. coli and

P. pastoris, the zeocin-resistant vectors are much smaller (3.3 kb) and easier to

manipulate than other P. pastoris expression vectors. These vectors also contain a

multiple cloning site (MCS) with several unique restriction sites for convenience of

foreign gene insertion and sequences encoding the polyhistidine (His6) and myc

epitopes so that foreign proteins can be easily epitope tagged at their carboxyl termini

for detection and purification of a recombinant fusion protein (Higgins and Cregg,

9

1998). This vector allows high-level, methanol inducible expression of the gene of

interest in Pichia, and can be used in any P. pastoris strain including X-33, GS115,

SMD1168H, and KM71H (Invitrogen corp, 1998).

Furthermore, selection of zeocin-resistant transformants at high concentration

of zeocin can be generated an enrichment in recombinant strains with multiple copies

of the integrated vector that may result in an increase in the level of heterologous

protein production (Higgins and Cregg, 1998).

2.1.3.2 The promoter in Pichia pastoris

In Pichia pastoris, the interested gene can be expressed under the control of

inducible and constitutive promoter.

Inducible promoter

AOX1 promoter is one of the strongest promoter and widely used for

heterologous protein expression. (Cereghino and Cregg, 2000; Leonardo et. al., 2004).

In the wild-type P. pastoris strain, AOX1 promoter controls the

expression of alcohol oxidase 1 which is the first enzyme in methanol metabolism.

There are two copies of the alcohol oxidase (AOX) gene in the genome of P. pastoris,

called AOX1 and AOX2. AOX1 is responsible for a vast majority of alcohol oxidase

activity in the cell. (Cereghino and Cregg, 2000) The regulation of the AOX1 gene is

a two step process: a repression/derepression mechanism plus an induction

mechanism (e.g. GAL1 gene in Saccharomyces). The AOX1 promoter is tightly

repressed by glucose and most other carbon sources but is induced >1000-fold in cells

shifted to methanol as a sole carbon source. With this promoter, expression of

recombinant proteins is highly repressed while cultures are grown to high density in

glucose or glycerol, which prevents selection for non-expressing mutant cells.

Cultures are then shifted to a methanol medium to induce rapid high-level expression

(Cereghino and Cregg, 1999).

For some applications, the high level of expression from AOX1

promoter may impact on the post-translational machinery of the cell, causing a

significant proportion of foreign protein to be misfolded, unprocessed, or mislocalized

(Thill et al., 1990; Brierley, 1998). For these and other applications, moderately

10

expressing promoters are desirable. Shen et al., 1998 described that there is the

promoter derived from the P. pastoris FLD1 gene, whose product is a glutathione-

dependent formaldehyde dehydrogenase, can be induced either by methanol or

methylamine (a nontoxic nitrogen source) in glucose containing media. Expression

levels from the methylamine-induced FLD1 promoter are comparable to those

obtained with the AOX1 promoter in methanol. After induction with either methanol

or methylamine, FLD1 promoter is able to express levels of an L-lactamase reporter

gene similar to those obtained with methanol induction from the AOX1 promoter. The

FLD1 promoter offers the flexibility to induce high levels of expression using either

methanol or methylamine, an inexpensive nontoxic nitrogen source. (Cereghino and

Cregg, 1999)

Constitutive promoter

Although the AOX1 promoter has been successfully used to express

numerous foreign genes, there are chances in which this promoter may not be

suitable. Methanol is a potential fire hazard, especially in quantities needed for large-

scale fermentations. Therefore, promoters that are not induced by methanol are

attractive for expression of certain genes. (Daly et al., 2005).

In any case, the P. pastoris glyceraldehyde 3-phosphate dehydrogenase

(GAP) gene promoter provides strong constitutive expression on glucose at a level

comparable to that seen with the AOX1 promoter (Hans et al., 1997; Waterham et al.,

1997). The glyceraldehyde-3-phosphate dehydrogenase gene was isolated from P.

pastoris by probing a genomic DNA library with the GAPDH gene of Saccharomyces

cerevisiae. The promoter region of this gene was subsequently cloned and used in an

expression vector to drive recombinant constitutive production of proteins. This

approach enables cells to be grown without the need of methanol induction and is

particularly attractive for protein production based on large-scale fermentors. Various

carbon sources have been tested for use with GAP promoters, such as glucose,

glycerol, oleic acid and methanol, with glucose found to result in the highest

expression levels. Furthermore, a comparison of the expression levels of β-lactamase

in P. pastoris using both the GAP and AOX1 promoters (single copy integrated at

HIS4) showed that the protein was produced at highest levels under the control of the

11

GAP promoter (grown on glucose) than the AOX1 promoter (grown on methanol)

(Waterham et al., 1997).

Another promoter that has been used successfully with P. pastoris

expression system is the constitutive YPT1 promoter, which is constructed from a

GTPase gene product that is involved in the secretory pathway. Its promoter provides

a low but constitutive level of expression in media containing glucose, methanol, or

mannitol as carbon sources. Toward this end, the P. pastoris PEX8 promoters may be

of use. The PEX8 gene encodes a peroxisomal matrix protein that is essential for

peroxisome biogenesis. It is expressed at a low but significant level on glucose and is

induced modestly when cells are shifted to methanol (Cereghino and Cregg, 2000).

2.1.3.3 Pichia pastoris expression strains

All P. pastoris expression strains are derived from NRRL-Y 11430 (Northern

Regional Research Laboratories, Peoria, IL) (Cereghino and Cregg, 2000). The choice

of a specific strain is determined by the required application. The genotype and

phenotype characteristics of a number of the more useful strains are summarized in

Table 2.1. Most have one or more auxotrophic genes [e.g., GS115 (his4)] to allow for

selection of expression containing biosynthetic gene (e.g. HIS4) upon transformation.

Prior to transformation, all of these strains grow on complex media but require

supplementation with appropriate nutrients for growth on minimal media (Higgins

and Cregg, 1998; Cereghino and Cregg, 2000).

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Table 2.1 Genotype and phenotype of Pichia pastoris strains

Strains

Genotype Phenotype Application

X-33 Wild type - Selection of Zeocin-

resistant expression

vectors

GS115 his4 Mut +, His - Selection of expression

vectors containing HIS4

KM71 his4, aox1 :

ARG4;arg4

Mut s ,His - Selection of expression

vectors containing HIS4

to generate strains with

MutS phenotype

MC100-3 arg4 his4 aox1

Δ::

SARG4

aox2 Δ:: Phis4

Mut -, His - Selection of expression

vectors containing HIS4

Protease-deficient

strains

- SMD1163

- SMD1165

- SMD1168

his4, prb 1,

pep4

His4, prb 1

His4, pep4

Mut +, His -,

pep4 , prb1-

Mut +, His -,

prb1-

Mut +, His -,

pep4-

Selection of expression

vectors containing HIS4

to generate strains

without protease A

activity

Note: This table was adapted from Invitrogen crop, 1998 and Daly et al., 2005

The most commonly used expression host is wild-type (X-33) or GS115 (his4)

which retains both AOX1 and AOX2 genes and grows on methanol at the wild-type

13

rate (methanol utilization plus or Mut+ phenotype). The X-33 strain is recommended

for expression of recombinant proteins from zeocin-resistant expression vectors such

as pPICZ (A, B, C) and pPICZα (A, B, C). Whilst GS115 is an auxotroph lacking

HIS4 gene and need expression vector with HIS4 gene for selection (Invitrogen corp.,

1998).

The KM71 (his4 arg4 aox1Δ::ARG4) is a strain in which the chromosomal

AOX1 gene is largely deleted and replaced with the S. cerevisiae ARG4 gene. This

strain must depend on the much weaker AOX2 gene for AOX and grows on methanol

at a slow rate (methanol utilization slow or Muts phenotype). Recombinant P. pastoris

with Muts phenotype can also be obtained from GS 115. With many P. pastoris

expression vectors, it is possible to insert an expression cassette that containing HIS4

and simultaneously delete the AOX1 gene, for example, pPIC3.5K, pPIC9K, pAO815,

pPIC9, pPIC3.5, pHIL-D2 and pHIL-S1 (Invitrogen corp., 1998).

Another strain, MC100-3 (his4 arg4 aox1Δ::SARG4 aox2Δ::Phis4), is deleted

for both AOX genes and is totally unable to grow on methanol (methanol utilization

minus or Mut- phenotype).

Several protease-deficient strains, SMD1163 (his4 pep4 prb1), SMD1165

(his4 prb1), or SMD1168 (his4 pep4) have been shown to be effective in reducing

degradation of some foreign protein. This is especially noticeable that major vacuolar

proteases appear to be a significant factor in degradation, particularly in fermenter

cultures, due to the high cell density environment combination with the lysis of a

small percentage of cells. The SMD1168, for example, are defective in the vacuole

peptidase (pep4) that required for the activation of other vacuolar proteases such as

carboxypeptidase Y and proteinase B (encoded by PRB1 genes). Therefore, pep4

mutants display a substantial decrease or elimination in proteinase A and

carboxypeptidase Y activities, and partial reduction in proteinase B activity. In the

prb1 mutant, only proteinase B activity is eliminated, while pep4 prb1 double mutants

show a substantial reduction or elimination in all three of these protease activities

(Higgins and Cregg, 1998; Cereghino and Cregg, 2000; Daly et al., 2005).

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2.1.3.4 Transformation and integration into the P. pastoris genome

Pichia pastoris is transformed by integration of the expression vectors into the

chromosome at a specific locus to generate genetically stable transformant (Daly et

al., 2005). This can be done in two ways. The simplest way is to restrict the vector at

a unique site in either the marker gene (e.g., HIS4) or the AOX1 promoter fragment

and then transform it into the appropriate auxotrophic mutant. The free DNA termini

stimulate homologous recombination events that result in single crossover-type

integration events (figure 2.5) into these loci at high frequencies (50-80% of His+

transformants) (Cereghino and Cregg, 2000).

Alternatively, P. pastoris expression vectors can be digested in such a way

that the expression cassette and marker gene are released, flanked by 5’and 3’AOX1

sequences. Approximately 10-20% of transformation events are the result of a gene

replacement event (figure 2.6) in which the AOX1 gene is deleted and replaced by the

expression cassette and marker gene (Cereghino and Cregg, 2000). The disruption of

the AOX1 gene forces these strains to rely on the transcriptionally weaker AOX2

gene for growth on methanol. These make transformants resulting from such an

AOX1 replacement even are phenotypically His+ and Muts. (Higgins and Cregg,

1998; Cereghino and Cregg, 2000).

Integration by gene insertion (either at the AOX1 or his4 loci) can result in

multiple gene insertion events due to repeated recombination events at a rate of 1–

10% of transformants. The number of integrated copies of the expression cassette can

affect the amount of protein expressed (Daly et al., 2005).

15

Figure 2.5 Gene insertions in Pichia pastoris (Invitrogen Corp., 1998).

Figure 2.6 Gene replacements in Pichia pastoris (Invitrogen Corp., 1998).

The introduction of the expression cassette into the yeast chromosome can be

achieved in a variety of ways including spheroplast formation, electroporation, PEG

1000 and lithium chloride treatments. In general, spheroplasting and electroporation

provide the highest efficiency of transformation for most researchers (103 to 104

transformants per μg DNA). The spheroplast transformation requires several steps

with the risk that contamination of the yeast may occur. Also, over-digestion with the

16

cell-lysing enzyme, zymolyase, can reduce cell viability. Electroporation has become

increasingly popular and can be used successfully with expression vectors. This

method requires fewer steps and the risk of contamination is reduced (Daly et al.,

2005). Other methods, the Polyethylene glycol (PEG) that is a member of a group of

membrane active compounds has recently been shown to be an efficient promoter of

genetic transformation in bacteria, yeast, and mammalian cells. This procedure is

better than LiCl, but not as good as spheroplasting or electroporation. However, it is

convenient for people who do not have an electroporation device (Invitrogen crop.,

1998)

2.1.3.5 Intracellular and secretory protein expression

Heterologous protein expression in Pichia pastoris can be produced either

secretion or intracellular expression. This choice will depend on the protein to be

expressed.

Secretion signals

Secretion requires the presence of a signal sequence on the foreign

protein to target it to the secretory pathway. While several different secretion signal

sequences have been used successfully, including the native secretion signal present

on some heterologous proteins, success has been variable (Higgins and Cregg, 1998).

The most commonly used signal sequence in P. pastoris secretion systems is the S.

cerevisiae α-mating factor pre–pro leader sequence (α-MF). This sequence comprises

a 19 amino acid signal peptide (pre-sequence), followed by a 60 amino acid pro-

region (Raemaeker et al., 1999).

The major advantage of expressing heterologous proteins as secreted

proteins is that P. pastoris secretes very low levels of native proteins. That combined

with the very low amount of protein in the minimal Pichia growth medium, means

that the secreted heterologous protein comprises the vast majority of the total protein

in the medium and serves as the first step in purification of the protein (Invitrogen

Crop., 1998).

Intracellular expression

In case that the secreted protein is not secreted in its native system and

may result in the protein being altered by glycosylation or other post-translational

17

modifications. Intracellular expression is therefore an alternative to secretion and

usually does not result in glycosylation, thus in some cases providing a more desirable

method. However, purification of intracellular expressed proteins can be more

difficult than for secreted proteins as the target protein typically represents less than

1% of the total intracellular proteins.

An advantage of intracellular expression of recombinant proteins with

P. pastoris is that generally an amino-terminal methionine residue is cleaved by

methionine amino-peptidase, unlike proteins expressed in E. coli. The efficiency of

this cleavage is further improved if the second amino acid is proline, valine or

cysteine. The amino terminal amino acid of proteins expressed in P. pastoris can also

be acetylated by N-acetyl-transferase. Indeed, many proteins have been produced

successfully in P. pastoris using intracellular expression systems, particularly

membrane-associated proteins such as the hepatitis B surface antigen (Daly R, 2005).

.

2.1.3.6 Posttranslational modification

The advantage of Pichia pastoris over bacterial expression systems is that the

yeast has the potential of performing many of the post-translational modifications

typically associated with higher eukaryotes, such as processing of signal sequences

(both pre and pre-pro type), folding, disulfide bridge formation, certain types of lipid

addition, and O and N-linked glycosylation (Higgins and Cregg, 1998).

Such modifications may be of critical importance to the function of an

expressed protein. Secreted proteins, membrane proteins, and proteins targeted to

vesicles or certain intracellular organelles are likely to be glycosylated. The most

common and best studied is N-linked glycosylation, where oligosaccharides are

uniquely added to asparagine found in Asn-X-Ser/Thr recognition sequences in

proteins. Another type is O-linked glycosylation, which involves either simple

oligosaccharide chains or glycosaminoglycan chains (Alberts, B. et al,. 1989). P.

pastoris is able to add both O- and N-linked carbohydrate moieties to secreted

proteins. The length of the oligosaccharide chains added posttranslationally to

proteins in Pichia (average 8-14 mannose residues per side chain) is much shorter

than those in Saccharomyces cerevisiae (50-150 mannose residues). Thus, in

comparison to S. cerevisiae, P. pastoris may have an advantage in the glycosylation

18

of secreted proteins because it may not hyperglycosylate. Very little O-linked

glycosylation has been observed in P. pastoris (Invitrogen crop., 1998).

In addition, differences in the number and type of sugar units added by

humans as compared with P. pastoris make a problem for the use of yeast-secreted

glycoproteins as therapeutic products. The yeast-secreted proteins can be extremely

antigenic if introduced into the bloodstream of mammals and are rapidly cleared from

the bloodstream (Cereghino et al., 2002).

2.1.4 Process of Pichia pastoris fermentation

The culture conditions used for Pichia pastoris expression systems are also an

important factor to be considered in order to improve the productivity of a correctly

processed protein. Shake-flask, small-scale expression methods are often the first

stage employed in optimizing protein levels and selecting culture conditions.

The small-scale expression conditions were developed to resemble the

requirements of fermentor systems by ensuring high cell densities were generated

through dramatic increases in the volume of the initial biomass generating cultures (1

liter) and resuspending this biomass into small volumes of methanol induction media

(50–75 ml) (Barr et al.1992). The higher product yields for recombinant proteins have

resulted by developing a model for methanol utilization and then using that model to

predict and control specific growth rates (Daly et al., 2005).

P. pastoris can be cultured at extremely high cell densities (>100 g/l dry cell

weight; >400 g/l wet cell weight; >500 OD600 units/ml) in the fermenter where

parameters such as pH, aeration and carbon source feed rate can be controlled.

However, fermentation growth is especially important for secreted proteins, as the

concentration of product in the medium is roughly proportional to the concentration of

cells in culture. Another feature of growing P. pastoris in fermenter cultures is that

the level of transcription initiated from the AOX1 promoter can be 3-5 times greater

in cells fed with methanol at growth-limiting rates compared to cells grown in excess

methanol. Thus, even for intracellularly expressed proteins, product yields are

significantly higher from fermenter cultured cells (Cereghino et al., 2002).

A three-stage process as shown in figure 2.7 is typically utilized for the

production of foreign proteins from P. pastoris in the fermenter. In the first stage, the

19

engineered strain is batch-cultured in a simple defined medium containing repressing

carbon source such as glycerol. The biomass accumulates but heterologous gene

expression is fully repressed. The second stage is a feed-batch transition phase in

which glycerol is fed to the culture at a growth-limiting rate to further increase the

biomass concentration and to prepare (derepress) the cells for induction. The third

stage, induction phase, is started by adding methanol to the culture at a slow rate,

which facilitates the culture’s acclimation to methanol and initiates the synthesis of

the recombinant protein. The methanol feed rate is then adjusted upwards periodically

until the desired growth rate is reached (Cereghino and Cregg, 2000; Cereghino et al.,

2002). A common alternative to straight methanol feeding for induction of

recombinant protein synthesis is mixed feeding, in which both glycerol and methanol

are continuously added at a preset ratio and rate. The potential advantages of this

strategy are improved cell-culture viability, a shorter induction phase, and a higher

recombinant protein production rate (Cereghino et al., 2002).

Figure 2.7 The process flow scheme of P. pastoris fermentation : day 1, preparation

and inoculation of primary seed medium, and preparation of the bioreactor; day 2,

inoculation of the secondary seed medium and bioreactor hook-up; day 3, starting the

fermentation; day 4/5, spike, limited feed start, samples and monitoring the

fermentation.(Berend et al., 2006)

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2.1.5 Application of Pichia pastoris

Nowadays, the species Pichia pastoris is extensively used for the expression

of heterologous proteins. The protein produced from P. pastoris are usually properly

folded and has no toxic cell wall pyrogens as E. coli; nor contains potentially

oncogenic or viral nucleic acids as mammalian cells (Freyre et al., 2000). Many

reviews describe the general features of this yeast expression system and examining

advances in its development and application

2.1.5.1 Therapeutics application

Pichia pastoris has been used widely for the production of therapeutically

relevant macromolecules. The system has gained interest compared with

Saccharomyces cerevisiae, and is now available in a commercial kit (Invitrogen, San

Diego, CA, U.S.A.). The cattle tick Bm86 antigen has been expressed in Pichia for

manufacturing a vaccine based on this antigen and a certified production and

downstream processing protocol has been described (Canales et al. 1997). HV-2, a

variant of hirudin, a blood-coagulation inhibitor, has been produced to high yield (1.5

g/l) as a secreted protein in Pichia (Rosenfeld et al. 1996) as well as a cytokine

consisting of a fusion of an interleukin with a soluble form of its receptor. Several

antibody fragments, such as single-chain antibodies (scFvs) have been expressed at

high yield in P. pastoris (Fischer et al. 1999). Recently reported, P. pastoris is used to

produce basic fibroblast growth factor (bFGF) which is a potent angiogenic molecule

stimulates smooth muscle cell growth, wound healing, and tissue repair (Mu et al.,

2008). And then, production of recombinant N-A1 to develop a tumor marker for

adenocarcinomas and as a target for antibody-directed therapeutics (Sainz-Pastor et

al., 2006).

2.1.5.2 Enzymes production

Enzymes for blood group conversion such as erythrocytes from blood group B

harbour oligosaccharide chains with terminal β-galactose residues. For conversion, β-

galactosidases from different sources can be used. A Pichia pastoris-based

production has been established for two enzymes from coffee beans (Zhu et al. 1995)

and from soybeans (Davis et al. 1996), respectively. Moreover, the methylotrophic

21

yeast species have been developed as high-yield production systems for recombinant

amylases and sugar converting enzymes from different sources. New examples of this

class of proteins are α-amylases 1 and 2 from barley that produced in P. pastoris was

similar with respect to structure and function to that isolates from malt extracts (Juge

et al. 1996). The recombinant production of the spinach phosphoribulokinase in P.

pastoris provided the possibility to explore its function (Brandes et al. 1996).

2.1.5.3 Recombinant methylotrophs as a biocatalyst

Pyruvic acid is a chemical intermediate in the preparation of fine chemicals,

agrochemicals and pharmaceuticals. Pyruvate salts and esters are chemical

intermediates in the manufacture of cosmetics and neutralizing agents for contact

lenses. Moreover, Dietary supplementation with pyruvate has been found to increase

the metabolism, accelerating weight loss. As a result, the demand for pyruvic acid as a

dietary supplement is increasing dramatically. Genetically modified strains of Pichia

pastoris, that express both the GO ((s)-2-hydroxyacid oxidase) from spinach and

endogenous catalase have been used as whole-cell biocatalysts for conversion of L-

lactic acid to pyruvic acid (Gough et al. 2005).

2.1.5.4 Methanol removing

Methanol is a ubiquitous substrate in nature, and is present in many industrial

waste streams. Biological treatment of condensate is an alternative means to remove

the methanol fraction. Its advantage over steam stripping is a much-reduced energy

cost as well as complete removal of the compound, although it is a slower and more

complex process to operate. The use of methylotrophic yeast in this application not

just to remove methanol but allows the potential to generate a valuable product. The

recent paper was to determine Pichia pastoris could be grown in condensates, and

whether engineered products could be produced in such an industrial media using

engineered strains of the yeast (Hoy et al. 2006).

2.1.5.5 Recombinant scFv production

To provide insight into the localization and function of specific protein, single-

chain antibodies (scFv) were generated to both the conserved region and to unique

22

variable domains for use in immunolocalization, biochemical, and biossay studies

(Xianzong et al., 2002). The high level of two murine scFv fragments produced by the

yeast P. pastoris (up to 250 mg/l) are reported by Eldin et al., 1997. The P. pastoris

expression system was used to produce functionalized single-chain antibody

fragments (scFv) directed against the ED-B domain of the B-fibronectin (B-Fn)

isoform which was found to be present only in newly formed blood vessels during

tumor angiogenesis (Marty et al., 2001). In addition, the P. pastoris was used to

produce an anti-CD33-scFv, with the intention of conjugation the scFv to a

radioisotope as a immunotherapeutic approach to treat acute myeloid leukaemia

(AML) (Emberson et al., 2005).

2.2 Single chain variable fragment (scFv) Single chain variable fragment (scFv) is a part of immunoglobulin (IgG) or

antibody (Ab). As shown in figure 2.8 (a), antibody contains two functionally and

molecularly separable modules. One module for antigen binding (Fab) and another for

triggering effector function (Fc). The structural basis of all antibody or

immunoglobulin molecules can be found in the variations of heavy chain (VL) and

light chain (VH). These two amino-terminal domains lie at each tip of the branches of

the immunoglobulin. The VL and VH hypervariable regions (CDR's) project from the

end of their domains, ready for antigen binding (Fischmann et al., 1991; Rini et al.,

1992).

The production of human monoclonal antibodies often has problems, mostly

related to the instability of the hybridoma cell line, and the therapeutic use of murine

monoclonal antibodies which is sometimes effective under laboratory conditions is at

present not economical. To avoid the most of these problems is to engineer novel

recombinant antigen binding proteins that have increased efficiency and new

properties. So, scFv fragments which are the minimal structure likely to retain the

binding characteristics of an antibody can be produced in heterologous systems

(Mousli et al., 1998).

The recombinant variable domains (VH and VL) are genetically linked in a

single-chain (scFv) construct via a short flexible peptide. This stabilizes their

23

association. scFv can be derived from hybridoma or combinatorial antibody libraries

by phage display technology (Billiald et al., 1995; Lafaye et al., 1997) and may be

very useful in many fields, such as research, immunodiagnosis and immunotherapy.

scFvs are cleared more rapidly from the blood to extravascular spaces than the much

larger molecules such as IgGs, F(ab’)2 and Fab, resulting in better tissue penetration.

Thus, scFvs are potentially very useful molecules for treating cancer and detoxication.

The small size of the scFv permits their rapid diffusion in the body for detoxication

and the fast in vivo clearance of immunocomplexes via the urine. This minimizes the

chance of late release of bound toxin and the reemergence of toxicity. Lastly, the

absence of the immunoglobulin constant domains decreases the risk of an

immunological response from the patient. (Mousli et al., 1998)

(a) (b)

Figure 2.8 Structure of the immunoglobulin and scFv

(Leong and Chen, 2008)

(a) A full-length antibody of the IgG subclass contains both heavy (VH)

and light (VL) chains.

(b) A scFv contains VH and VL domains connected via a polypeptide linker.

24

2.2.1 Molecular design of single chain antibodies

A single chain antibody is made up of an antibody variable light (VL) and

heavy (VH) regions that are linked together. (Raag and Whitlow, 1995; Eldin et al,

1997), and hence also commonly known as a single chain variable fragment (scFv).

The reduced size of scFv fragment (27,000 - 30,000 Da) makes them to be the

smallest antibody fragments encoded by a single gene that carries the entire antigen-

binding region, for a given antibody (Bird et al., 1988). The carboxyl terminus of one

variable fragment is linked to the amino group of the other fragment via a polypeptide

linker of typically 10–25 amino acid residues in length (Fig. 2.8(b)). The choice of

polypeptide linker depends on the suitability of the linker’s conformation and length

in joining the two variable chains without imposing any major steric obstruction

(Takkinen et al., 1991), and can also vary according to the intended application of the

scFvs (Raag and Whitlow, 1995). In generally, the linker is designed with glycine and

serine residues to provide flexibility and protease resistance, and the linker sequence

can be optimized by phage display (Hadson and Kortt, 1999). To increase antigen-

binding selectivity, non-specific interactions between long linkers and variable

fragments must be eliminated. Proteolytic stability of the linkers is also critical to

minimize protein aggregation, which will result in function and yield losses.

2.2.2 Recombinant scFv production

There are various expression systems that can be used for the production of

antibody fragments include bacterial or mammalian cell culture and transgenic

animals or plants. The expression system of choice is partially dependent upon the

intended use of the antibody, as well as the antibody yield derived from each system.

This yield affects the cost of goods, which is comprised principally of two factors: the

cost of the upstream process that generates the antibody, and the cost of the

downstream process, that is, purification and final fill of the active pharmaceutical

ingredient (Chadd and Chamow, 2001).

Recombinant production of scFv is undoubtedly more efficient and increases

the stability of the Fv fragments produced. The production of bioactive recombinant

scFv was first reported by Skerra and Pluckthun (1988) using E. coli. The insertion of

25

a polypeptide linker that joins the VL chain to the VH chain was found to generally

improve scFv expression (Bird et al., 1988). Optimization of the linkers’ sequence

and length were later found to significantly influence the overall product yield (Arndt

et al., 1998; Trinh et al., 2004).

2.2.2.1 Production of scFv in Escherichia coli

Immunoglobulin fragments are commonly expressed in E. coli. This

system is the ability to produce protein in large quantities and tends to be inexpensive.

In addition, transformation of E. coli cells with the foreign DNA is easy and requires

minimal amounts of DNA. Importantly, well-established knowledge of the basic

cellular processes of E. coli facilitates easy genetic manipulation and characterization

of protein expression to improve the quality and yield of the target protein (Verma et

al., 1998; Leong and Chen, 2008). However, E. coli is not capable of glycosylating

proteins, thus if glycosylation is required (e.g. for scFv-Fc fusion proteins) other

expression systems may be preferred.

Bacterial expression of scFv fragments can occur via cytoplasmic

secretion (i.e., soluble expression in the cytoplasm), periplasmic secretion or inclusion

body formation in the cytoplasm (Kujau et al., 1998; Martineau et al., 1998;

Rippmann et al., 1998;Wu et al., 2002; Lee et al., 2002). Expression in the reducing

environment of the cytoplasm can be achieved at high concentrations but often results

in the formation of inclusion bodies (i.e. reduced and unfolded Ab) that require

solubilization with denaturing agents (e.g. 8 M urea) and subsequent refolding, by

dialysis, of the denaturing agent in the presence of a redox pair (e.g. reduced and

oxidized glutathione). As an alternative to Ab recovery from inclusion bodies,

cytoplamic expression can be done in E. coli strains that promote proper folding and

oxidation in vivo. Using such strains, scFv have been produced at levels equal to or

greater than those achieve with periplasmic expression (Levy et al., 2001).

2.2.2.2 Production of scFv in Yeast

The main advantages of yeast are related to the fact that it has the

advantage of both prokaryote and eukaryote. Unlike Escherichia coli, yeast systems

provide advanced protein folding pathways for heterologous proteins and, when yeast

26

signal sequences are used, yeast can secrete correctly folded and processed proteins.

Therefore functional and fully folded heterologous proteins can be secreted into

culture media. Additionally, yeast systems eliminate the presence of toxic pyrogen

found in E. coli. Unlike mammalian expression systems, yeast can be rapidly grown

on simple growth media and nor contains potentially oncogenic or viral nucleic acids

as found in mammalian cells. For the expression of clinically and industrially

important proteins, yeast is an attractive option as industrial scale fermentation

technology is widely used (Cregg et al., 1993; Verma et al., 1998).

The scFv production in yeast systems often involves direct secretion

into the cell cytoplasm in the correctly folded form, or direct extracellular secretion to

the medium. Single-chain antibodies have also been successfully expressed in yeast

systems, for example, an anti-fluorescein scFv has been produced in

Schizosaccharomyces pombe (Davis et al., 1991) and anti-recombinant human

leukaemia inhibitory factor scFv has been expressed in Pichia pastoris (Ridder et al.,

1995). Proteins which accumulate as insoluble inclusion bodies in E. coli are often

soluble when expressed in yeast (Ridder et al., 1995). In recent report, scFv-based

fragments, including diabody and diabody-immunotoxin conjugates (Kim et al., 2007)

and scFv-Fcs (Powers et al., 2001; Ren et al., 2008), have been produced in yeast and

filamentous fungal species including Sacchromyces cerevisiae, P. pastoris,

Aspergillus awamori and Kluyveromyces lactis, reviewed in detail by Gasser et al.

(2007) (Gasser and Mattanovich, 2007).

However, although scFv production in yeast is possible, the shorten of

process time to increase the volumetric productivity is still require (Gasser and

Mattanovich, 2007). A recent comparison of the fed-batch and continuous culture

methods in P. pastoris has shown that the continuous culture with methanol control

feeding has increased scFv productivity and thus looks promising for the industrial

scale Ab production (Yamawaki et al., 2007).

In a comparison of S. cerevisiae, P. pastoris, and E. coli expression

systems, it can be concluded that E. coli was the fastest and most consistent way to

obtain and characterize purified scFv (Miller et al., 2005). However, the recent

advance in the humanization of N-glycosylation in yeast (Wildt and Gerngross, 2005)

may obviate the need for mammalian expression of glycosylated scFv-Fc type

27

fragments. In the future, fungal expression systems will likely be used to complement

the Ab-expression field by providing Ab fragments that are not easily or routinely

produced by bacterial or mammalian cells culture and possibly as a replacement

system for the expression of glycosylated mAb and scFv-Fc minibody fragments.

2.2.2.3 Production in mammalian cells.

The use of eukaryotic systems to produce recombinant scFv proteins is

attractive when post-translational modifications of the product are required, where the

signals for synthesis, processing and secretion of eukaryotic proteins are adequately

recognized by eukaryotic cells.

Although the use of mammalian expression systems may be inevitable

for complex proteins, this is rarely the case for scFv which are relatively simple in

structure. Microbial production of antibody fragments can prove more favourable in

terms of processing economics and throughput. Furthermore, in the large-scale

production of scFv include the need for manipulation of specific cellular pathways

(for example, those that control cell growth) to improve large-scale performance with

respect to product titres, and the inevitable high costs of maintaining mammalian cells

at industrial bioreactor scale (Leong and Chen, 2008).

In summary, eukaryotic expression systems including, yeast,

mammalian or plant cells are favored hosts for high-yield expression of full length

mAb and minibody Ab fragments that require glycosylation, whereas bacterial

expression systems are the preferred format for small non-glycosylated Ab fragments

(Holliger and Hudson, 2005).

2.2.3 Applications of single chain antibodies

2.2.3.1 Therapeutic and diagnostic applications

The scFv have now become important molecules for improved targeted

delivery of drugs and other biomolecules for therapeutic and diagnostic applications

and have shown to be extremely effective against a wide range of different proteins,

receptors, haptens and tumour antigens (Yokota et al., 1992; Huston et al., 1993).

28

Radioimmunodetection and immunotherapy

The scFv fragment is ideal for tumor therapy because they achieve

higher total tumor uptake, faster clearance and better tumor-to-blood ratios than IgG.

The radioimmunotherapy techniques have been tested and show promise including the

use of radiolabeled scFv-albumin fusion proteins (Yazaki et al., 2008), and

radiolabeled (scFv)2 that are co-injected with cell penetrating peptides for increased

tumor penetration (Jain et al., 2005). Furthermore, the administration of radiolabeled

scFv dimers linked to magnetic iron oxide nanoparticles (NPs) can result in tumor cell

lysis following the application of an external alternative magnetic field (Natarajan et

al., 2008).

Immunotoxins

Immunotoxins are a class of anti-tumor agents that consist of Ab-toxin

(or ligandtoxin) hybrid molecules that are composed of an anti-tumor Ab or ligand

linked to a tumor cell-killing moiety, such as a toxin. Several preclinical studies and

clinical trials of immunotoxins have been conducted or are underway for cancer

therapies (Yang et al., 2007). For example, scFv toxin fusion proteins have reportedly

facilitated immunotargeting of the effector protein (Huston et al., 1993). Recombinant

antibody toxin scFv(FRP5)-ETA, which specifically recognizes Erb2 receptor, was

recently reported to show promising clinical potential in reducing disease progression

in patients with advanced solid malignomas (Minckwitz et al., 2005).

Intrabodies

The scFv fragments are remain to be the most common intrabody

fragments used. Some Ab libraries have been designed to specifically selecting scFv

that will be expressed at high levels under the reducing conditions of the cytoplasm

(Philibert et al., 2007). On neurodegenerative diseases, intrabody-mediated in vivo

suppression of neuropathology, using a Drosophila model of Huntington’s (HD)

resulted in a 23% to 100% increase in the proportion of HD flies surviving to

adulthood when in the presence of the scFv intrabody (Wolfgang et al., 2005).

Additionally, intrabodies have also been used in the functional inhibition of virus

envelope proteins and receptors on the surface of host cells include human papilloma

29

virus (HPV) (Griffin et al., 2005), Kaposi sarcoma-associated herpesvirus (KSHV)

(Corte-Real et al., 2005) and HIV (Silva et al., 2004). And then, intrabodies have also

been investigated as mechanisms to inhibit tumor growth. For example, the blockade

of both VEGF-R2 and Tie-2 pathways resulted in a 92% inhibition of tumor growth

and angiogenesis (Jendreyko et al., 2005). Moreover, the in situ expression and

secretion of a bispecific diabody (anti-CEA and CD3) in vivo reduced tumor growth

(Blanco et al., 2003).

2.2.3.2 scFv engineering for improved pharmacokinetics

The scFv-type fragments have short in vivo half-lives due to their low

MW and the absence of an Fc region, thus negative binding to the FcRn that is

receptors are expressed on the surface of phagocytic cells of the reticuloendothelial

system and prevent the rapid elimination of IgG.

Thus, to prolong the pharmacokinetic circulating half-lives of small

rAbs, most attempts have been directed at increasing the apparent molecular size of

the recombinant protein (e.g., via PEGylation) (Krinner et al., 2006) or to engineer the

fusion to heavy chain fragments (i.e. to CH3 or Fc) (Hu et al., 1996; Kenanova et al.,

2005). For example, the PEGylated scFv molecules have been constructed (Krinner et

al., 2006; Xiong et al., 2006; Yang et al., 2003) and have shown a prolonged in vivo

half-life of ca. 30 folds (Krinner et al., 2006) to 100 folds (Yang et al., 2003)

compared to the unconjugated scFv. Another reported is 125I-labeled anti-CEA scFv-

albumin fusion demonstrated rapid tumor uptake that reached a plateau of 22.7%

injected dose (ID)/g body weight at 18 h compared to 4.9% ID/g for the scFv (Yazaki

et al., 2008).

2.3 CD147

CD147, also known as M6 Ag (Kasinrerk et al., 1992), basigin (Miyauchi et

al., 1991), and EMMPRIN (extracellular matrix metalloproteinase inducer) (Biswas

et al., 1995), is a 50–60 kDa type 1 transmembrane glycoprotein that is a member of

immunoglobulin (Ig) superfamily and contains two extracellular Ig domains

(Kasinrerk et al., 1992). The CD147 molecule is broadly expressed on hemopoietic

and non-hemopoietic cell lines. Within peripheral blood cells, CD147 is expressed on

30

all leukocytes, red blood cells, platelets and endothelial cells (Stockinger et al., 1997).

High levels of CD147 expression were also observed on human tumor cells (Ellis et

al., 1989; Muraoka et al., 1993; Polette et al., 1997). Elevated CD147 expression is

correlated with tumor progression of gliomas (Sameshima et al., 2000a), hepatomas

(Jiang et al., 2001), squamous cell carcinomas (Bordador et al., 2000), and melanomas

(van den Oord et al., 1997). In addition to cancer, EMMPRIN has been implicated in

many other pathological processes. Its up-regulation has been identified in tissues

such as lung injury (Foda et al., 2001), rheumatoid arthritis (Konttinen et al., 2000),

chronic liver disease (Shackel et al., 2002), heart failure (Spinale et al., 2000) and

atherosclerosis (Major et al., 2002).

As the presence of several different names for emmprin/CD147 implies,

multiple functions of this molecule have been demonstrated (Figure 2.9):

emmprin/CD147 can also affect the multiple matrix metalloproteinases (MMPs)

induction that leading to extracellular matrix degradation and increased tumor growth

and metastasis, the activation and development of T cells, escort monocarboxylate

transporters (MCT) to the plasma membrane, act as a receptor for cyclophilin A and is

associated with Blood-brain barrier function of cerebral endothelial cells. These lines

of evidence suggest that emmprin is a multifunctional transmembrane protein that

mediates molecular events involved in intercellular interactions that are critical for

many pathological and physiological processes (Nabeshima et al., 2006).

31

Figure 2.9 Multiple functions of EMMPRIN/CD147. (Nabeshima et al., 2006)

2.3.1 Structural organization of CD147

Emmprin/CD147 consists of a 185 amino acid (aa) extracellular region

containing two immunoglobulin (Ig) domains, a 24 aa residue transmembrane domain

and a 39 aa cytoplasmic domain as shown in figure 2.10 (Biswas et al., 1995;

Miyauchi et al., 1991 ; Muramatsu and Miyauchi, 2003) The extracellular region

contains three N-glycosylation sites (Muramatsu and Miyauchi, 2003), but the glycan

portion of the molecule differs according to source of EMMPRIN/CD147, the

different glycosylation pattern of the native 27-kDa protein accounts for its variable

molecular weight, ranging between 45 and 65 kDa (Biswas et al., 1995).

Glycosylation was shown to determine its MMP stimulating activity (Guo et al., 1998;

Sun and Hemler, 2001), the purified deglycosylated EMMPRIN/CD147 not only

failed to induce MMP activity but also antagonized the activity of the native molecule

(Sun and Hemler, 2001). Treatment with different endoglycosidases indicated that N-

linked oligosaccharide chains may contribute to almost half the size of the mature

molecule (Kanekura et al, 1991). A stretch of 24 aa in the transmembrane region is

32

completely conserved among human, mouse and chicken, indicating the importance

of this region in the function of the molecule (Miyauchi et al., 1991). The presence of

the charged amino acid glutamic acid in the middle of this domain is not commonly

encountered in other membrane proteins and implies that CD147 can associate with

other transmembrane proteins (Muramatsu and Miyauchi, 2003). The intracellular

domain CD147 is also well conserved between species (Miyauci et al., 1991) and may

transduce signals into the cells.

Figure 2.10 Scheme of EMMPRIN/CD147 molecule. ECI, first extracellular Ig

domain; ECII, second extracellular Ig domain; TD, transmembrane domain; CD,

cytoplasmic domain. Three N-linked oligosaccharides are shown by helixes. ECI is

involved in matrix metalloproteinase (MMP) induction, binding to counter-receptors,

emmprin in trans and cis manners, and high-mannose-type L3 epitope, and

association with integrins. ECII is required for association with caveolin-1. Pro211

and Glu218 of TD are involved in association with Cyp60 and membrane targeting of

emmprin, respectively. CD is required for association with monocarboxylate

transporter 1 (MCT1) (Nabeshima et al., 2006).

33

2.3.2 Regulation of CD147 expression

2.3.2.1 Regulation of CD147 in cancer

In the first functional approach, the researcher discovered

CD147/EMMPRIN, then called tumor celled-derived collagenase stimulatory factor

(TCSF), as a surface molecule on tumor cells that stimulated nearby fibroblasts to

produce matrix metalloproteinases (MMPs) (Biswas et al., 1995; Ellis et al., 1989;

Guo et al., 1998; Kataoka et al., 1993). Further studies revealed that CD147 is capable

of inducing the expression of several MMPs other than MMP-1, including MMP-2,

MMP-3, MMP-9 and MMP-11 (Kataoka et al., 1993; Biswas et al., 1995; Guo et al.,

1998). It was determined that either CD147 expressing tumor cells or conditioned

media from the same cells were equally capable of inducing MMP production in co-

cultured fibroblast cells (Biswas, 1982, 1984).

CD147 was initially identified that interstitial collagenase (MMP-1)

production was induced during the co-culturing of tumor cells and fibroblasts

(Biswas, 1982, 1984). CD147 was also shown to bind to MMP-1 at the tumor cell

surface, potentially concentrating and localizing this collagen degrading enzyme at

the tumor cell surface to promote cell invasion. The association of MMP-1 with tumor

cell surface has already been described. Recently, vesicular shedding has been

proposed as a mechanism for CD147 release. The soluble CD147 may well be of

profound biological importance as it would be able to exert its effect on fibroblasts or

endothelial cells at distant sites (Gabison et al., 2005). A schematic summary of the

potential mechanisms modulating CD147 action is presented in Figure 2.11

In view of the high expression of CD147 in malignant tissues and its

potential as a target for therapy, it is surprising that relatively little is currently known

on the way it is regulated in normal and pathological tissues and the nature of the

factors that may induce its expression during tumor progression.

34

Figure 2.11 Possible modes of modulating MMP inducing activity of

EMMPRIN/CD147 in the tumor situation. Although the receptor for CD147 on

fibroblasts has not been identified (a), it is thought to act as its own receptor in

neighboring tumor cells (b). Soluble CD147 released either as fulllength molecule or

as a proteolytically cleaved ectodomain were shown to retain their MMP inducing

activity (c) and can therefore act at a distance. Full-length soluble CD147 was

suggested to be released via microvesicular membrane shedding (d). On the other

hand, CD147 can increase pericellular collagen degradation by binding MMP-1 and

so focusing its activity to the invasion front (e). CD147 synthesis and MMP induction

are upregulated by the activation of the EGFR signaling pathways (f), while CD147

glycosylation and MMP induction are inhibited by its association with caveolin-1 (g).

(Gabison et al., 2005)

2.3.2.2 Role in lymphocyte migration and activation

Within the thymus, CD147 expression is highest on late stage,

immature thymocytes, which correlates with transition to the next maturational level.

The requirement of CD147 in T cell maturation was illustrated following the

treatment of immature fetal lymphocytes with an anti-CD147 monoclonal antibody,

which arrested any further development (Renno et al., 2002).

35

Within circulating immune cell populations, CD147 is highly

expressed on activated T and B lymphocytes, as well as dendritic cells, monocytes

and macrophage (Koch et al., 1999). CD147 was identified as an activation associated

antigen on PHA activated T cells (Kasinrerk et al., 1992). Recently, increased

expression of CD147 was observed on CD3+T cells from systemic lupus

erythematosus patients compared to healthy donor T cells, likely indicating their

increased activation (Pistol et al., 2007). Engagement of CD147 with the MEM-M6/6

anti-CD147 monoclonal antibody was found to inhibit anti-CD3 induced T cell

proliferation (Koch et al., 1999), presumably by inhibiting the reorganization and

clustering of GPI-anchored co-receptors within lipid rafts which in turn alters

downstream signaling events (Staffler et al., 2003).

For T cells, the level of CD147 expression also correlates with the state

of differentiation, whereby immature thymocytes express higher CD147 levels than

do mature resting T cells (Kirsch et al., 1997). Blocking CD147 expressed on antigen

presenting cells inhibits T cell activation. Studies on antibody pre-pulsed

monoblastoid cells or peripheral blood dendritic cells cocultured with T cells revealed

inhibition of anti-CD3 induced T cell proliferation as well as inhibition of alloantigen

induced T cell responses, suggesting that CD147-dependent T cell activation occurs at

the level of the antigen presenting cell (APC) rather than the T cell (Stonehouse et al.,

1999; Woodhead et al., 2000). This may be attributable to CD147’s function as an

adhesion molecule.

Studies performed with anti-CD147 treated human monoblastoid U937

cells demonstrated the importance of CD147-induced cellular adhesion and

highlighted the involvement of the LFA-1/ICAM-1 pathway in this process

(Kasinrerk et al., 1999). These findings, coupled with the fact that CD147 is

expressed on both APCs and T cells, suggest a more general function for CD147 as a

mediator of cell–cell interaction and regulator of antigen presentation (Woodhead et

al., 2000).