The role of the glycan moiety on the structure–function relationships

of PD-L1, type 1 ribosome-inactivating protein from P. dioica leaves

Valeria Severino,a Angela Chambery,*a Antimo Di Maro,a Daniela Marasco,b

Alessia Ruggiero,cRita Berisio,

cFrancesco Giansanti,

dRodolfo Ippoliti

dand

Augusto Parentea

Received 24th September 2009, Accepted 9th December 2009

First published as an Advance Article on the web 21st January 2010

DOI: 10.1039/b919801f

N-glycosylation is one of the major naturally occurring covalent co-translational modifications of

proteins in plants, being involved in proteins structure, folding, stability and biological activity. In

the present work the influence of carbohydrate moieties on the structure–function relationships of

type 1 ribosome-inactivating proteins (RIPs) was investigated. To this aim, PD-Ls, RIPs isolated

from Phytolacca dioica L. leaves, differing for their glycosylation degree, were used as an

experimental system. In particular, comparative structural and biological analyses were performed

using native and unglycosylated recombinant PD-L1, the most glycosylated P. dioica RIP

isoform. The glycans influence on protein synthesis inhibition and adenine polynucleotide

glycosidase activity was investigated. The interaction with adenine, the product of the

de-adenylation reaction, was also investigated for native and recombinant PD-L1 by fluorescence

spectroscopy. Furthermore, the crystal structure of PD-L1 in complex with adenine was

determined. Our data confirm that the absence of glycan moieties did not affect the biological

activity in terms of protein synthesis inhibition. However, the removal of carbohydrate chains

significantly increased the deadenylation capability, likely as a consequence of the increased

accessibility of substrates to the active site pocket. Furthermore, preliminary data on cellular

uptake showed that all PD-L isoforms were internalized and, for the first time, that the vesicular

distribution within cells could be influenced by the protein glycosylation degree.

Introduction

Ribosome-inactivating proteins (RIPs) are N-b-glycosidaseswhich remove a specific adenine residue in the major rRNA of

prokaryotic and eukaryotic ribosomes (A4324 in rat liver 28S

rRNA).1–4 RIPs are divided into type 1, single-chain proteins

with molecular mass around 30 kDa and basic pI, and type 2,

two chain proteins with Mr around 60 kDa and pI between 4.5

and 8.0, in which an enzymatically active A chain is linked to a

B chain with lectin properties. A type 3 has been proposed,

including some peculiar RIPs,5–7 synthesized as inactive

precursors (pro-RIPs), and then enzymatically activated by

proteolytic cleavages. RIPs act as adenine polynucleotide

glycosylases (APG),8 being also active on substrates other

than rRNA, such as viral RNA, poly(A), and DNA.9 The

lectinic B chain of type 2 RIPs preferentially binds to receptors

on the cell surface carrying a terminal galactose at their

carbohydrate moiety, allowing the internalization of the

enzymatic A chain into cells. Type 1 RIPs, being devoid of

the B chain, cannot bind to cells and consequently their cell

toxicity is much lower than that of type 2 RIPs. The wide

spectrum of biological activities, beyond the main rRNA

N-b-glycosidase action, increased the interest on the physiological

role of RIPs in planta and on their potential biotechnological

applications, mainly focused on their use as immunotoxins

against tumors. RIPs have also applications in agriculture as

antiviral and antifungal agents against plants pathogens.

Several type 1 and type 2 RIPs are glycoproteins. Although

both RIP structure and rRNA N-b-glycosidase activity have

been studied in great detail, few studies have been focused on

the potential influence of the glycan moiety on the structure of

type 1 RIPs and in turn on their biological activities.

Phytolacca dioica L. contains several RIP isoforms which

constitute an excellent experimental system for such studies.

Indeed, their characterization revealed that fully expanded

leaves of adult plants express four isoforms, named PD-L1/

PD-L2 and PD-L3/PD-L4.10,11 The interest in these RIPs

stems from the fact that PD-L1 and PD-L2 have the same

primary structure, but are differently glycosylated. In particular,

PD-L1 presents three N-glycosylation sites (Asn10, Asn43 and

Asn255), occupied by a paucimannosidic-type N-glycan

[(Man)3 (GlcNAc)2 (Fuc)1 (Xyl)1], while PD-L2 lacks the

glycan chain on Asn255. Similarly, PD-L3 and PD-L4 have

the same primary structure (with 81.6% primary structure

identity with PD-L1/PD-L2). PD-L3 has the glycan moiety

aDipartimento di Scienze della Vita, Seconda Universita di Napoli,Via Vivaldi 43, I-81100 Caserta, Italy.E-mail: angela.chambery@unina2.it; Fax: +39 0823 274571

bDipartimento delle Scienze Biologiche, Sez. Biostrutture, Universitadegli Studi di Napoli ‘‘Federico II’’, Via Mezzocannone 16,I-80134 Napoli, Italy

c Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone 16,I-80134, Napoli, Italy

dDipartimento di Biologia di Base ed Applicata, Universita degli Studidi L’Aquila, Via Vetoio, I-67010 Coppito, Italy

570 | Mol. BioSyst., 2010, 6, 570–579 This journal is �c The Royal Society of Chemistry 2010

PAPER www.rsc.org/molecularbiosystems | Molecular BioSystems

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only on Ans10, while PD-L4 is not glycosylated.11 Although

some care must be taken in interpreting the results of the

modeling approach, molecular simulations of glycan chains of

PD-L1, PD-L2 and PD-L3 have been reported by Di Maro

et al., 2009.11 In this work, the spatial position of the

paucidomannosidic glycan moieties, including their mobility

and H-bonds interaction network with the protein chain, were

obtained. The standard plant paucimannosidic-type N-glycan

structure was also found in PD-S2, type 1 RIP isolated from

P. dioica seeds.12

Even not considering the implications on the enzymatic

N-b-glycosidase activity and/or the resulting protein synthesis

inhibition, it could not be excluded that the glycan moiety of

type 1 RIPs could be responsible of different internalization

mechanisms and routing into animal cells.

In the present work the potential influence of the glycan

moiety on RIP structure and biological activities was

investigated. To this aim, the PD-L1/2 gene was cloned and

expressed in the E. coli heterologous system to obtain the

unglycosylated recombinant protein. Comparative structural

and functional analyses were performed to study the implications

of the lack of PD-L1/2 carbohydrate chains. In addition, a

preliminary study reporting evidences of RIP differential

cellular uptake is also reported.

Experimental

Materials

Restriction endonucleases and T4 DNA ligase were purchased

from Boehinger Mannheim GmbH (Mannheim, Germany).

The kit for plasmid purification was from Promega (Milan,

Italy). Expression vector pET22b(+) and E. coli strain

BL21(DE3) were from AMS Biotechnology (Lugano,

Switzerland). DNA manipulation, transformation and plasmid

purification were performed according to previously published

methods.13 Native PD-L1, PD-L2 and PD-L4 were purified

from P. dioica leaves as described.10

Synthesis and cloning of PD-L1 synthetic gene

PD-L1/2 amino acid sequence was used for the backtranslation

to the DNA sequence by using the GeneOptimizer software

(GENEART GmbH, Germany). The codon usage was

adapted to the codon bias of Escherichia coli genes to allow

high and stable expression rates. In addition, regions of very

high (480%) or very low (o30%) GC content were avoided

where possible. The PD-L1 optimized synthetic gene was

produced by GENEART GmbH Company (Regensburg,

Germany). A NdeI restriction site was added at the 50 end of

the coding sequence. A stop codon, followed by a EcoRI

restriction site was introduced at the 30 end. The synthetic

gene was assembled from synthetic oligonucleotides using a

PCR-based strategy. The 810 bp long PD-L1/2 synthetic gene

was first cloned into pGA4 (ampR) vector using KpnI and

SacI restriction sites (Stratagene, CA, USA). All constructs

were verified by sequencing, showing 100% sequence

congruence to the designed gene. For heterologous expression,

the PD-L1/2 coding sequence was subcloned into pET22b

(ampR) using NdeI and EcoRI restriction sites and trans-

formed into E. coli BL21(DE3) strain by electroporation.

Expression, folding, and purification of recombinant PD-L1

Expression, folding, and purification of recombinant PD-L1/2,

hereafter named rPD-L1, were mainly performed according to

previously reported protocols.13 The induction of the expression

of rPD-L1 was carried out on with 0,4 mM IPTG at 37 1C for

16 h in Luria-Bertani (LB) broth. Considering the glycosylation

of native PD-L1 (nPD-L1), it was necessary to introduce some

modifications into the rPD-L1 refolding strategy. In particular,

a higher ratio (1 : 10) of the redox-pair oxidized : reduced

glutathione, a higher ionic strength and the addition of

0.4 M sucrose were used in the refolding buffer (10 mM

NaP/0.5 M L-Arg/0.6 mM GSSG/6 mM GSH/0.4 M

sucrose/0.2 M NaCl/10 mM KCl/2 mM CaCl2/2 mM MgCl2,

pH 8.4). The refolded PD-L1 was concentrated by ultra-

filtration, dialyzed against 5 mM Na phosphate buffer (NaP)

pH 7.2 at 4 1C and purified by FPLC, using a cation exchange

chromatography on a Source 15S column.13 The column

was equilibrated in and washed with 5 mM NaP, pH 7.2 at

1 mL min�1. Protein elution was achieved by increasing the

NaCl concentration from 0 to 0.3 M in the same buffer. Each

step of the expression and purification procedure was

monitored by 12% SDS-PAGE. The eluted protein peaks

were collected, dialyzed, and kept frozen until use.

N-terminal amino acid sequencing

rPD-L1, separated by SDS-PAGE, was transferred onto a

PVDF membrane (Applied Biosystems) and directly subjected

to Edman degradation on a Procise Model 491 sequencer

(Applied Biosystems) for N-terminal sequencing.13

Mass spectrometry analysis

The relative molecular mass (Mr) of rPD-L1 was determined

using a Q-TOF Micro mass spectrometer (Waters, Manchester,

UK) as previously reported.12 The capillary source voltage

and the cone voltage were set at 3000 V and 35 V, respectively.

The source temperature was kept at 80 1C and nitrogen was

used as a drying gas (flow rate 50 L h�1). RP-HPLC purified

protein at a concentration of about 1 pmol mL�1 was infusedinto the system at a flow rate of 5 mL min�1. The acquisition

and deconvolution of data were performed by Mass Lynx

software (Waters, Manchester, UK).

Circular dichroism spectroscopy and thermal denaturation

analysis

CD spectra were recorded on a Jasco J-810 spectropolarimeter

(JASCO Corp, Milan, Italy) in the far UV region from 190 to

260 nm. Each spectrum was obtained averaging three scans,

subtracting contributions from corresponding blanks and

converting the signal to mean residue ellipticity in units of

deg cm2 dmol�1 res�1.

Measures were performed with a protein concentration of

0.8 mM in 20 mM phosphate buffer at pH 7.0, using a 0.1 cm

path-length quartz cuvette. Data were collected at 0.2 nm

resolution, 20 nm min�1 scan speed, 1.0 nm bandwidth and 4s

response. Thermal denaturation profiles were obtained by

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measuring the temperature dependence of the ellipticity at

222 nm in the 40–90 1C range with a resolution of 0.5 1C and

1.0 nm bandwidth. The heating rate was 1 1C min�1 and the

response at 16 s with a Peltier temperature controller.

Fluorescence analysis

Fluorescence spectroscopy investigations were carried out at

room temperature using a Varian Cary Eclipse spectro-

fluorimeter, with excitation at 280 nm and emission at 300 nm

using 5 nm slits. Spectra were recorded in the 300–400 nm

wavelength range. Adenine titration with nPD-L1, rPD-L1

and PD-L4 was performed using protein concentrations of 3,

4.5 and 3 mM, respectively in 20 mM phosphate buffer at

pH 7.0. Adenine equivalents ranged from 0 to 50.

Biological assays

Protein synthesis inhibition was determined with a rabbit

reticulocyte lysate as previously described.14 Adenine poly-

nucleotide glycosidase activity was assayed on herring sperm

DNA (hsDNA) and poly(A) substrates as described

elsewhere.9,13 Briefly, 100 pmol of rPD-L1, native PD-L1 or

PD-L2 were incubated with 100 mg of substrates at 37 1C for

1 h in 50 mM sodium acetate buffer pH 4.0, containing 100 mM

KCl in a final volume of 300 mL. Reactions were always run in

duplicate and results expressed as the mean of three different

experiments. After incubation, the reaction was stopped by

cooling samples in ice. Polynucleotides were removed by

ethanol/sodium acetate precipitation. Adenine release was

measured spectrophotometrically at 260 nm.

Crystallization, data processing and structure refinement

Best crystals of PD-L1 were obtained using the macro-seeding

technique as described.15 Crystals suitable for X-ray diffraction

experiments were obtained after the transfer of needles to a

pre-equilibrated solution containing 10 mg mL�1 PD-L1,

0.16 M sodium acetate trihydrate, 0.1 M sodium cacodylate

trihydrate (pH 6.5), 24% (w/v) polyethylene glycol 8000,

3 mM adenine.

Diffraction data (1.65 A) were collected at the BW7A

beamline, DESY, Hamburg. Crystals were flash-cooled after

the addition of increasing concentrations, ranging from 4 to

14% (v/v) of ethylene glycol to the crystallisation buffer. Data

processing and scaling was performed using the HKL2000

package.16

Crystallographic refinement was carried out starting from

the structure of nPD-L1 (PDB code 3H5K) against 95% of the

measured data, in the range 15–1.65 A resolution, using the

ccp4i program suite.17 The remaining 5% of the observed

data, which was randomly selected, was used in Rfree

calculations to monitor the progress of refinement. Non

crystallographic restraints were applied in REFMAC with

medium restraints for main-chain atoms and loose restraints

for side-chain atoms. Water molecules were incorporated into

the structure in several rounds of successive refinement, using

ARP/wARP followed by REFMAC runs.18,19 The final

protein model included 522 residues, and presented R and

Rfree values of 19.6 and 25.1%, respectively. The pertinent

refinement details along with the necessary statistics for the

final protein model are given in Table 1. Atomic coordinates

have been deposited in the Protein Data Bank (PDB) with

PDB-ID 3LE7.

PD-Ls and rPD-L1 labelling and cell internalization by

fluorescence analysis

Native PD-Ls and rPD-L1 were labelled using Texas-red

fluorescent dye by incubation with 5 fold excess dye in

20 mM phosphate buffer, pH 7.0 for 1 h at room temperature.

The reaction was stopped by desalting the mixture on a

Sephadex G25 column equilibrated in the same buffer.

Labelled proteins were analysed by SDS-PAGE.

For cell internalization experiments the NIH3T3 mouse

embryonic fibroblasts (ATCC, LGC Promochem, Middlesex,

UK) were cultured in DMEM containing 10% foetal calf

serum. For microscopy experiments, cells (2 � 105 mL) were

grown on glass coverslips for two days before use. PD-Ls and

rPD-L1 were incubated on the coverslips with fluorescent

proteins (2 mM final concentration). After 24, 48 and 72 h,

the coverslips were washed with PBS and fixed with 4%

p-formaldehyde in the same buffer for 20 min at 4 1C.

Coverslips were mounted onto microscope slides and sealed

with nail polish, for observation on a Zeiss Axiophot

fluorescence microscope. Monochrome images have been

taken for each sample using the filter sets for texas red and

DAPI fluorescence. All experiments were carried out in triplicates.

Results

Synthesis and expression of PD-L1 synthetic gene

The PD-L1/2 amino acid sequence was used for the back-

translation to the DNA sequence, in turn utilized for the

assembling of a synthetic gene using a PCR-based strategy.

The 810 bp PD-L1/2 synthetic gene was cloned into pET22b

and transformed into E. coli BL21(DE3) strain for hetero-

logous expression. The SDS-PAGE analysis of lysates from

induced and non-induced cells showed that a protein with a

molecular mass of about 29 kDa, the expected molecular size

for recombinant PD-L1/2 (hereafter named rPD-L1), was

present only in the induced cells (data not shown). The

expression of the rPD-L1 was found to be mostly in the

inclusion bodies with an average yield of about 0.02 mg of

protein per mL of bacterial culture. Its identity was confirmed

by direct Edman degradation of the first 11 N-terminal

residues, including the initial methionine at the N-terminal

(NH2-MINTITYDAGN-COOH).

Folding and purification of recombinant PD-L1

Despite the high percentage of sequence identity (81.6%), a

first attempt to use a folding strategy already reported for the

renaturation of PD-L412 revealed to be unsuccessful for

rPD-L1 folding (data not shown). Protein aggregates were

present at the end of the folding procedure. We first hypothesized

that this failure could be due to the presence of the glycan

chains on native PD-L1 and decided to modify the rPD-L1

folding strategy, increasing both redox-pair GSH/GSSH ratio,

and salt concentration. In particular, the addition of sucrose

to the folding buffer was determinant to achieve rPD-L1

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renaturation as revealed by structural and biological assays

(see later). The refolded protein was concentrated by ultra-

filtration and purified by cation exchange chromatography on

a Source 15S column (Fig. 1A). Peak 3, the major component,

was found to be homogeneous by SDS-PAGE. Minor peaks

(peaks 1 and 2), whose amount was found to be variable among

folding experiments, contained refolding intermediates, as

confirmed bymass spectrometry analysis (see later) and according

to a previously reported study on the folding of PD-L4.13

Mass spectrometry analysis

ESI/Q-TOF MS analysis of peaks 1 and 2 revealed the

presence of heterogeneous protein species whose molecular

masses corresponded to that of rPD-L1 with an extra methionine

and one or more glutathione moieties (data not shown). Thus,

these forms were not further analysed. The accurate relative

molecular mass (Mr) of peak 3 (29349, 32 � 0, 24) was found

to be in good agreement with the theoretical Mr of PD-L1

(29349.28, D=0.04 Da), calculated on the basis of its amino

acid sequence, including the extra methionine, and considering

the two disulfide bridges present in native PD-L1.11 The

ESI-MS spectra, deconvoluted using maximum entropy

algorithm,20 is reported in Fig. 1B. Minor MS peak, corres-

ponding to the [M + H3PO4]+ adduct (+98 amu) was also

detected.

Circular dichroism

A comparative study of the secondary structures of nPD-L1

and rPD-L1 was performed by circular dichroism (CD)

analysis. The CD spectra of both proteins exhibited, at 20 1C,

minima at 222 and 208 nm and a positive maximum at 190 nm,

typical of a-helices (insets of Fig. 2, plain line). This indicated,

as expected, that the two proteins adopted the same secondary

structure. To investigate the potential influence of the carbo-

hydrate moiety on protein stability, thermal denaturation

curves of rPD-L1 and nPD-L1 were performed. We observed

that unfolding of rPD-L1 was fully cooperative (Fig. 2A),

whereas cooperativity of unfolding was not observed for

nPD-L1 (Fig. 2B). The thermal denaturation of nPD-L1

displays a smaller variation of the CD signal at 222 nm,

compared to rPD-L1. This indicates that nPD-L1 is composed

of two unfolding units which display different thermal

stabilities and demonstrates that the major part of nPD-L1

is resistant to thermal unfolding. Consistently, measures of

CD spectra at 90 1C showed that both rPD-L1 and nPD-L1

retained residual secondary structure upon thermal denaturation

(insets of Fig. 2, dotted lines). Both spectra recorded at 90 1C

for nPD-L1 and for rPD-L1 exhibited a clear minimum at

214 nm (insets of Fig. 2, dotted lines), which is indicative of a

residual b-sheet structure. Furthermore, a deeper minimum at

214 nm, indicative of a larger amount of residual b-sheet, wasobserved for nPD-L1. This result well agrees with previous

studies showing that glycosylation often has a stabilising effect

on protein structures.21

Biological and enzymatic activity assays

Both recombinant and native (as positive control) PD-L1 were

assayed for their biological activities by measuring their

abilities to inhibit protein synthesis in a rabbit reticulocyte

cell-free system as described in the Method section. The ability

of rPD-L1 and nPD-L1 to inhibit protein synthesis was

comparable, as the IC50 values were 8.9 pM and 8.5 pM,

respectively.

Furthermore, the adenine polynucleotide glycosidase

activity of both recombinant and native PD-L1 was determined

on hsDNA and poly(A) substrates (Fig. 3). PD-L2 and PD-L4

were also assayed for comparison. As previously reported

for native PD-Ls, the deadenylation activity was found to

be higher when the glycosylation decreases11 with both

substrates. Interestingly, a higher adenine release was obtained

for rPD-L1 with respect to native PD-Ls when DNA was used

as substrate (Fig. 3A). This finding was further confirmed

when the smaller and homogeneous poly(A) was used as

substrate (Fig. 3B). In this instance, an increase of the

deadenylation activity of more than two-fold was measured

for rPD-L1 with respect to the native protein.

Table 1 Data collection and refinement statistics

nPD-L1 adenine

A. Data collectionSpace group C2Unit-cell parameters a,b,c (A); b (1) 161.72, 34.74, 121.29; 128.01

Resolution (A) 1.65N. of unique reflections 56763Average redundancy 4.2 (2.5)Rmerge (%) 9.0 (7.0)Completeness (%) 92.8 (85.3)Mean I/s(I) 6.7 (2.0)

B. RefinementResolution shell (A) 10.00–1.65Rwork (%) 19.6Rfree (%) 25.1No. residues Protein 522

Water 650Average Bfactors (A2) Protein 18.5

Water 28.5r.m.s. deviations Bond lengths (A) 0.008

Bond angles (1) 1.2

Values in parentheses are for the highest resolution shell (1.69–1.65 A).

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Fig. 1 (A) Elution profile from the cation exchange chromatography on a Source 15S column of recombinant PD-L1. The dotted line

indicates the increase of the conductivity gradient (mS cm�1). The SDS-PAGE analysis of the eluted peaks is reported in the inset.

Lane 1: molecular weight markers; lanes 2–4: peaks 1–3 from FPLC. (B) Deconvoluted mass spectrum of rPD-L1 by ESI/Q-TOF mass

spectrometry.

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Interaction of PD-L1 with adenine

In an attempt to understand the differences in the enzymatic

properties of nPD-L1 and rPD-L1, we investigated the

interaction between the two enzymes and adenine, the major

product of their enzymatic reaction. PD-L1 contains two

tryptophan residues, one of which (Trp 207) is situated in

the protein active site (Fig. 4). Therefore, we used fluorescence

spectroscopy to investigate the protein–adenine interaction

by exciting the tryptophan fluorescence at the maximum

quantum yield (280 nm). As a result, we observed that adenine

titration of nPD-L1 produced a remarkable shift in the

fluorescence emission spectrum toward lower wavelengths.

Indeed, in the presence of 5 adenine equivalents (moles of

adenine per mole of protein), the wavelength corresponding to

the fluorescence spectrum maximum moved from lmax 347 nm

(in the absence of adenine) to lmax 320 nm. Further adenine

addition produced a clear quenching of the fluorescence

intensity in nPD-L1, corresponding to about 50% of the

global fluorescence (Fig. 5A). A parallel fluorescence

experiment showed that adenine titration produced a different

effect on rPD-L1, as only the fluorescence intensity quenching

was observed, without the lmax shift (Fig. 5B). A similar result

was observed titrating PD-L4 with adenine (Fig. 5C). The

blue-shift observed upon binding of adenine to nPD-L1 is

compatible with either a different binding mode of adenine or

with a participation of glycan chains to the adenine binding.

To validate these hypotheses, we determined the crystal

structure of nPD-L1 in complex with adenine and compared

it to the available structure of its non-glycosylated homolog

PD-L4. The crystal structure was refined at high resolution

(1.65 A) and provided well-defined electron density maps

(Table 1 and Fig. 6). Analysis of the catalytic site showed that

adenine exhibited the same binding mode observed in the

structure of PD-L4, and it was anchored through several

hydrogen bonding interactions (Fig. 6). Similar to what

observed in the structure of PD-L4, adenine forms a stacking

interaction with the phenol ring of Tyr72 (Fig. 6), consistent

with the role of adenine carrier attributed to this residue.22 The

architecture of nPD-L1 catalytic site shows that, beside Tyr72,

another Tyr residue (Tyr122) composes the adenine binding

cleft (Fig. 6). This suggests that the shift in fluorescence spectra

upon adenine addition (Fig. 5A) reflects changes in accessibility

of interacting Trp and Tyr residues, given the overlapping

excitation wavelengths of Trp (280 nm) and Tyr (275 nm).

Despite the high quality of the density maps, no structure of

the glycan chains bound to Asn43 and to Asn255, which are

located just aside the active site cleft (Fig. 4), could be

modelled. As for the sugar chain bound to Asn10, on the

opposite side of the active site cleft, we modelled only one

N-acetyl-glucosamine sugar whereas other sugar rings were

not detectable in the electron density according to a large

conformational mobility of sugar chains.

Cellular uptake of rPD-L1 and native PD-Ls

It has been reported that single chain type 1 RIPs and the free

A subunit of type 2 RIPs can be internalized within cells.23

With the aim to investigate the cellular uptake of native PD-Ls

and rPD-L1, internalization experiments in NIH3T3 cells were

performed.

All native PD-Ls and rPD-L1 were internalized as attested

by the protein associated fluorescence present throughout the

Fig. 2 Thermal denaturation profile of (A) rPD-L1 and (B) of nPD-L1.

Insets report overlays of CD spectra at 20 1C (plain line), 40 1C

(dashed line) and 90 1C (dotted line).

Fig. 3 Adenine polynucleotide glycosidase activity of native PD-L1,

PD-L2 and recombinant PD-L1 assayed on hsDNA (A) and poly(A)

(B). Released adenine was measured spectrophotometrically at

260 nm. BSA was used as negative control.

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cytoplasm after 72 h of incubation (Fig. 7). A time course

experiment revealed a similar behaviour also after 24 and 48 h

of incubation (data not shown). It could be noted that

fluorescent proteins were in some instances distributed as

grain, likely indicative of a vesicular distribution. This peculiar

distribution appeared to be more evident when the glyco-

sylation degree decreased, as in the case of PD-L3 and

PD-L4 (Fig. 7, panels D and E, respectively). More interestingly,

a vesicular distribution was also revealed for rPD-L1

compared to native glycosylated PD-L1 and PD-L2 (Fig. 7,

panels F, B and C, respectively).

Discussion

N-glycosylation, a widely occurring co-translational modification

of plant proteins, starts in the endoplasmic reticulum (ER)

by the transfer of a core oligosaccharide unit to specific

asparagine residue(s) of the nascent polypeptide chain.

Fig. 4 3D model of PD-L1. The two tryptophanyl residues (Trp207

and Trp236) are reported in red and the three glycosylation sites on

Asn10, Asn43 and Asn255 are reported in green.

Fig. 5 Overlay of fluorescence spectra upon adenine addition, in the 0–50 equivalent range. Adenine titrations were performed with (A) nPD-L1,

(B) rPD-L1 and (C) PD-L4.

Fig. 6 (2Fo-Fc) electron density map, contoured at 2s, of the

catalytic site of nPD-L1 complexed with adenine.

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Processing of this oligosaccharide into high-mannose-type,

paucimannosidic-type or complex-type N-glycans occurs in

the secretory pathway as the glycoprotein moves from the ER,

through the Golgi complex, to its final destination.24,25

It has become evident that protein glycosylation is of utmost

importance for the structure and biological function of several

proteins, being involved in proteins conformation, stability,

solubility and activity. In addition, N-linked oligosaccharides

may contain targeting information for protein sorting within

the appropriate cell compartments.24,25 The effect of glycan

chains on protein folding, stability and biological activity has

been studied for several glycoproteins from various sources,

giving extremely diverse and sometime conflicting results.25–27

Some type 1 and type 2 RIPs, isolated from different

biological sources, are glycosylated. Several studies aimed to

investigate the potential influence of carbohydrate moieties on

RIP biological activity have been reported for type 2 RIPs.

It has been reported that sugars have no effect on ribo-

some-inactivating activities, as recombinant proteins, expressed

in heterologous systems (i.e. ricin and volkensin A chains),

retained full biological activity.28,29 The structural character-

ization of the oligosaccharide chains of several type 1 RIPs

has also been widely exploited, revealing the occurrence of a

well-defined paucimannosidic N-glycosylation pattern, observed

for most of known vacuolar glycoproteins.11,12,30–32

Despite the intensive research on RIP biochemical and

functional characterization, also finalized to their interesting

application in therapy against viral infections or malignancies,

little is known about the role of glycan chains in their

conformation and stability, likely due to the difficulty in

obtaining high resolution diffracting crystals for glycosylated

proteins. Furthermore, structural characterization of glycan

chains is usually hampered by their large conformational

freedom. Only in rare cases, like the recently determined

structure of cucurmosin, a novel type 1 RIP isolated from

Cucurbita moschata, the structure of glycan chains is stabilized

by intermolecular contacts in the crystal.31

Three glycosylated variants (glycoforms) with the standard

paucimannosidic oligosaccharide and a fourth unglycosylated

isoform have been isolated and characterized from P. dioica

leaves, as previously discussed. The two primary differentially

glycosylated structures (PD-Ls1/2 and PD-Ls 3/4) have 81.6%

sequence identity.11 The most glycosylated isoform, PD-L1,

has been used as a model to specifically investigate the

influence of carbohydrate moieties on type 1 RIP structural

conformation and functional activities. To this aim, the PD-L1

Fig. 7 Cellular uptake of rPD-L1 and native PD-Ls by NIH3T3 cells. For each isoform, a schematic representation of primary structure and

glycan moiety is reported on the right. Magnification 1600�.

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synthetic gene was expressed in the E. coli heterologous

system. The expressed recombinant PD-L1 was renaturated,

purified and assayed for protein synthesis inhibition in a cell

free system and for adenine polynucleotide glycosidase activity

on hsDNA and poly (A). We did not find a reduction of

activity measured as IC50 (i.e. the amount of enzyme to inhibit

protein synthesis by 50%). However, the lack of carbohydrate

moieties in PD-L1 affects the adenine polynucleotide

glycosidase activity measured as adenine release from DNA

and poly (A), in comparison with both native PD-L1 and

PD-L2. The observed catalytic activity increase is more

evident with the poly (A) substrate, likely due to the higher

abundance of adenines in this substrate and/or to its smaller

dimension.13 Whereas total removal of glycans results in the

62% increase of adenine release activity on poly(A), the lack

of only one glycan in PD-L2 (Asn255), produces a 37%

adenine release increase compared to PD-L1. To further

investigate this issue, we analyzed the interactions of

nPD-L1 and of its recombinant form rPD-L1 with adenine,

the product of the de-adenylation reaction. Using fluorescence

spectroscopy, we observed that binding of adenine to nPD-L1

induces a remarkable blue-shift (17 nm) of the fluorescence

emission wavelength, and a strong intensity quenching. This

wavelength shift was not observed upon binding of adenine to

either rPD-L1 or PD-L4, which is not glycosylated (Fig. 5). By

determining the crystal structure of nPD-L1 in complex with

adenine, we show that adenine binding pocket includes two

Tyr residues (Tyr72 and Tyr122) and Trp207 (Fig. 6). The

binding mode of adenine as well as the conformation of

residues belonging to the catalytic site are superimposable to

those revealed in the complex of the homologue PD-L4 with

adenine.22 Therefore, the blue-shift observed for nPD-L1 but

not for PD-L4 (Fig. 5) is not to be ascribed to a change in the

adenine interaction with the catalytic site residues. Blue-shifts

are often associated with an increase in hydrophobicity

around aromatic rings. Consistently, a similar blue-shift in

tryptophan emission maximum was recently observed upon

binding of glycolipids to the human glycolipid binding protein,

an event which severely limits the solvent accessibility of the

tryptophan.33 The crystal structure of nPD-L1 also shows that

glycan chains of nPD-L1 are endowed with a large structural

mobility, as electron density did not allow sugar modelling.

Altogether, these results suggest that although flexible and not

detectable by crystallographic methods, glycan chains severely

hamper solvent accessibility to the aromatic residues located

in the catalytic site (Fig. 6, Trp207, Tyr72 and Tyr122).

Therefore, it is reasonable to hypothesize that the presence

of multiple glycosylation sites surrounding the active site could

be responsible of a reduced accessibility to substrates and,

therefore, of the observed reduced enzymatic efficiency

measured as adenine release of nPD-L1.

It has been reported that single chain type 1 RIPs and the

free A subunits of type 2 RIPs can be internalized by cells.23

To further investigate the cellular uptake of native

glycosylated PD-Ls and rPD-L1, preliminary internalization

experiments in NIH3T3 cells were performed. Under the used

experimental conditions we found that both native PD-Ls and

rPD-L1 were internalized. Interestingly, a peculiar distribution

in grains, indicative of a vesicular compartmentalization was

revealed. From the comparison of native PD-L1 and PD-L2

with rPD-L1, this distribution appeared to be related to the

glycosylation degree. Notably, this behaviour is independent

from the recognition of the mannosylated glycoproteins,

as NIH3T3 cells lack mannose receptors,34,35 known to be

responsible for their differential uptake by macrophages and

non-parenchymal cells of liver.36–38 It has been shown that

type 1 RIPs and the free A subunits of type 2 RIPs can be

cytotoxic at high concentrations.23 Initially it was thought that

the single chain toxins might enter cells via passive mechanisms

such as fluid phase uptake.37 This hypothesis has been revised

as different type 1 RIPs display an organ-specific toxicity and

cell types show a variable sensitivity to RIPs. These evidences

suggest that specific mechanism(s) occur to allow type 1 RIPs

uptake. A study of the uptake of gelonin has suggested that

this RIP, when internalised by pinocytosis, is released from

endosomes and lysosomes,38,39 although the mechanism by

which this is achieved is completely unknown. Furthermore it

is also known that saporin, a type 1 unglycosylated RIP, can

be internalized by the interaction with specific receptors (alpha

2-macroglobulin or LRP receptors40) leading to an intra-

cellular pathway that involves the endo-lysosomal system but

never the classical Golgi pathway followed by whole ricin.41,42

Further studies are needed to elucidate the mechanisms of

the cellular entry pathway of PD-Ls. Although a detailed

study of PD-Ls internalization is beyond the scope of the

present work, it will be of interest to perform subcellular

localization experiments aimed to identify the intracellular

compartments involved in the delivery of type 1 RIPs

within cells by using specific markers and laser scanning

microscopy.43 These studies could be useful to understand

the differences at the basis of type 1 internalization mechanisms,

important for the design of approaches for targeted cell

delivery.

Overall, the presented data confirm that the removal of

glycan moieties from type 1 RIPs does not affect the biological

activity in terms of protein synthesis inhibition. However, the

removal of carbohydrate chains significantly increases the

deadenylation capability, likely as a consequence of the

increased accessibility of substrates to the active site pocket.

Furthermore, preliminary data on cellular uptake showed that

all PD-Ls isoforms are internalized and, for the first time, that

the vesicular distribution within cells could be influenced by

the protein glycosylation degree.

Abbreviations

RIPs Ribosome-inactivating proteins;

IPTG Isopropyl-b-thiogalactopyranoside;APG Adenine polynucleotide glycosylase;

Man Mannose;

GlcNAc N-acetyl-D-glucosamine;

Fuc Fucose;

Xyl Xylose;

GSH Reduced glutathione;

GSSG Oxidized glutathione;

NaP Na phosphate buffer;

PVDF Polyvinylidene difluoride;

CD Circular Dichroism;

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hsDNA Herring sperm DNA;

ER Endoplasmic reticulum;

DAPI 40,6-diamidino-2-phenylindole.

Acknowledgements

This study was supported by funds from the Second University

of Naples and University of L’Aquila and by grants from the

Ministero Istruzione, Universita e Ricerca (MIUR, Italy) and

by FIRB no. RBRN07BMCT.

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