Engineering a versatile tandem repeat-type α2-6sialic acid-binding lectin

Previously, we developed an α2-6-sialic acid (Sia)-specific lectin (SRC) starting from an R-type galactose-specific lectin C-terminal domain. However, it showed relatively low affinity because of its monovalency. Here, we engineered a tandem repeat construct (SRC2) showing substantial affinity for α2,6-sialylated N-glycans (in the order of 10⁻⁶ M in K_d), almost comparable to a natural α2-6Sia-specific lectin from Sambucus sieboldiana (SSA). Notably, its binding to branched N-glycans was found to be more selective than SSA. Nevertheless, SRC2 showed no apparent hemagglutinating activity, while it exerted strong erythrocyte-binding activity. This unique feature will help flow cytometry analysis, where usual lectins including SSA agglutinate cells. Some other biochemical properties investigated for SRC2, e.g., high productivity in bacteria and easy release of captured glycoproteins with lactose have demonstrated versatility of this mutant protein as a powerful tool for sialoglycomics.     

Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model

Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas.     

Combined biochemical and cytological analysis of membrane trafficking using lectins

We have tested the application of high-mannose-binding lectins as analytical reagents to identify N-glycans in the early secretory pathway of HeLa cells during subcellular fractionation and cytochemistry. Post-endoplasmic reticulum (ER) pre-Golgi intermediates were separated from the ER on Nycodenz–sucrose gradients, and the glycan composition of each gradient fraction was profiled using lectin blotting. The fractions containing the post-ER pre-Golgi intermediates are found to contain a subset of N-linked α-mannose glycans that bind the lectins Galanthus nivalis agglutinin (GNA), Pisum sativum agglutinin (PSA), and Lens culinaris agglutinin (LCA) but not lectins binding Golgi-modified glycans. Cytochemical analysis demonstrates that high-mannose-containing glycoproteins are predominantly localized to the ER and the early secretory pathway. Indirect immunofluorescence microscopy revealed that GNA colocalizes with the ER marker protein disulfide isomerase (PDI) and the COPI coat protein β-COP. In situ competition with concanavalin A (ConA), another high-mannose specific lectin, and subsequent GNA lectin histochemistry refined the localization of N-glyans containing nonreducing mannosyl groups, accentuating the GNA vesicular staining. Using GNA and treatments that perturb ER–Golgi transport, we demonstrate that lectins can be used to detect changes in membrane trafficking pathways histochemically. Overall, we find that conjugated plant lectins are effective tools for combinatory biochemical and cytological analysis of membrane trafficking of glycoproteins.     

1,4-Butanediol diglycidyl ether coupling of carbohydrates to sepharose: Affinity adsorbents for lectins and glycosidases

The use of 1,4-butanediol diglyeidyl ether to couple carbohydrate ligands to Sepharose [Sundberg, L., and Porath, J. (1975) J. Chromatogr. 90, 87; Vretblad, P. (1976) Biochim. Biophys. Acta. 434, 169] has been explored with a number of carbohydrate derivatives, such as amino sugars (N-acetylglucosamine, N-acetylgalactosamine, nonacetylated short oligomers of glucosamine), methyl glycosides (O-methyl-α-mannoside and O-methyl-β-galactoside), di- and oligosaccharides (lactose and stachyose), and polysaccharides (yeast mannan and dextrin). Using model reactions it has been established that all but the reducing sugars are sufficiently stable to withstand the alkaline conditions required for the coupling reaction, and that the primary alcohol at the C₆ position of the sugars is the primary site for the coupling reaction. In the case of glucosamine, the amino group is six times more reactive than is the 6-hydroxy group. The following affinity adsorbents have been tested and found useful in the purification of lectins and glycosidases: a yeast mannan adsorbent binds jack bean α-mannosidase (0.15 mg/ml of adsorbent) and concanavalin A (0.5 mg/ml); a dextrin adsorbent binds bovine pancreatic α-amylase (2 mg/ml of adsorbent); a lactose adsorbent binds peanut lectin (5 mg/ml of adsorbent); a (GlcNAc)_3–4 adsorbent binds wheat germ lectin (50 mg/ml of adsorbent); and, as previously reported [Vretblad, P. (1976) Biochim. Biophys. Acta. 434, 169], a GlcNAc adsorbent binds wheat germ lectin (10 mg/ml of adsorbent) and a GalNAc adsorbent binds soybean lectin (18 mg/ml of adsorbent).     

The Lectins of Sophora japonica: II. Purification, Properties, and N-Terminal Amino Acid Sequences of Five Lectins from Bark

Five N-acetyl-galactosamine-specific lectins were isolated from the bark of the legume tree Sophora japonica. These lectins are immunologically and structurally very similar, but not identical, to the Sophora seed and leaf lectins. The carbohydrate specificities and hemagglutinin activities of these lectins are indistinguishable at pH 8.5 but their activities differ markedly at pH values below 8. All five lectins are tetrameric glycoproteins made up of different combinations of subunits of about 30,000, 30,100, 33,000 M_r containing 3% to 5% covalently attached sugar. These lectins are the overwhelmingly dominant proteins in bark, but they do not appear to be present in other tissues. Amino terminal sequence analysis indicates that at least two distinct lectin genes are expressed in bark.     

Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)_n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis^x and sialyl-Lewis^x determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.     

High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120

We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and cDNA cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per a litter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide-binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48–1.61?×?10⁹ M^?1). Native KAAs and the recombinants inhibited the HIV-1 entry at IC₅₀s of low nanomolar levels (7.3–12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).     

Site specific <Emphasis Type="Italic">N</Emphasis>-glycan profiling of NeuAc(α2-6)-Gal/GalNAc-binding bark <Emphasis Type="Italic">Sambucus nigra</Emphasis> agglutinin using LC–MS<Superscript>n</Superscript> revealed differential glycosylation

The bark of Sambucus nigra contains a complex mixture of glycoproteins that are characterized as chimeric lectins known as type II ribosome inactivating proteins and holo lectins. These type II ribosome inactivating proteins possess RNA N-glycosidase activity in subunit A and lectin activity associated with subunit B exhibiting distinct sugar specificities to NeuAc(α2-6)-Gal/GalNAc and Gal/GalNAc. In the present study we have determined the N-glycosylation pattern of type II ribosome inactivating protein specific to NeuAc(α2-6)-Gal/GalNAc (Sambucus nigra agglutinin I) by subjecting it to digestion with multiple proteases. The resulting mixture of peptides and N-glycopeptides were analyzed on liquid chromatography coupled to electro spray ionization-iontrap mass spectrometry in MSⁿ mode. MS² of precursor ions was carried out using CID which provided information on glycan sequence. In subsequent MS³ of Y₁/Y_1α ions (peptide + HexNAc)⁺ⁿ of corresponding N-glycopeptides, resulted in the fragmentation of peptide backbone confirming the site of attachment. We observed microheterogeneity in each glycan occupied site with subunit A possessing four N-glycans out of six sites with complex and paucimannose types while subunit B comprises occupancy of two sites with a paucimannose and a high mannose type. The differential N-glycosylation of subunits in SNA is discussed in the context of other type II RIPs glycans.     

Differential recognition of natural and remodeled glycotopes by three Diocleae lectins

The carbohydrate specificities of Dioclea grandiflora lectins DGL-I¹ and DGL-II, and Galactia lindenii lectin II (GLL-II) were explored by use of remodeled glycoproteins as well as by the lectin hemagglutinating activity against erythrocytes from various species with different glycomic profiles. The three lectins exhibited differences in glycan binding specificity but also showed overlapping recognition of some glycotopes (i.e. Tα glycotope for the three lectins; IIβ glycotope for DGL-II and GLL-II lectins); in many cases the interaction with distinct glycotopes was influenced by the structural context, i.e., by the neighbouring sugar residues. Our data complement and expand the existing knowledge about the binding specificity of these three Diocleae lectins, and taken together with results of previous studies, allow us to suggest a functional map of the carbohydrate recognition which illustrate the impact of modification of basic glycotopes enhancing, permiting, or inhibiting their recognition by each lectin.     

Purification and Characterization of Griffonia simplicifolia Leaf Lectins

Leaves from mature Griffonia simplicifolia plants were examined for the presence of leaf lectins possessing sugar binding specificities similar to the four known seed lectins (GS-I, GS-II, GS-III, GS-IV). Three (GS-I, -II, -IV) of the four known G. simplicifolia seed lectins were present in the leaves. Leaf G. simplicifolia lectins I and IV were similar to the respective seed lectins. Leaf GS-II, however, was composed of two types of subunits (M_r = 33,000 and 19,000), whereas the seed lectin consists of only one type of subunit (M_r 32,500). Seed and leaf GS-II lectins also had different isoelectric points. All leaf and seed lectins were similar with respect to their hemagglutination and glycoconjugate precipitation properties and all subunits contained covalently bound carbohydrate. Leaf GS-IV appeared slightly under-glycosylated compared to seed GS-IV.

The fate of GS-I and GS-II seed lectins in aging cotyledons was investigated. GS-I isolectins usually contain isolectin subtypes associated with each main isolectin. Upon inbibition and germination, these GS-I isolectin subtypes disappeared. Over time, GS-II lectin did not change its disc gel electrophoretic properties.

     

N-Acetyl-D-glucosamine binding lectins. A model system for the study of binding specificity

Synthetic glycoconjugates prepared by the direct reductive amination of di-N-acetylchitobiose and tetra-N-acetyl-chitotetraose to poly-l-lysine with sodium cyanoborohydride have been used to explore the binding specificities of the lectins wheat germ agglutinin and Bandeiraea simplicifolia II. These conjugates are effective precipitating antigens for these lectins, and hapten inhibition experiments, employing the per-N-acetylated oligomers of chitin as inhibitors, demonstrate that wheat germ agglutinin and Bandeiraea simplicifolia II lectin have binding sites complementary to three and two contiguous β 1,4-linked N-acetyl-d-glucosamine residues, respectively, in agreement with conclusions reached using other methods. Conjugates prepared by this technique should be useful for examining the binding specificities of other lectins, and the results of a study of the effect of chain length of the hapten on the affinity of the lectin for these conjugates should provide guidance in selection of the hapten most appropriate for these studies.     

High mannose-specific lectin (KAA-2) from the red alga Kappaphycus alvarezii potently inhibits influenza virus infection in a strain-independent manner

The carbohydrate binding profile of the red algal lectin KAA-2 from Kappaphycus alvarezii was evaluated by a centrifugal ultrafiltration–HPLC method using pyridylaminated oligosaccharides. KAA-2 bound exclusively to high mannose type N-glycans, but not to other glycans such as complex type, hybrid type, or the pentasaccharide core of N-glycans. This lectin exhibited a preference for an exposed α1–3 Man on a D2 arm in a similar manner to Eucheuma serra agglutinin (ESA-2), which shows various biological activities, such as anti-HIV and anti-carcinogenic activity. We tested the anti-influenza virus activity of KAA-2 against various strains including the recent pandemic H1N1-2009 influenza virus. KAA-2 inhibited infection of various influenza strains with EC₅₀s of low nanomolar levels. Immunofluorescence microscopy using an anti-influenza antibody demonstrated that the antiviral activity of KAA-2 was exerted by interference with virus entry into host cells. This mechanism was further confirmed by the evidence of direct binding of KAA-2 to a viral envelope protein, hemagglutinin (HA), using an ELISA assay. These results indicate that this lectin would be useful as a novel antiviral reagent for the prevention of infection.     

Lectin binding in the anterior segment of the bovine eye

Summary Eleven different fluorescent lectin-conjugates were used to reveal the location of carbohydrate residues in frozen sections of the anterior segment of bovine eyes. The lectins were specific for the following five major carbohydrate groups: (1) glucose/mannose group (Concanavalin A (Con A)); (2)N-acetylglucosamine group (wheat germ agglutinin (WGA)); (3) galactose/N-acetylgalactosamine group (Dolichos biflorus agglutinin (DBA),Helix pomatia agglutinin (HPA),Helix aspersa agglutinin (HAA),Psophocarpus tetragonolobus agglutinin (PTA),Griffonia simplicifolia agglutinin-I-B₄ (GSA-I-B₄),Artocarpus integrifolia agglutinin (JAC), peanut agglutinin (PNA) andRicinus communis agglutinin (RCA-I)); (4)l-fucose group (Ukex europaeus agglutinin (UEA-I)); (5) sialic acid group (wheat germ agglutinin (WGA)). All the studied lectins except UEA-I reacted widely with different structures and the results suggest that there are distinct patterns of expression of carbohydrate residues in the anterior segment of the bovine eye. UEA-I bound only to epithelial structures. Some of the lectins reacted very intensely with apical cell surfaces of conjunctival and corneal epithelia suggesting a different glycosylation at the glycocalyx of the epithelia. Also, the binding patterns of conjunctival and corneal epithelia differed with some of the lectins: PNA and RCA-I did not bind at all, and GSA-I-B₄ bound only very weakly to the epithelium of the cornea, whereas they bound to the epithelium of the conjunctiva. In addition, HPA, HAA, PNA and WGA did not bind to the corneal basement membrane, but bound to the conjunctiva and vascular basement membranes. This suggests that corneal basement membrane is somehow different from other basement membranes. Lectins with the same carbohydrate specificity (DBA, HPA, HAA and PTA) reacted with the sections almost identically, but some differences were noticed: DBA did not bind to the basement membrane of the conjunctiva and the sclera and did bind to the basement membrane of the cornea, whereas other lectins with same carbohydrate specificities reacted vice versa. Also, the binding of PTA to the trabecular meshwork was negligible, whereas other lectins with the same carbohydrate specificities reacted with the trabecular meshwork. GSA-I-B₄ reacted avidly with the endothelium of blood vessels and did not bind to the stroma, so that it made blood vessels very prominent and it might be used as an endothelial marker. This lectin also reacted avidly with the corneal endothelium. Therefore, GSA-I-B₄ appears to be a specific marker in bovine tissues for both blood vessel and corneal endothelium cells.     

Comparative analysis of oligosaccharide specificities of fucose-specific lectins from Aspergillus oryzae and Aleuria aurantia using frontal affinity chromatography

Aleuria aurantia lectin (AAL) is widely used to estimate the extent of α1,6-fucosylated oligosaccharides and to fractionate glycoproteins for the detection of specific biomarkers for developmental antigens. Our previous studies have shown that Aspergillus oryzae lectin (AOL) reflects the extent of α1,6-fucosylation more clearly than AAL. However, the subtle specificities of these lectins to fucose linked to oligosaccharides through the 2-, 3-, 4-, or 6-position remain unclear, because large amounts of oligosaccharides are required for the systematic comparative analysis using surface plasmon resonance. Here we show a direct comparison of the dissociation constants (K_d) of AOL and AAL using 113 pyridylaminated oligosaccharides with frontal affinity chromatography. As a result, AOL showed a similar specificity as AAL in terms of the high affinity for α1,6-fucosylated oligosaccharides, for smaller fucosylated oligosaccharides, and for oligosaccharides fucosylated at the reducing terminal core GlcNAc. On the other hand, AOL showed 2.9-6.2 times higher affinity constants (K_a) for α1,6-fucosylated oligosaccharides than AAL and only AAL additionally recognized oligosaccharides which were α1,3-fucosylated at the reducing terminal GlcNAc. These results explain why AOL reflects the extent of α1,6-fucosylation on glycoproteins more clearly than AAL. This systematic comparative analysis made from a quantitative viewpoint enabled a clear physical interpretation of these fucose-specific lectins with multivalent fucose-binding sites.     

A Comparative Study of Lectin Affinity Based Plant N-Glycoproteome Profiling Using Tomato Fruit as a Model

Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with different affinities can be particularly effective. A multi-lectin approach has also been reported to provide a significant benefit for the analysis of plant N-glycoproteins; however, it has yet to be determined whether certain lectins, or combinations of lectins are optimal for plant N-glycoproteome profiling; or whether specific lectins show preferential association with particular N-glycosylation sites or N-glycan structures. We describe here a comparative study of three mannose-binding lectins, concanavalin A, snowdrop lectin, and lentil lectin, to profile the N-glycoproteome of mature green stage tomato (Solanum lycopersicum) fruit pericarp. Through coupling lectin affinity chromatography with a shotgun proteomics strategy, we identified 448 putative N-glycoproteins, whereas a parallel lectin affinity chromatography plus hydrophilic interaction chromatography analysis revealed 318 putative N-glycosylation sites on 230 N-glycoproteins, of which 100 overlapped with the shotgun analysis, as well as 17 N-glycan structures. The use of multiple lectins substantially increased N-glycoproteome coverage and although there were no discernible differences in the structures of N-glycans, or the charge, isoelectric point (pI) or hydrophobicity of the glycopeptides that differentially bound to each lectin, differences were observed in the amino acid frequency at the −1 and +1 subsites of the N-glycosylation sites. We also demonstrated an alternative and complementary in planta recombinant expression strategy, followed by affinity MS analysis, to identify the putative N-glycan structures of glycoproteins whose abundance is too low to be readily determined by a shotgun approach, and/or combined with deglycosylation for predicted deamidated sites, using a xyloglucan-specific endoglucanase inhibitor protein as an example.N-glycosylation is one of the most heterogeneous and common post-translational modifications of eukaryotic proteins and one that affects many aspects of protein targeting, enzymatic properties, stability and intermolecular interactions (1–3). There is therefore considerable interest in developing robust and sensitive high throughput analytical pipelines to isolate and structurally characterize N-glycoprotein populations (2, 4–8), allowing glycoprotein identification and analysis of the glycosylation site occupancy and N-glycan structure. To this end, lectin affinity chromatography (LAC)¹ is increasingly popular: specifically, various lectins are known to have different binding affinities for N-glycans and so the selective binding of N-glycoproteins in complex protein extracts to these lectins and their subsequent release allows a critical enrichment step before sequencing and glycan analysis by MS (9).As a refinement of this approach, the use of multiple lectin affinity chromatography (MLAC) in yeast and animal studies (4, 6, 10), using different proteomic platforms, has been shown to increase the numbers of isolated N-glycoproteins or N-glycopeptides. Collectively, these studies of taxonomically diverse eukaryotic N-glycoproteomes suggest a general conservation of the glycosylation site (N-X-S/T, where X can be any amino acid except proline), as well as conserved features of three-dimensional protein structure (4, 6, 10). Although there have been several studies to determine the structural basis of the binding specificity of specific lectins to yeast and animal N-glycoproteins (11), larger scale N-glycoproteomic analyses have typically not attempted to determine whether a particular combination of lectins provides optimal enrichment, or whether specific features, such as N-glycan structure or amino acid sequence at and around the N-glycosylation site, are associated with different lectins. Therefore, systematic comparative studies are essential to determine whether particular lectins can be optimal for specific tissues, organs, and organisms.LAC has also been used in plant N-glycoprotein analyses to enrich for populations of cell wall localized proteins (8, 12, 13) and a recent report (13) described the application of MLAC to map substantial numbers of N-glycosylation sites in a range of key experimental model organisms, included the plant Arabidopsis thaliana. Using a LTQ-Orbitrap Velos mass spectrometer, the authors identified 2186 unique N-glycosylation sites in proteins extracted from five different arabidopsis organs (13), which represents a substantial increase in the number of identified N-glycosylation sites that have resulted from previous plant N-glycoprotein studies. However, the particular analytical platform that was used did not allow the structural characterization of the N-glycans or N-glycopeptides (13). Indeed, certain features of N-glycopeptides, such as poor fragmentation, heterogeneity and a large dynamic range in most complex mixtures often limits the structural analysis of N-glycans in high throughput systematic analyses (2). It is also important to note that the structures of plant N-glycans differ from those of animals and yeast, as exemplified by the presence of β-1, 2-xylose and α-1, 3-fucose and the complete absence of multiantennary N-glycans and sialic acid in plant N-glycoproteins (1, 14). Therefore, assumptions that are made with regard to the lectin binding of animal and yeast proteins do not necessarily apply to those from plants. Consequently, there is a need to investigate the structural basis of lectin binding to plant N-glycoproteins. Moreover, the limitations of typical shotgun based profiling approaches in identifying and characterizing low abundance N-glycoproteins in complex protein extracts need to be addressed to allow more comprehensive plant N-glycoproteome profiling.In the present study we address both these issues using mature green stage tomato (Solanum lycopersicum) fruit pericarp as an experimental model to carry out a comparative analysis of N-glycoproteins associated with each of three mannose-binding lectins: concanavalin A (ConA), snowdrop lectin (GNA), and lentil lectin (LCH). Fruit development is associated with substantial cell wall metabolism and the expression of many wall localized N-glycoproteins (8) and tomato in particular represents an excellent model for studies of fleshy fruits and cell wall N-glycoproteins (15). We established an MLAC analytical pipeline that included shotgun proteomic profiling and deglycosylation and deamidation analysis, to allow the determination of N-glycoprotein protein identity, N-glycosylation site and N-glycan structures. This information was then used to establish whether a combination of lectins is indeed advantageous for the study of plant N-glycoproteomes, and to assess whether any of these structural characteristics result in predictable preferential binding to specific lectins. From these studies it became evident that large dynamic range of N-glycoprotein abundance was a significant limiting factor in the structural determination of the tomato, and that there was a bias toward the detection of highly abundant N-glycopeptides. We therefore evaluated the use of an in planta recombinant expression strategy, combined with affinity purification MS (AP-MS), as a means to characterize the N-glycan structures of glycoproteins whose abundance is too low to be readily determined via the primary shotgun pipeline, using a tomato xyloglucan-specific endoglucanase inhibitor protein (XEGIP) as a test case.     

Terminal N-Acetylgalactosamine-Specific Leguminous Lectin from Wisteria japonica as a Probe for Human Lung Squamous Cell Carcinoma

Millettia japonica was recently reclassified into the genus Wisteria japonica based on chloroplast and nuclear DNA sequences. Because the seed of Wisteria floribunda expresses leguminous lectins with unique N-acetylgalactosamine-binding specificity, we purified lectin from Wisteria japonica seeds using ion exchange and gel filtration chromatography. Glycan microarray analysis demonstrated that unlike Wisteria floribunda and Wisteria brachybotrys lectins, which bind to both terminal N-acetylgalactosamine and galactose residues, Wisteria japonica lectin (WJA) specifically bound to both α- and β-linked terminal N-acetylgalactosamine, but not galactose residues on oligosaccharides and glycoproteins. Further, frontal affinity chromatography using more than 100 2-aminopyridine-labeled and p-nitrophenyl-derivatized oligosaccharides demonstrated that the ligands with the highest affinity for Wisteria japonica lectin were GalNAcβ1-3GlcNAc and GalNAcβ1-4GlcNAc, with K _a values of 9.5 × 10⁴ and 1.4 × 10⁵ M^-1, respectively. In addition, when binding was assessed in a variety of cell lines, Wisteria japonica lectin bound specifically to EBC-1 and HEK293 cells while other Wisteria lectins bound equally to all of the cell lines tested. Wisteria japonica lectin binding to EBC-1 and HEK293 cells was dramatically decreased in the presence of N-acetylgalactosamine, but not galactose, mannose, or N-acetylglucosamine, and was completely abrogated by β-hexosaminidase-digestion of these cells. These results clearly demonstrate that Wisteria japonica lectin binds to terminal N-acetylgalactosamine but not galactose. In addition, histochemical analysis of human squamous cell carcinoma tissue sections demonstrated that Wisteria japonica lectin specifically bound to differentiated cancer tissues but not normal tissue. This novel binding characteristic of Wisteria japonica lectin has the potential to become a powerful tool for clinical applications.     

The Lectins of Sophora japonica: I. Purification Properties and N-Terminal Amino Acid Sequences of Two Lectins from Leaves

Two lectins, Leaf Lectin I and Leaf Lectin II (LLI and LLII) were purified from the leaves of Sophora japonica. Like the Sophora seed lectin, LLI and LLII are tetrameric glycoproteins containing a single subunit with respect to size. The subunits of LLI (32 kilodaltons) and LLII (34 kilodaltons) are slightly larger than those of the seed lectin (29.5 kilodaltons). The three Sophora lectins display indistinguishable specificities, amino acid compositions, specific hemagglutinin activities, and extinction coefficients. Although very closely related to the seed lectin, the leaf and seed lectins are not immunologically identical and they differ in subunit molecular weights, carbohydrate content, and in the pH sensitivity of their hemagglutinin activities. N-terminal amino acid sequence analysis shows that although they are homologous proteins, the three Sophora lectins are products of distinct genes.     

Interaction of the Laburnum anagyroides lectin with fucoantigens

We studied interaction of the lectin from the bark of Golden Rain shrub (Laburnum anagyroides, LABA) with a number of basic fucose-containing carbohydrate antigens by changes in its tryptophan fluorescence. The strongest LABA binding was observed for the trisaccharide H of type 6 [α-L-Fucp-(1-2)-β-D-Galp-(1-4)-D-Glc, K _a = 4.2 × 10³ M^?1]. The following antigens were bound with a weaker affinity: H-disaccharide α-L-Fucp-(1-2)-D-Gal, a glucoanalogue of tetrasaccharide Le^y α-L-Fucp-(1-2)-β-D-Galp-(1-4)-[α-L-Fucp-(1-3)]-D-Glc, and 6-fucosyl-N-acetylglucosamine, a fragment of core of the N-glycans family (K _a 1.1?1.7 × 10³ M^?1). The lowest binding was observed for L-fucose (K _a = 2.7 × 10² M^?1) and trisaccharide Le^a, (β-Galp-(1-3)-[α-L-Fucp-(1-4)]-GlcNAc (K _a = 6.4 × 10² M^?1). The Le^d, Le^a, and Le^x pentasaccharides and Le^b hexasaccharide were not bound to LABA.     

Strict Binding Specificity of Small-Sized Lectins from the Red Alga Hypnea japonica for Core (α1-6) Fucosylated N-Glycans

Small-sized isolectins (9 KDa) from Hypnea japonica belong to a new lectin family. Here, we describe the carbohydrate-binding properties of the three isolectins (hypninA1, A2, and A3) and the amino acid sequence of hypninA3 (P85888). In frontal affinity chromatography with about 100 pyridylaminated oligosaccharides, the isolectins, which had no affinity for monosaccharides, commonly bound only core (α1-6) fucosylated N-glycans, and did not the other oligosaccharides examined, including (α1-2), (α1-3), and (α1-4) fucosylated glycans. The specific binding of hypninA3 with the fucosylated N-glycans (K _a; 0.52–7.58×10⁶ M^?1) was confirmed by surface plasmon resonance analyses on an immobilized glycoprotein with and without core (α1-6) fucose. Such specificity of hypninA is clearly distinct from those of other known fucose-binding lectins, making it a valuable tool for cancer diagnosis and quality control of medicinal antibodies. HypninA3 is a polypeptide composed of 90 amino acids containing four half-cystines.     

A lectin from Platypodium elegans with unusual specificity and affinity for asymmetric complex N-glycans

Lectin activity with specificity for mannose and glucose has been detected in the seed of Platypodium elegans, a legume plant from the Dalbergieae tribe. The gene of Platypodium elegans lectin A has been cloned, and the resulting 261-amino acid protein belongs to the legume lectin family with similarity with Pterocarpus angolensis agglutinin from the same tribe. The recombinant lectin has been expressed in Escherichia coli and refolded from inclusion bodies. Analysis of specificity by glycan array evidenced a very unusual preference for complex type N-glycans with asymmetrical branches. A short branch consisting of one mannose residue is preferred on the 6-arm of the N-glycan, whereas extensions by GlcNAc, Gal, and NeuAc are favorable on the 3-arm. Affinities have been obtained by microcalorimetry using symmetrical and asymmetrical Asn-linked heptasaccharides prepared by the semi-synthetic method. Strong affinity with K_d of 4.5 μm was obtained for both ligands. Crystal structures of Platypodium elegans lectin A complexed with branched trimannose and symmetrical complex-type Asn-linked heptasaccharide have been solved at 2.1 and 1.65 Å resolution, respectively. The lectin adopts the canonical dimeric organization of legume lectins. The trimannose bridges the binding sites of two neighboring dimers, resulting in the formation of infinite chains in the crystal. The Asn-linked heptasaccharide binds with the 6-arm in the primary binding site with extensive additional contacts on both arms. The GlcNAc on the 6-arm is bound in a constrained conformation that may rationalize the higher affinity observed on the glycan array for N-glycans with only a mannose on the 6-arm.