Báo cáo khoa học: Expression analysis of the nucleocytoplasmic lectin ‘Orysata’ from rice in Pichia pastoris

Expression analysis of the nucleocytoplasmic lectin ‘Orysata’ from rice in Pichia pastoris Bassam Al Atalah1, Elke Fouquaert1, Dieter Vanderschaeghe2, Paul Proost3, Jan Balzarini4, David F. Smith5, Pierre Rouge´ 6, Yi Lasanajak5, Nico Callewaert2 and Els J. M. Van Damme1

1 Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Belgium 2 Unit for Medical Biotechnology, Department for Molecular Biomedical Research, Ghent, Belgium 3 Laboratory of Molecular Immunology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium 4 Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium 5 Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA 6 Signaux et Messages Cellulaires chez les Ve´ ge´ taux, Castanet-Tolosan, France

Keywords antiviral activity; glycan array; lectin; nucleus; Orysata

Correspondence E. J. M. Van Damme, Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure links 653, B-9000 Gent, Belgium Fax: +32 92646219 Tel: +32 92646086 E-mail: elsjm.vandamme@ugent.be

(Received 24 December 2010, revised 5 March 2011, accepted 1 April 2011)

The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin- related lectin expressed in rice plants after exposure to certain stress condi- tions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pasto- ris with the Saccharomyces a-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the med- ium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS ⁄ PAGE and western blot analysis showed that the recombi- lectin exists in two molecular forms. Far western blot analysis nant revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9–11 glycans being the most abundant. Glycan array analysis showed that Orys- ata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.

doi:10.1111/j.1742-4658.2011.08122.x

Introduction

Carbohydrate-binding proteins or lectins are wide- spread in the plant kingdom. These proteins have the ability to recognize and reversibly bind to well defined carbohydrate structures in plants or on the surface of

pathogens and predators. In the past, research was concentrated on lectins that are expressed at high con- centrations especially in storage tissues and hence were easy to purify. For many of these lectins it was shown

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Abbreviations AOX1, alcohol oxidase 1; BY2, Bright Yellow 2; Calsepa, Calystegia sepium agglutinin; EGFP, enhanced green fluorescent protein; GlcNAc, 2-amino-2-N-acetylamino-D-glucose; GNA, Galanthus nivalis agglutinin; HHA, Hippeastrum hybrid agglutinin; JRL, jacalin related lectin; Morniga M, mannose binding Morus nigra agglutinin; Nictaba, Nicotiana tabacum agglutinin; Orysata, Oryza sativa agglutinin; PHA, Phaseolus vulgaris agglutinin; PNGase F, peptide N-glycosidase F; PVDF, poly(vinylidene difluoride); RSV, respiratory syncytial virus.

brasiliensis, a homolog of the classical vacuolar conca- navalin A, was also expressed by the yeast P. pastoris [15]. Oliveira et al. described the expression of the JRL from breadfruit seeds (Artocarpus incisa) in Pichia [16]. In 2007 the first nucleocytoplasmic lectin from tobacco (Nicotiana tabacum agglutinin, Nictaba) related to the Cucurbitaceae lectins was expressed and purified from P. pastoris [17]. More recently, the first nucleocytoplas- (GNAmaize) was mic GNA homolog from plants expressed in P. pastoris [18].

that they could play a role in plant defense. In the last decade evidence has accumulated that plants also express certain carbohydrate-binding proteins after exposure to abiotic stress situations like drought and salinity. In contrast to the abundant lectins that are mostly located in the plant vacuole, these lectins are present in the nucleus and the cytoplasm of the plant cell. A novel concept was developed that these lectins probably play a role in the stress physiology of the plant [1].

galactose-binding

JRLs

that

the

to

The family of jacalin-related lectins (JRLs) groups all proteins that possess one or more domains equiva- lent to ‘jacalin’, a galactose-binding protein from jack fruit (Artocarpus integrifolia) seeds [2]. In the last dec- ade many JRLs have been identified which resulted in a subdivision of this family into two groups: the galac- tose-binding and the mannose-binding lectins. In con- trast are synthesized on the endoplasmic reticulum and follow the secretory pathway to accumulate in protein storage vacuoles, the mannose-binding JRLs are synthesized and located in the cytoplasm [3].

In this paper we describe the heterologous expres- sion of Orysata, a JRL from rice. Based on a detailed analysis of its sequence, this lectin was predicted to locate to the nucleocytoplasmic compartment of plant cells, as shown by expression of a fusion protein in tobacco cells. Furthermore, the successful expression the His-tagged Orysata in the yeast P. pastoris of allowed sufficient amounts of the lectin to be purified to study in detail the molecular structure of the pro- tein, its carbohydrate-binding specificity and its antivi- ral activity. Interestingly, antiviral assays showed that Orysata is active against HIV as well as respiratory syncytial virus (RSV), indicating that the lectin may qualify as a microbicide agent.

B. Al Atalah et al. Expression of nucleocytoplasmic Orysata

Results

Orysata is located in the cytoplasmic/nuclear compartment

The very first inducible lectin to be purified and characterized was a mannose-specific JRL from NaCl- treated rice seedlings, called Oryza sativa agglutinin or Orysata [4]. Sequence analysis revealed that Orysata corresponded to a previously described salt-inducible protein (SalT) [5] and can be classified in the group of JRLs. Orysata cannot be detected in untreated plants but is rapidly expressed in roots and sheaths after exposure of whole plants to salt or drought stress, or upon jasmonic acid and abscisic acid treatment [5–7]. Interestingly, the lectin is also expressed in excised leaves after infection with an incompatible Magnapor- the grisea strain [8,9] as well as during senescence [10]. Since Orysata is expressed at very low levels in certain plant tissues and only after exposure to stress, the purification of the lectin is cumbersome and requires huge amounts of plant material.

fusion construct

reported to be

transformation

points

after

time

Analysis of the amino acid sequence of Orysata (Gen- Bank accession number CB632549) using the signalp 3.0 tool (http://www.cbs.dtu.dk/services/SignalP) indi- cated the absence of a signal peptide, suggesting that the corresponding rice protein is synthesized on free polysomes. Furthermore the psort program (http:// psort.nibb.ac.jp) predicted a subcellular localization of Orysata in the cytoplasmic compartment of the plant cell. The localization of Orysata was corroborated by expression of an enhanced green fluorescent protein (EGFP) for the lectin in tobacco cells. Therefore the lectin sequence was fused in-frame to the C-terminus of EGFP and the fusion protein was transiently expressed in tobacco Bright Yellow 2 (BY2) cells. Confocal microscopy of EGFP-Orysata at different time points after particle bombardment revealed that the rice lectin is located in the nucleus and the cytoplasm of the plant cell. No fluorescence emission was seen in the nucleolus or the vacuole. A very similar distribution pattern was observed at and different fluorescence was detectable until (cid:2) 80 h after trans- formation (Fig. 1).

In the last decades the methylotrophic yeast Pichia pastoris has become the leading yeast vehicle for the production of a broad range of proteins [11]. Heterolo- gous protein expression in Pichia is controlled by the alcohol oxidase 1 (AOX1) promoter. Expression of the AOX1 gene is tightly regulated and induced by metha- nol to high levels [12,13]. A variety of lectins were successfully among the proteins expressed in P. pastoris. For example, Raemakers et al. [14] described the successful expression of the legume lectin Phaseolus vulgaris agglutinin (PHA) and the GNA-related lectin from snowdrop (Galanthus nivalis agglutinin, GNA) in P. pastoris. A glucose-mannose- binding legume lectin from the seeds of Canavalia

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EGFP

EGFP

Orysata

cytoplasm and the nucleoplasm, including the nucleo- lus (Fig. 1).

Purification and characterization of recombinant Orysata expressed in Pichia pastoris

B. Al Atalah et al. Expression of nucleocytoplasmic Orysata

24 h

n

the presence of

N

48 h

m

v

c

recombinant

of

Cloning of the coding sequence of Orysata into the Escherichia coli ⁄ P. pastoris shuttle vector pPICZaB yielded a fusion construct whereby the Orysata sequence was linked to a C-myc epitope and a C-ter- minal histidine tag (Fig. 2). The fusion protein was successfully expressed in the Pichia strain X-33. the a-mating sequence Because of from Saccharomyces cerevisiae at the N-terminus of the construct, the recombinant Orysata was secreted into the medium. Transformed Pichia colonies that yielded a positive result after analysis of the total protein by SDS ⁄ PAGE and subsequent western blot analysis were grown in 1 L cultures. Afterwards the recombinant Orysata was purified from the medium using a combination of ion exchange chromatogra- phy, metal affinity chromatography on a Ni-Sepha- rose column and affinity chromatography on a mannose-Sepharose 4B column. Starting from a 1 L culture (cid:2) 12 mg protein was obtained.

A construct for the native 27 kDa EGFP under the control of the 35S promoter was used as a control. Expression of this protein in tobacco cells yielded an even distribution of the fluorescence pattern over the

SDS ⁄ PAGE analysis of the purified Orysata from Pichia revealed two bands of (cid:2) 18.5 and 23 kDa (Fig. 3A). A very similar result was obtained after western blot analysis and detection of the recombi- nant proteins using a monoclonal antibody directed

Fig. 1. Confocal transiently trans- images collected from living, formed tobacco BY2 cells expressing free EGFP and EGFP-Orysata. Expression of EGFP-Orysata or EGFP was analyzed at different time points after transformation. Scale bars represent 25 nm. Cell compartments: n, nucleolus; N, nucleus; m, cell membrane; c, cyto- plasm; v, vacuole.

A

B

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Fig. 2. (A) Sequence of recombinant Orysata expressed in Pichia, preceded by an N-terminal signal peptide (residues 1–89) necessary for secretion and a C-terminal tag containing a c-myc epitope and a (His)6 tag (residues 254–259). The cleavage sites for the signal peptide are indicated (Kex2 protease site at position 86 and Ste13 protease sites at positions 87 and 89). The N-terminal sequence of recombinant Orys- ata determined by Edman degradation is underlined. The putative N-glycosylation site is shown in bold. (B) Sequence alignment for the three mannose-binding JRLs from Oryza sativa, Calystegia sepium and Morus nigra. Identical residues are shown in white with a black background and similar residues are boxed. The amino acid residues forming the monosaccharide-binding site are indicated by dots.

B. Al Atalah et al. Expression of nucleocytoplasmic Orysata

A

B

C

D

Agglutination activity and carbohydrate-binding properties of recombinant Orysata

against the polyhistidine tag (Fig. 3B). The deduced molecular mass of the lower band is in good agree- ment with the calculated molecular mass of Orysata fused to the c-myc epitope and the polyhistidine tag (18.46 kDa).

(Table 1).

and

To study the biological activity of the recombinant lec- tin expressed in Pichia, the recombinant Orysata was tested for agglutination activity towards rabbit ery- throcytes. Agglutination was observed after adding the red blood cells to the purified lectin, the minimal con- centration of lectin necessary to obtain agglutination activity being 5 lgÆmL)1 whereas it was 0.12 lgÆmL)1 for the native Orysata [4]. Preliminary carbohydrate inhibition assays revealed that the agglutination activ- ity of the recombinant Orysata was similar to that of the native lectin in that the agglutination of rabbit ery- throcytes was inhibited by mannose, methyl a-manno- Several trehalose pyranoside glycoproteins also inhibited the agglutination activity of recombinant Orysata, although at higher concentra- tion than required for inhibition of the native lectin.

More detailed carbohydrate-binding studies were performed using a screening of the lectin on a glycan array (Table 2). The carbohydrate-binding properties of recombinant Orysata were investigated on glycan array v4.2, and compared with the sugar-binding speci- ficities of two other mannose-binding JRLs from Caly- stegia sepium and Morus nigra, further referred to as Calsepa and Morniga M, respectively (Fig. 2B). At first sight all three JRLs show similar interaction pat- terns with the glycan array (Fig. 5). All lectins react with both high-mannose and complex N-glycans. How- ever, more detailed analyses of the glycan array data

N-terminal sequence analysis of both polypeptides yielded an identical sequence EAEAAAMTLVKI GLW. Since the six N-terminal amino acid residues in this sequence correspond to the yeast a-mating sequence it can be concluded that part of the signal peptide sequence was not cleaved properly (Fig. 2). the amino acid sequence for Detailed analysis of Orysata revealed the presence of a putative glycosyla- tion site NNT (Fig. 2). Far western blot analysis whereby the blotted proteins were incubated with the N-glycan binding lectin Nictaba [17] revealed interac- tion of Nictaba with the Orysata polypeptide of (cid:2) 23 kDa, suggesting that this polypeptide is glycosy- lated (Fig. 3C). Indeed, only one polypeptide band of 18.5 kDa remains after removing the N-glycans of Orysata using peptide N-glycosidase F (PNGase F) treatment (Fig. 3D). Subsequent N-glycan analysis (Fig. 4) revealed that the carbohydrate structures are high-mannose (Man9–11) glycans which are typically produced by wild-type P. pastoris [19]. Molecular modeling of the mature Orysata sequence with an N-glycan at the position of the putative N-glycosyla- tion side revealed that the glycan is located at the the carbohydrate-binding site and opposite side of hence is unlikely to interfere with the carbohydrate- binding properties of the lectin (results not shown).

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Fig. 3. Crude protein extract from the medium of Pichia cell culture and purified Orysata were analyzed by SDS ⁄ PAGE (A), western blot analysis with a monoclonal anti-His antibody (B), far western blot analysis using Nictaba (1 lgÆmL)1) (C) and PNGase F treatment (D). Sam- ples are loaded as follows: lane M1, protein ladder (increasing molecular mass 10, 17, 26, 34, 43, 55, 72, 95, 130, 170 kDa); lane M2, unstained protein ladder (increasing molecular mass 14.4, 18.4, 25, 35, 45, 66.2, 116 kDa) (Fermentas, St Leon-Rot, Germany); lanes 1 and 4, crude extract from Pichia cells expressing Orysata (15 lg); lanes 2 and 5, purified recombinant Orysata (2.5 lg) analyzed in the presence of mercaptoethanol; lanes 3 and 6, purified recombinant Orysata (2.5 lg) analyzed in the absence of mercaptoethanol; lanes 7 and 8, posi- tive controls (Nictaba); lane 9, recombinant Orysata (2.5 lg); lane 10, pure Orysata (2.5 lg); lane 11, pure Orysata (2.5 lg) digested with PNGase F (3.8 IUB mU); lane 12, positive control RNase B (2.5 lg); lane 13, RNase B (2.5 lg) digested with PNGase F (3.8 IUB mU).

B. Al Atalah et al. Expression of nucleocytoplasmic Orysata

show that Orysata and Morniga M show a higher reactivity towards high-mannose N-glycans than Cal- sepa, which interacts primarily with galactosylated and sialylated bi-antennary complex N-glycans.

Antiviral activity of recombinant Orysata, compared with Calsepa and Morniga M

at a 50% effective concentration of 1.7–5.6 lgÆmL)1, corresponding to a concentration which is (cid:2) 10-fold higher than required for HHA. In contrast, Calsepa was marginally inhibitory against HIV-1 (EC50 ‡ 100 lgÆmL)1). Morniga M could not be evaluated at compound concentrations higher than 4 lgÆmL)1 due to cytotoxicity in the cell cultures at a concentration of ‡ 20 lgÆmL)1.

The lectins have also been investigated for their inhibitory activity against syncytia formation between persistently HIV-1(IIIB)-infected HUT-78 ⁄ HIV-1 cells and uninfected Sup T1 cells. The three lectins pre- vented giant cell formation at 18–38 lgÆmL)1 by 50%.

The three JRLs were evaluated for their antiviral activ- ity against HIV-1(IIIB) and HIV-2(ROD) in CEM cell (Table 3). The a1,3 ⁄ a1,6-mannose-specific cultures Hippeastrum hybrid agglutinin (HHA) was included as a control. Orysata efficiently suppressed HIV infection

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Fig. 4. Identification of the N-glycans pres- ent on recombinant Orysata. N-glycans were released using PNGase F (C) and to identify aspecific peaks (*) we also omitted the enzyme as a negative control (B). Alpha- 1,2-mannosidase (D) and a broad-specific a-mannosidase (E) were added to the PNGase F treated Orysata to identify the N-glycan structures. The result of a malto- dextrose reference is also given (A). Sugar code used: green circles indicate mannose residues; red circles are a-1,2-mannoses that cannot be cleaved by the a(1,2)-man- nosidase due to steric hindrance. Blue squares indicate GlcNAc residues and yellow circles indicate galactose residues as suggested by the Consortium for Functional Glycomics.

can accommodate

B. Al Atalah et al. Expression of nucleocytoplasmic Orysata

Table 1. Comparison of the carbohydrate-binding specificities of native and recombinant Orysata. IC50 is the concentration required to give a 50% inhibition of the agglutination of trypsin-treated rabbit erythrocytes at a lectin concentration of 12 lgÆmL)1. The results for native Orysata are taken from [4].

IC50

Native Orysata Recombinant Orysata

binding cavity largely open at both extremities, and thus extended oligosaccharide chains (Fig. 6B,E). The binding site of Morniga M possesses a totally different shape due to the bulki- ness of loop L2 which closes up the cavity at one extremity and considerably decreases its size (Fig. 6E). However, the carbohydrate-binding cavity of Morniga M remains largely open at the opposite extremity which should allow a3-O-linked saccharides to inter- act with the lectin but prevent the correct accommo- dation of a1-O-linked saccharides.

Sugar

12 12 12 50 25 25 Mannose (mM) Trehalose (mM) Methyl a-mannopyranoside (mM) Glycoprotein

Discussion

lectins

recombinant

This concentration proved to be 10- to 20-fold higher than required for HHA under similar experimental conditions (Table 3). Interestingly, when exposed to RSV-infected HeLa cell cultures Orysata and Calsepa (EC50 1.6–2.1 lgÆmL)1) but not Morniga M and HHA (EC50 ‡ 20 lgÆmL)1) efficiently inhibited viral infection.

Molecular modeling of carbohydrate-binding sites

We describe the characterization of Orysata, a man- nose-binding JRL from rice (Oryza sativa) expressed in P. pastoris. Recombinant Orysata was successfully expressed in Pichia strain X-33 with the addition of a signal sequence for secretion of the recombinant pro- tein into the medium. Approximately 12 mg of the recombinant lectin was purified from the medium of a 1 L culture (BMMY medium, pH 6) induced with methanol for 72 h. Compared with the yield reported for other that were expressed extracellularly in Pichia, the amount of lectin obtained for Orysata is considered to be rather low. However, it should be mentioned that the yield obtained for the nucleocytoplasmic lectin from tobacco was even lower, being only 6 mgÆL)1 [17]. To our knowledge only one JRL has been previously expressed in Pichia. The galactose-binding lectin frutalin from breadfruit seeds was successfully expressed at 18–20 mgÆL)1 [16]. Much higher yields of recombinant protein can be obtained when Pichia cultures are grown in a bioreactor under controlled conditions, as reported for the recombinant lectins from Aleuria aurantia (67 mgÆL)1) [22], snow- drop (80 mgÆL)1) [23] and the bean lectin PHA-E (100 mgÆL)1) [24].

sequences,

suggesting that

JRLs

the carbohydrate-binding cavity of

is unglycosylated,

indicating that

Although the JRLs Orysata, three Man-specific Morniga M and Calsepa accommodate both Man and methyl mannose (MeMan) in a very similar way (Fig. 6A,D,G), they display a rather different affinity towards more complex saccharides as shown from the reported glycan array experiments (Table 2) and the anti-HIV activity (Table 3). In this respect, Orysata resembles Morniga M, since both lectins predomi- nantly interact with high-mannose N-glycans, whereas Calsepa exhibits a higher affinity for complex N-gly- cans. These discrepancies most probably depend on differences in the shape and size of their carbohy- drate-binding cavities. The carbohydrate-binding cav- ity of Man-specific (Calsepa, Morniga M, Orysata) consists of three loops L1, L2 and L3 con- taining two conserved Gly (L1) and Asp (L3) residues and two other variable residues (Thr134 and Leu135 in Orysata, Phe150 and Val151 in Calsepa, Tyr141 and Tyr142 in Morniga M) that also belong to loop L3 (Fig. 6C,F,I). Depending on the bulkiness of loop the lectins L2, exhibits considerable differences in shape and size [20,21]. Orysata and Calsepa exhibit a crescent-shaped

After purification, two molecular forms of the lectin were detected by SDS ⁄ PAGE and western blot analy- sis. Edman degradation revealed them to have identical N-terminal the higher fraction might be glycosylated. molecular weight Indeed a careful analysis of the amino acid sequence revealed one putative N-glycosylation site at position 102 of the mature Orysata sequence (NNT). Far wes- tern blot analysis using Nictaba, a lectin with well defined specificity towards high-mannose and complex N-glycans [25], confirmed that the 23 kDa polypeptide for Orysata is glycosylated whereas the 18.5 kDa the polypeptide recombinant Orysata obtained from the Pichia culture further is partially glycosylated. This

result was

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2 8 250 60 250 500 Thyroglobulin (lgÆmL)1) Ovomucoid (lgÆmL)1) Asialomucin (lgÆmL)1)

B. Al Atalah et al. Expression of nucleocytoplasmic Orysata

Table 2. Comparative analysis of glycan array results for Orysata, Morniga M and Calsepa. The glycan with the highest relative fluorescence unit (RFU) is assigned a value of 100. The rank is the percentile ranking.

Morniga M 50 lgÆmL)1 Calsepa 50 lgÆmL)1 Orysata 25 lgÆmL)1

RFU Rank RFU Rank RFU Rank Glycan no. Structure

360 42 939 100 29 317 76 18 912 86

212 41 305 96 31 139 81 6814 31

342 34 647 81 33 653 87 10 507 48

321 34 083 79 28 240 73 12 119 55

56 32 258 75 33 609 87 19 389 88

361 305 31 759 30 801 74 72 35 422 30 973 92 80 11 095 75 86 51 35

399 29 008 68 25 848 67 5930 27

358 28 743 67 19 812 51 8588 39

316 28 510 66 33 022 86 14 593 67

51 27 612 64 29 277 76 6775 31

346 458 27 579 27 178 64 63 37 958 30 338 98 79 13 309 11 613 61 53

53 26 984 63 31 648 82 13 724 63

393 26 515 62 24 029 62 11 719 53

52 26 286 61 38 115 99 15 111 69

345 323 25 287 25 059 59 58 33 568 32 351 87 84 18 242 15 692 83 72

49 343 24 991 24 979 58 58 38 600 29 082 100 75 12 609 12 118 58 55

a

a

317 24 343 57 24 806 64 12 033 55

418 23 801 55 23 280 60

425 23 714 55 16 526 43 5874 27

315 23 432 55 24 349 63 1325 6

368 23 094 54 30 841 80 5745 26

a No reactivity.

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50 213 477 21 861 21 621 21 471 51 50 50 34 978 26 316 28 412 91 68 74 21 918 7179 2475 100 33 11 Gala1-3Galb1-4GlcNAcb1-2Mana1-3(Gala1-3Galb1-4GlcNAcb1-2Mana1- 6)Manb1-4GlcNAcb1-4GlcNAcb-Sp20 Mana1-6(Mana1-3)Mana1-6(Mana1-2Mana1-3)Manb1-4GlcNAcb1- 4GlcNAcb-Sp12 Mana1-3(Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1- 4GlcNAc-Sp12 Galb1-3GlcNAcb1-2Mana1-3(Galb1-3GlcNAcb1-2Mana1-6)Manb1- 4GlcNAcb1-4GlcNAcb-Sp19 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb1-4GlcNAcb1- 2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp13 Mana1-3(Galb1-4GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6)Manb1-4Glc- NAcb1-4GlcNAcb-Sp12 Gala1-4Galb1-3GlcNAcb1-2Mana1-3(Gala1-4Galb1-3GlcNAcb1- 2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp19 Fuca1-2Galb1-4GlcNAcb1-2Mana1-3(Fuca1-2Galb1-4GlcNAcb1-2Mana1- 6)Manb1-4GlcNAcb1-4GlcNAcb-Sp20 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Galb1-4GlcNAcb1-2Mana1- 6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 GlcNAcb1-2Mana1-3(GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1- 4GlcNAcb-Sp12 Galb1-4GlcNAcb1-2Mana1-3Manb1-4GlcNAcb1-4GlcNAc-Sp12 Galb1-4GlcNAcb1-6(Galb1-4GlcNAcb1-2)Mana1-6(Galb1-4GlcNAcb1- 2Mana1-3)Manb1-4GlcNAcb1-4GlcNAcb-Sp19 Galb1-4GlcNAcb1-2Mana1-3(Galb1-4GlcNAcb1-2Mana1-6)Manb1- 4GlcNAcb1-4GlcNAcb-Sp12 Galb1-4GlcNAcb1-2Mana1-3(GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1- 4GlcNAc-Sp12 GlcNAcb1-2Mana1-3(GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1- 4GlcNAcb-Sp13 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3Manb1-4GlcNAcb1-4GlcNAc-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Neu5Aca2-3Galb1- 4GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 Mana1-3(Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Mana1-6)Manb1-4GlcNAcb1- 4GlcNAc-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(GlcNAcb1-2Mana1-6) Manb1-4GlcNAcb1-4GlcNAcb-Sp12 GlcNAcb1-2Mana1-3(GlcNAcb1-2(GlcNAcb1-6)Mana1-6)Manb1-4GlcNAcb1- 4GlcNAcb-Sp19 Galb1-3GlcNAcb1-2Mana1-3(Galb1-3GlcNAcb1-2(Galb1-3GlcNAcb1- 6)Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp19 Neu5Aca2-3Galb1-4GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb1-4GlcNAcb1- 2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 Gala1-3(Fuca1-2)Galb1-4GlcNAcb1-2Mana1-3(Gala1-3(Fuca1-2)Galb1-4Glc- NAcb1-2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp20 Mana1-3(Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp13 Mana1-6(Mana1-3)Mana1-6(Mana1-3)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 Mana1-6(Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAcb-Sp19

B. Al Atalah et al. Expression of nucleocytoplasmic Orysata

A

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35 000

35 000

30 000

30 000

25 000

25 000

20 000

20 000

15 000

15 000

l

l

10 000

10 000

t i n u e c n e c s e r o u l f e v i t a e R

t i n u e c n e c s e r o u l f e v i t a e R

5000

5000

0

0

1 21 41 61 81 101 121 141 161 181 201 221 241 261 281 301 321 341 361 381 401 421 441 461 481 501

Glycan no.

1 21 41 61 81 101 121 141 161 181 201 221 241 261 281 301 321 341 361 381 401 421 441 461 481 501 Glycan no.

C

30 000

25 000

20 000

15 000

10 000

l

t i n u e c n e c s e r o u l f e v i t a e R

5000

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1 21 41 61 81 101 121 141 161 181 201 221 241 261 281 301 321 341 361 381 401 421 441 461 481 501 Glycan no.

the

Fig. 5. Comparative analysis of binding of recombinant Orysata, Morniga M and Calsepa on the glycan array. (A–C) Interaction of recombi- nant Orysata (25 lgÆmL)1), Morniga M (50 lgÆmL)1) and Calsepa (50 lgÆmL)1), respectively. The complete primary data set for each protein is available on the website of the Consortium for Functional Glycomics (http://www.functionalglycomics.org). Sugar code used: green circles indicate mannose residues, yellow circles indicate galactose residues, blue squares indicate GlcNAc residues, purple diamonds indicate Neu- Ac and red triangles indicate fucose.

Table 3. Inhibitory activity of the lectins against HIV-1 and HIV-2 in human T-lymphocyte (CEM) cell cultures and against syncytium for- mation between HUT-78 ⁄ HIV-1 and Sup T1 cells. EC50 is the effec- tive concentration or the concentration required to protect CEM cells against the cytopathogenicity of HIV by 50% or to prevent syncytia formation in co-cultures of persistently HIV-1-infected HUT-78 cells and uninfected Sup T1 lymphocyte cells.

EC50 (lgÆmL)1)

HUT-78 ⁄ HIV-1 + HIV-2(ROD) Sup T1 Compound HIV-1(IIIB)

1.7 ± 0.14 5.6 ± 3.7

tioned that JRL frutalin was also partially glycosylated after secreted expression in Pichia with a very similar size difference between the glycosylated and the non-glycosylated lectin polypeptides [16]. Fur- thermore N-terminal sequence analysis of recombinant Orysata showed that the processing of the a-mating sequence was not fully completed. It has been reported before that cleavage of EA repeats by Ste13 protease is an inefficient process, but these repeats are necessary to enhance proper function of the Kex2 protease [26]. In the case of Nictaba and frutalin incomplete process- ing of the signal peptide was also reported [16,17]. The uncleaved part of the a-mating sequence at the N-ter- minus as well as the histidine tag at the C-terminus of the recombinant lectin apparently do not influence the biological activity of Orysata, since the recombinant lectin reacted with carbohydrate structures and aggluti- nated red blood cells.

confirmed by PNGase F treatment of the recombinant Orysata which resulted in a shift of the 23 kDa poly- peptide to 18.5 kDa. In this respect it should be men-

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‡ 100 > 4 > 100 > 4 38 ± 6.7 26 ± 10 18 ± 4.0 Orysata Calsepa MornigaM HHA 0.17 ± 0.021 0.49 ± 0.47 1.7 ± 0.8

B. Al Atalah et al. Expression of nucleocytoplasmic Orysata

A

B

C

E

F

D

G

H

I

lectins with preferential

nose (as in the case of Calsepa). In the last decade several so-called mannose-binding JRLs have been identified and characterized from different plant species [1]. Structural analyses as well as detailed studies of the carbohydrate-binding properties have shown that both the galactose-binding and the

Molecular cloning and characterization of the lectin from rhizomes of Calsepa unambiguously showed that some JRLs show specificity towards mannose [27]. Since then the family of JRLs has been subdivided into two classes of specificity towards galactose (as in the case of jacalin) and man-

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Fig. 6. Molecular modeling of the carbohydrate-binding sites of Orysata, Calsepa and Morniga M. (A), (D), (G) Network of hydrogen bonds anchoring Man to the saccharide binding sites of Orysata (A), Calsepa (D) and Morniga M (G). Hydrogen bonds are represented as blue dot- ted lines. Aromatic residues that create a stacking interaction with the sugar are colored orange. (B), (E), (H) Topography of the saccharide binding cavity at the surface of the Orysata (B), Calsepa (E) and Morniga M (H) protomers. Cavities are delineated by red dotted lines and the curved blue arrows indicate the overall orientation of the cavities. (C), (F), (I) Ribbon diagrams at the top of the Man-binding lectins show- ing the overall topography of the carbohydrate-binding sites of Orysata (C), Calsepa (F) and Morniga M (I). L1, L2 and L3 correspond to the loops forming the carbohydrate-binding cavity of the lectins. Strands of b-sheet participating in the binding cavities are numbered.

formation)

(syncytia

Taking all data together, the lectin may qualify as a candidate microbicide agent since it not only blocks T- cell infection by cell-free HIV but it also prevents virus transmission between HIV- infected cells and uninfected cells. However, additional studies are required to further explore the microbicide potential of Orysata.

Expression of the less abundant rice lectin Orysata in Pichia allowed us to compare its biological activity with that of other JRLs such as Calsepa and Morniga M which are expressed in high amounts in plants. Gly- can array analyses confirmed earlier reports on the polyspecificity of Calsepa and Morniga M [28,29]. Data from molecular modelling suggest that subtle dif- ferences in the carbohydrate-binding site of the differ- ent JRLs could explain the different specificities and antiviral activities of the JRLs under study.

B. Al Atalah et al. Expression of nucleocytoplasmic Orysata

Materials and methods

Construction of the EGFP-fusion vector for expression analysis in tobacco cells

Until

The coding sequence for Orysata (GenBank accession num- ber CB632549) was amplified by PCR using the cDNA clone encoding Orysata as a template. The primers for amplification of Orysata were ORY-f1 (5¢-AAAAAG CAGGCTTCACGCTGGTGAAGATTGGCCTG-3¢) and ORY-r1 (5¢-AGAAAGCTGGGTGTCAAGGGTGGACGT AGATGCC-3¢). The PCR program was as follows: 5 min 94 (cid:2)C, 25 cycles (15 s 94 (cid:2)C, 30 s 65 (cid:2)C, 24 s 72 (cid:2)C), 5 min 72 (cid:2)C. PCR was performed in a 50 lL reaction volume containing 40 ng DNA template, 10 · DNA polymerase buffer, 10 mm dNTPs, 5 lm of each primer and 0.625 U Platinum Pfx DNA Polymerase (Invitrogen, Carlsbad, CA, USA) using an AmplitronIIR Thermolyne apparatus (Dubuque, IA, USA). The PCR product was 1 : 10 diluted and used as a template in an additional PCR, using attB primers EVD 2 (5¢-GGGGACAAGTTTGTACAAAAA AGCAGGCT-3¢) and EVD 4 (5¢-GGGGACCACTTTG TACAAGAAAGCTGGGT-3¢) in order to complete the attB recombination sites. The reaction mixture was as described for previous PCR. The cycle conditions were as follows: 2 min at 94 (cid:2)C, five cycles each consisting of 15 s at 94 (cid:2)C, 30 s at 50 (cid:2)C, 30 s at 72 (cid:2)C, 20 cycles with 15 s at 94 (cid:2)C, 30 s at 55 (cid:2)C, 30 s at 72 (cid:2)C, and a final incubation of 5 min at 72 (cid:2)C. Subsequently, the BP reaction was per- formed using the pDONR221 vector (Invitrogen). After sequencing of the resulting entry clone, the LR reaction was done with the pK7WGF2 destination vector [35] to fuse the rice sequence C-terminally to EGFP. Overexpression of EGFP alone was achieved using the pK7WG2 destination vector [35]. Tobacco BY2 cells were transiently trans- formed with the EGFP-fusion construct by particle

mannose-binding JRLs are polyspecific lectins with a preference for galactose and mannose, respectively [28,29]. Analysis of the carbohydrate-binding specificity of three mannose-binding JRLs on the glycan array revealed differences in their specificity. Clearly Orysata and Morniga M interact much better with high-man- nose binding glycans than Calsepa does. These results are in agreement with the analyses of the sugar-binding specificity of Morniga M and Calsepa by frontal affin- ity chromatography where it was shown that although Morniga M and Calsepa both react with high-mannose structures (especially of Man2–6 type), Calsepa showed a much better interaction with complex N-glycans with bisecting 2-amino-2-N-acetylamino-d-glucose (GlcNAc) [30]. Although the frontal affinity chromatography indicated that Morniga M and Calsepa did not react with tri- and tetra-antennary glycans, some interac- tions with these glycan structures have been observed on the array. Molecular modeling studies suggest sub- tle differences in the carbohydrate-binding sites of JRLs. The shortening of the carbohydrate-binding cav- ity in Morniga M could account for the differences in specificity of the different Man-specific JRLs towards extended oligosaccharide chains, e.g. the a1-O-linked, a3-O-linked and a6-O-linked oligosaccharides. lectins now especially mannose-binding belonging to the group of GNA-related lectins such as snowdrop (GNA) and amaryllis (HHA) lectin have been shown to exhibit significant activity against HIV as well as some other viruses such as hepatitis C virus [31–33]. Since very little is known with respect to the antiviral activity of JRLs the anti-HIV activity of three mannose-binding JRLs was tested and compared. Detailed analysis showed that Orysata has potent anti- HIV and anti-RSV activity. Only recently the man- nose-binding JRL isolated from the fruit of banana Musa acuminata BanLec was also reported to exhibit potent anti-HIV activity [34]. It was shown that HHA and BanLec interact with gp120 and can inhibit HIV replication. It is intriguing, however, to notice that the a1,3 ⁄ a1,6-mannose-specific HHA is 10- to 20-fold more inhibitory to HIV but more than 10-fold less inhibitory to RSV than Orysata. This may point to subtle differences in carbohydrate recognition of the two lectins, and is in agreement with the modeling and glycan arrays suggesting that Orysata also recognizes complex-type glycans in addition to high-mannose type glycans. Although the nature of the glycans on the envelope of RSV is not unambiguously determined, they most probably predominantly consist of complex- type glycans since mannose-specific lectins such as GNA and HHA have never been found to be endowed with significant anti-RSV activity in cell culture.

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bombardment and the expression was analyzed by confocal laser microscopy as described by Fouquaert et al. [36].

Expression of Orysata in Pichia pastoris

(2% final concentration)

1.34% yeast nitrogen base with ammonium sulfate and without amino acids, 4 · 10)5% biotin, 100 mm potassium phosphate (pH 6.0) and 1% glycerol, and grown at 30 (cid:2)C in a shaker incubator at 200 r.p.m. for 24 h. Afterwards, Pichia cells were washed with sterilized water and trans- ferred to the BMMY medium (BMGY medium supple- mented with 1% of methanol instead of 1% of glycerol). Induction of the culture was achieved by adding 100% for three successive methanol days, once in the morning and once in the evening. Protein profiles in the medium and the cell pellet were compared. Proteins in the culture medium were analyzed after trichlo- roacetic acid precipitation (10% final concentration) by SDS ⁄ PAGE and western blot analysis.

Large-scale culture and purification of Orysata

The EasySelect Pichia Expression Kit from Invitrogen was used to clone and express Orysata in the P. pastoris strain X-33. To achieve secretion of the recombinant protein into the culture medium the E. coli ⁄ P. pastoris shuttle vector pPICZaB containing an a-mating sequence from Saccharo- myces cerevisiae was used. This vector contains a polyhisti- dine tag located downstream from the multiple cloning site. The coding sequence for Orysata was amplified by PCR starting from the Bluescript vector containing the cDNA encoding Orysata (GenBank accession number CB632549) using primers evd 519 (5¢-GGCGGACTGCAGCAAT GACGCTGGTGAAGATTGGCCTGT-3¢) and evd 518 (5¢-CCCGCTTTCTAGAATAGGGTGGACGTAGATGC CAATTGCG-3¢). The PCR conditions were as follows: 2 min denaturation at 94 (cid:2)C, 25 cycles of 15 s 94 (cid:2)C, 30 s 55 (cid:2)C, 1 min 72 (cid:2)C, ending with an additional 5 min elon- gation at 72 (cid:2)C. The amplified Orysata sequence was cloned as a PstI ⁄ XbaI fragment in the shuttle vector pPICZaB and transformed in E. coli Top10F cells using heat shock transformation. Afterwards, E. coli transformants were selected on LB agar plates containing zeocin (25 lgÆmL)1). The plasmids were purified using the E.Z.N.A. Plasmid Mini kit I (Omega Bio-Tek, Norcross, GA, USA). Finally, the sequence of the fusion construct was verified by sequencing using 5¢ and 3¢ AOX1 specific primers (for- ward evd 21, 5¢-GACTGGTTCCAATTGACAAGC-3¢, and reverse evd 22, 5¢-GCAAATGGCATTCTGACATCC-3¢, carried out by LGC Genomics, Berlin, Germany).

Pichia transformation and expression analysis on a small scale

restriction enzyme SacI

Transformed P. pastoris X-33 colonies were inoculated into 5 mL BMGY medium and grown for 24 h at 30 (cid:2)C in a rotary shaker at 200 r.p.m. Afterwards, cultures were trans- ferred to 50 mL BMGY in 250 mL Erlenmeyer flasks and allowed to grow until the culture reached an optical density between 2 and 6 at 595 nm. Pichia cells were washed with sterilized water and resuspended in 200 mL of BMMY medium. The culture was allowed to grow for 72 h in a 500 mL Erlenmeyer flask under the same conditions as before. Every 24 h 100% methanol was added to the cul- ture twice a day as indicated above (2% final concentra- tion). After 3 days of methanol induction, the culture was centrifuged for 10 min at 3000 g and the supernatant was brought to 80% ammonium sulfate for protein precipita- tion and stored at 4 (cid:2)C. Five 200 mL cultures were pooled for one purification of recombinant Orysata. Purification of the lectin was achieved in three chromatographic steps. After precipitating the protein by centrifugation for 15 min at 5000 g the resulting pellet was resuspended in 150 mL 20 mm 1,3 diaminopropane. After overnight dialysis against 20 mm 1,3 diaminopropane, the supernatant was loaded on a Q Fast Flow column (GE Healthcare, Uppsala, Sweden) equilibrated with 20 mm 1,3 diaminopropane. After wash- ing the column with 20 mm 1,3 diaminopropane, elution of the bound proteins was achieved using 100 mm Tris ⁄ HCl (pH 8.7) containing 0.5 m NaCl. Subsequently, the eluted fractions were pooled and imidazole was added to a final concentration of 25 mm. The protein sample eluted from the Q Fast Flow column was applied on a Ni-Sepharose column (GE Healthcare) equilibrated with start buffer (0.1 m Tris pH 7 containing 0.5 m NaCl and 25 mm imidaz- ole) to purify the His-tagged protein. After washing the Ni-Sepharose column using the start buffer proteins were eluted using the elution buffer (0.1 m Tris pH 7 containing 0.5 m NaCl and 250 mM imidazole). Finally, fractions eluted from Ni-Sepharose were diluted five times with phos- phate buffered saline (1 · PBS: 1.5 mm KH2PO4, 10 mm Na2HPO4, 3 mm KCl, 140 mm NaCl, pH 7.4) and applied

The plasmid DNA from E. coli cells was purified and line- arized using the (Fermentas, incubation at St Leon-Rot, Germany) with overnight 37 (cid:2)C. After linearization, 10 lg of the expression vector was transformed into the Pichia strain X-33 via electropo- ration (Bio-Rad, Hercules, CA, USA) using the following pulse settings: 25 lF, 1.5 kV and 200 X. Transformants were selected on YPDS plates (1% yeast extract, 2% pep- tone, 2% dextrose, 1 m sorbitol, 2% agar) containing 100 lgÆmL)1 zeocin. Genomic DNA was extracted from Pichia transformants as reported before [37]. The integra- tion of the Orysata sequence in the chromosomal AOX1 locus of P. pastoris was confirmed by PCR using the AOX1 primers evd 21 and evd 22, and the following parameters: 2 min 95 (cid:2)C, 30 cycles of 1 min 95 (cid:2)C, 1 min 55 (cid:2)C, 1 min 72 (cid:2)C, ending with an elongation step of 7 min at 72 (cid:2)C. For expression analysis, several colonies were inoculated in 5 mL BMGY medium, i.e. 1% yeast extract, 2% peptone,

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on a mannose-Sepharose 4B column equilibrated with PBS. After washing the column with PBS, the lectin fraction was eluted using 20 mm 1,3 diaminopropane. The purity of the protein samples was verified by SDS ⁄ PAGE and ⁄ or wes- tern blot analysis after each purification step.

in binding buffer

N-terminal sequence analysis

A sample from the affinity purified Orysata was analyzed by SDS ⁄ PAGE, electroblotted onto a ProBlot(cid:3) polyviny- lidene difluoride membrane (Applied Biosystems, Foster City, CA, USA) and visualized by staining with a 1 : 1 mix of Coomassie Brilliant Blue and methanol. Bands of inter- est were excised from the membrane and the N-terminal sequence was determined by Edman degradation on a capil- lary Procise 491cLC protein sequencer without alkylation of cysteines (Applied Biosystems).

Agglutination assay

thetic glycan sequences representing major glycan structures of glycoproteins and glycolipids. Recombinant Orysata con- taining a His tag was purified from P. pastoris and detected using a fluorescent-labeled anti-His monoclonal antibody (Qiagen, Valencia, CA, USA). The lectin was diluted to desired concentrations (Tris-buffered saline containing 10 mm CaCl2, 10 mm MgCl2, 1% BSA, 0.05% Tween 20) and 70 lL of the lectin solution was applied to separate microarray slides. After 60 min incuba- tion under a cover slip in a humidified chamber at room temperature, the cover slip was gently removed in a solu- tion of Tris-buffered saline containing 0.05% Tween 20 and washed by gently dipping the slides four times in successive washes of Tris-buffered saline containing 0.05% Tween 20, and Tris-buffered saline. To detect bound lectin, the labeled anti-His antibody (70 lL at 1 lgÆmL)1 in binding buffer) was applied to the slide under a coverslip. After removal of the coverslip and gentle washing of the slide as described above, the slide was finally washed in deionized water and spun in a slide centrifuge for (cid:2) 15 s to dry. The slide was immediately scanned in a PerkinElmer ProScanArray MicroArray Scanner using an excitation wavelength of 488 nm and ImaGene software (BioDiscovery, El Segundo, CA, USA) to quantify fluorescence. The data are reported as average relative fluorescence units (RFU) of six repli- cates for each glycan presented on the array after removing the highest and lowest values. The results for Orysata were compared with the glycan array data obtained for the mannose-binding JRLs purified from Calystegia sepium rhi- zomes (Calsepa) and Morus nigra bark (Morniga M) [30].

Antiviral assays

To examine the lectin activity, an agglutination assay was performed using trypsin-treated rabbit red blood cells (Bio- Me´ rieux, Marcy l’Etoile, France). Therefore 10 lL of the purified protein (165 lgÆmL)1), 10 lL of 1 m ammonium sulfate and 30 lL of trypsinized erythrocytes were mixed in a glass tube. The negative control contained 10 lL PBS, 10 lL 1 m ammonium sulfate and 30 lL trypsinized ery- throcytes. After a few minutes agglutination was observed as clumping of the cells at the bottom of the glass tube. Samples that yielded no visible agglutination activity after incubation for 1 h were regarded as lectin negative. Dilu- tion series of the lectin were analyzed to determine its agglutination titer.

Carbohydrate inhibition test

Several carbohydrates (mannose, trehalose, glucose, galac- tose, GlcNAc or methyl mannopyranoside, at 0.5 m) and gly- coproteins (ovomucoid, asialomucin or thyroglobulin, at 10 mgÆmL)1) were used to test the carbohydrate specificity of the recombinant Orysata. Therefore 10 lL aliquots of a seri- ally twofold diluted purified lectin were mixed with 10 lL of carbohydrate or glycoprotein solution. After incubation for 10 min at room temperature, 30 lL trypsin-treated erythro- cytes were added. Agglutination activity was assessed visually after incubation for 1 h at room temperature.

Glycan array screening

to the

Human lymphocyte CEM cells (5 · 105 cells per mL) were suspended in fresh culture medium [RPMI-1640 (Gibco, Paisley, UK), supplemented with 10% fetal bovine serum, 2 mm l-glutamine and 0.075% NaHCO3] and exposed to HIV-1(IIIB) (provided by R. C. Gallo at that time at the NIH, Bethesda, MD, USA) or HIV-2(ROD) (provided by L. Montagnier at that time at the Pasteur Institute, Paris, France) at 100 · the CCID50 per mL of cell suspension. Then, 100 lL of the infected cell suspension was transferred to 200 lL microplate wells, mixed with 100 lL of the appropriate dilutions of the test compounds, and further incubated at 37 (cid:2)C. After 4 days, giant (syncytium) cell for- mation was recorded microscopically in the CEM cell cul- tures, and the number of giant cells was estimated as the percentage of the number of giant cells present in the non- treated virus-infected cell cultures ((cid:2) 50–100 giant cells in one microscopic field when examined at a microscope mag- nitude of 100 ·). The 50% effective concentration (EC50) corresponds to the compound concentration required to prevent syncytium formation by 50%. The 50% cytostatic compound corresponds concentration (CC50)

The microarrays are printed as described previously [38] and version 4.2 with 511 glycan targets was used for the analyses reported here (https://www.functionalglycomics. org/static/consortium/resources/resourcecoreh8.shtml). The printed glycan array contains a library of natural and syn-

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banana lectin 1X1V used as a template) exhibited an energy over the threshold value.

and weak

[2.5 A˚ < dist(D–A) < 3.5 A˚

concentration required to inhibit CEM cell proliferation by 50%. In the co-cultivation assays, 5 · 104 persistently HIV- 1-infected human lymphocyte HUT-78 cells (designated HUT-78 ⁄ HIV-1(IIIB) were mixed with 5 · 104 human lym- phocyte Sup T1 cells, along with appropriate concentra- tions of the test compounds. After 24–36 h, marked syncytium formation was reached in the control cell cul- tures, and the number of syncytia was determined under the microscope. The anti-respiratory syncytial virus (RSV strain Long) assay was based on inhibition of virus-induced cytopathicity in human cervix carcinoma HeLa cell cul- tures. Confluent cell cultures were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) in the presence of varying concen- trations of the test compounds. Viral cytopathicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds.

Molecular modeling and docking

The docking of MeMan into the carbohydrate-binding sites of Orysata and other JRLs was performed with the program insightii. The lowest apparent binding energy (Ebind expressed in kcalÆmol)1) compatible with the hydro- gen bonds (considering van der Waals interactions and strong [2.5 A˚ < dist(D–A) < 3.1 A˚ and 120(cid:2) < ang(D–H– and A)] 105(cid:2) < ang(D–H–A) < 120(cid:2)] hydrogen bonds, with D donor, A acceptor and H hydrogen) found in the Man– banana lectin complex (RCSB Protein Data Bank code 1X1V) [39] was calculated using the forcefield of discover3 and used to anchor the pyranose ring of the sugars into the binding sites of the lectin. The position of mannose observed in the Man–banana lectin complex was used as the starting position to anchor mannose in the carbohy- drate-binding sites of Orysata. Mannose (Man) was simi- larly docked into the saccharide-binding site of Calsepa (RCSB Protein Data Bank code 1OUW) [28]. Cartoons showing the docking of Man ⁄ MeMan in the mannose-bind- ing sites of the lectins were drawn with pymol (http:// www.pymol.org).

Analytical methods

tetrahydrochloride

The protein content was estimated using the Coomassie (Bradford) Protein Assay Kit (Thermo Fischer Scientific, Rockford, IL, USA), based on the Bradford dye-binding procedure [48]. SDS ⁄ PAGE was performed using 15% polyacrylamide gels under reducing conditions as described by Laemmli [49]. Proteins were visualized by staining with Coomassie Brilliant Blue R-250. For western blot analysis, samples separated by SDS ⁄ PAGE were electrotransferred to 0.45 lm poly(vinylidene difluoride) (PVDF) membranes (Biotrace(cid:3) PVDF, PALL, Gelman Laboratory, Ann Arbor, MI, USA). After blocking the membranes in Tris- buffered saline (TBS: 10 mm Tris, 150 mm NaCl and 0.1% (v ⁄ v) Triton X-100, pH 7.6) containing 5% (w ⁄ v) milk powder, blots were incubated for 1 h with a mouse mono- clonal anti-His (C-terminal) antibody (Invitrogen), diluted 1 ⁄ 5000 in TBS. The secondary antibody was a 1 ⁄ 1000 diluted rabbit anti-mouse IgG labeled with horseradish per- oxidase (Dako Cytomation, Glostrup, Denmark). Immun- odetection was achieved by a colorimetric assay using 3,3¢-diaminobenzidine (Sigma-Aldrich, St Louis, MO, USA) as a substrate. For far western blot analysis the blot was incubated with purified Nictaba (1 lgÆmL)1, diluted in Tris ⁄ HCl pH 7.6) for 1 h prior to incubation with the primary antibody against Nictaba, the secondary antibody and the detection buffer. All washes and incubations were conducted at room temperature with the purified Orysata gentle shaking. The N-glycans of (16 lg) were released using the on-membrane deglycosyla- tion method as described earlier [50]. Briefly, the sample

Homology modeling of Orysata was performed on a Silicon Graphics O2 10000 workstation, using the programs insightii, homology and discover (Accelrys, San Diego, CA, USA). The atomic coordinates of banana lectin com- plexed to mannose (code 1X1V) [39] were taken from the RCSB Protein Data Bank [40] and used to build the three- dimensional model of Orysata. The amino acid sequence alignment was performed with clustal-x [41] and the hydrophobic cluster analysis [42] plot was generated by the mobile server (http://mobyle.rpbs.univ-paris-diderot.fr/cgi- bin/portal.py?form=HCA) to recognize the structurally conserved regions common to Orysata and banana lectin. Steric conflicts resulting from the replacement or the inser- tion of some residues in the modeled lectin were corrected during the model building procedure using the rotamer library [43] and the search algorithm implemented in the homology program [44] to maintain proper side chain ori- entation. Energy minimization and relaxation of the loop regions were carried out by several cycles of steepest des- cent using discover3. After correction of the geometry of the loops using the minimize option of turbofrodo (Bio- Graphics, Marseille, France), a final energy minimization step was performed by 150 cycles of steepest descent using discover3, keeping constrained the amino acid residues forming the carbohydrate-binding site. The program tur- bofrodo was used to draw the Ramachandran plots [45] and perform the superimposition of the models. procheck [46] was used to check the stereochemical quality of the three-dimensional model: 82.8% of the residues were assigned to the favorable regions of the Ramachandran plot (84.6% for banana lectin), except for three residues Ser20, Glu61 and Tyr105, which occur in the non-allowed region of the plot. Using anolea [47] to evaluate the model, only seven residues over 146 (versus three over 137 for the

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jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) plants. Planta 210, 970–978.

5 Claes B, Dekeyser R, Villarroel R, Van den Bulcke M, Bauw G, Van Montagu M & Caplan A (1990) Charac- terization of a rice gene showing organic-specific expres- sion in response to salt stress and drought. Plant Cell 2, 19–27.

6 De Souza Filho GA, Ferreira BS, Dias JMR, Queiroz

KS, Branco AT, Bressan-Smith RE, Oliveira JG & Gar- cia AB (2003) Accumulation of SALT protein in rice plants as a response to environmental stresses. Plant Sci 164, 623–628.

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incubated for 1 h at 50 (cid:2)C in denaturing buffer was (360 mm Tris ⁄ HCl, pH 8.6, containing 8 m urea and 3.2 mm EDTA) and subsequently loaded on a 96-well Mul- tiscreen-ImmobilonP plate containing a PVDF membrane (Millipore). Then, the bound proteins were reduced and carboxymethylated using dithiothreitol and iodeacetic acid, respectively. Next, the N-glycans were released using PNG- ase F (in the negative control we omitted the enzyme). After labeling the N-glycans with 8-aminopyrene-1,3,6-tri- sulfonic acid, the excess of label was removed by size exclu- sion chromatography using Sephadex G-10. The samples were finally reconstituted in 10 lL of ultrapure water, and 10 lL of a 1 : 10 dilution was analyzed by capillary electro- phoresis on an ABI 3130 DNA sequencer (Applied Biosy- tems) as described earlier [50]. To identify the structures, exoglycosidase digests were performed overnight at 37 (cid:2)C by adding 66 ng of Trichoderma reseii a-1,2-mannosidase [51] or 20 mU of jack bean a-mannosidase (Sigma, St Louis, MO, USA) to 1.5 lL of sample in a total reaction volume of 3 lL containing 5 mm NH4Ac pH 5.

8 Kim ST, Cho KS, Yu S, Kim SG, Hong JC, Han CD, Bae DW, Nam MH & Kang KY (2003) Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells. Proteomics 3, 2368–2378.

B. Al Atalah et al. Expression of nucleocytoplasmic Orysata

Acknowledgements

9 Qin QM, Zhang Q, Zhao WS, Wang YY & Peng YL (2003) Identification of a lectin gene induced in rice in response to Magnaporthe grisea infection. Acta Bot Sin 45, 76–81.

10 Lee RH, Wang CH, Huang LT & Chen SC (2001) Leaf senescence in rice plants: cloning and characterization of senescence up-regulated genes. J Exp Bot 52, 1117– 1121.

11 Cereghino JL & Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24, 45–66.

12 Elias SB, Brust PF, Koutz PJ, Waters AF, Harpold

funded

by

MM & Gingeras TR (1985) Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol Cell Biol 5, 1111–1121.

This work was funded primarily by the Fund for Sci- entific Research – Flanders (FWO grants G.0022.08 and G.485.08), the Research Council of Ghent Uni- versity (projects BOF2005 ⁄ GOA ⁄ 008 and BOF2007 ⁄ - GOA ⁄ 0017), the Hercules Foundation and the Center of Excellence project (PF 10 ⁄ 08) of the K.U. Leuven. Bassam Al Atalah is recipient of a doctoral grant from the Special Research Council of Ghent Univer- sity. The authors want to thank the Consortium for Functional Glycomics the NIGMS GM62116 for the glycan array analysis. We are grateful to the Arizona Genomics Institute (Univer- sity of Arizona, Arizona, USA) for providing the cDNA clone encoding Orysata.

13 Hartner FS & Glieder A (2006) Regulation of methanol utilisation pathway genes in yeasts. Microb Cell Fact 5, 39.

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