Deciphering the Glycan Preference of Bacterial Lectins by Glycan Array and Molecular Docking with Validation by Microcalorimetry and Crystallography

Recent advances in glycobiology revealed the essential role of lectins for deciphering the glycocode by specific recognition of carbohydrates. Integrated multiscale approaches are needed for characterizing lectin specificity: combining on one hand high-throughput analysis by glycan array experiments and systematic molecular docking of oligosaccharide libraries and on the other hand detailed analysis of the lectin/oligosaccharide interaction by x-ray crystallography, microcalorimetry and free energy calculations. The lectins LecB from Pseudomonas aeruginosa and BambL from Burkholderia ambifaria are part of the virulence factors used by the pathogenic bacteria to invade the targeted host. These two lectins are not related but both recognize fucosylated oligosaccharides such as the histo-blood group oligosaccharides of the ABH(O) and Lewis epitopes. The specificities were characterized using semi-quantitative data from glycan array and analyzed by molecular docking with the Glide software. Reliable prediction of protein/oligosaccharide structures could be obtained as validated by existing crystal structures of complexes. Additionally, the crystal structure of BambL/Lewis x was determined at 1.6 Å resolution, which confirms that Lewis x has to adopt a high-energy conformation so as to bind to this lectin. Free energies of binding were calculated using a procedure combining the Glide docking protocol followed by free energy rescoring with the Prime/Molecular Mechanics Generalized Born Surface Area (MM-GBSA) method. The calculated data were in reasonable agreement with experimental free energies of binding obtained by titration microcalorimetry. The established predictive protocol is proposed to rationalize large sets of data such as glycan arrays and to help in lead discovery projects based on such high throughput technology.     

The fucose-binding lectin from Ralstonia solanacearum. A new type of beta-propeller architecture formed by oligomerization and interacting with fucoside, fucosyllactose, and plant xyloglucan

Plant pathogens, like animal ones, use protein-carbohydrate interactions in their strategy for host recognition, attachment, and invasion. The bacterium Ralstonia solanacearum, which is distributed worldwide and causes lethal wilt in many agricultural crops, was shown to produce a potent L-fucose-binding lectin, R. solanacearum lectin, a small protein of 90 amino acids with a tandem repeat in its amino acid sequence. In the present study, surface plasmon resonance experiments conducted on a series of oligosaccharides show a preference for binding to alphaFuc1-2Gal and alphaFuc1-6Gal epitopes. Titration microcalorimetry demonstrates the presence of two binding sites per monomer and an unusually high affinity of the lectin for alphaFuc1-2Gal-containing oligosaccharides (KD = 2.5 x 10(-7) M for 2-fucosyllactose). R. solanacearum lectin has been crystallized with a methyl derivative of fucose and with the highest affinity ligand, 2-fucosyllactose. X-ray crystal structures, the one with alpha-methyl-fucoside being at ultrahigh resolution, reveal that each monomer consists of two small four-stranded anti-parallel beta-sheets. Trimerization through a 3-fold or pseudo-3-fold axis generates a six-bladed beta-propeller architecture, very similar to that previously described for the fungal lectin of Aleuria aurantia. This is the first report of a beta-propeller formed by oligomerization and not by sequential domains. Each monomer presents two fucose binding sites, resulting in six symmetrically arranged sugar binding sites for the beta-propeller. Crystals were also obtained for a mutated lectin complexed with a fragment of xyloglucan, a fucosylated polysaccharide from the primary cell wall of plants, which may be the biological target of the lectin.     

Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by mannan-binding protein. A highly fucosylated polylactosamine type N-glycan

Mannan-binding protein (MBP) is a C-type serum lectin and activates complement through the lectin pathway when it binds to ligand sugars such as mannose, N-acetylglucosamine, and fucose on microbes. In addition, the vaccinia virus carrying the human MBP gene was shown to exhibit potent growth inhibitory activity toward human colorectal carcinoma, SW1116, cells in nude mice. We have proposed calling this activity MBP-dependent cell-mediated cytotoxicity (MDCC) (Ma, Y., Uemura, K., Oka, S., Kozutsumi, Y., Kawasaki, N., and Kawasaki, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 371-375). In this study, the MBP ligands on the surface of SW1116 cells were characterized. Initial experiments involving plant lectins and anti-Lewis antibodies as inhibitors of MBP binding to SW1116 cells indicated that fucose plays a crucial role in the interaction. Subsequently, Pronase glycopeptides were prepared from whole cell lysates, and oligosaccharides were liberated by hydrazinolysis. After being tagged by pyridylamination, MBP ligand oligosaccharides were isolated with an MBP affinity column, and then their sequences were determined by mass spectrometry and tandem mass spectrometry after permethylation, in combination with endo-beta-galactosidase digestion and chemical defucosylation. The MBP ligands were shown to be large, multiantennary N-glycans carrying a highly fucosylated polylactosamine type structure. At the nonreducing termini, Le(b)/Le(a) or tandem repeats of the Le(a) structure prevail, a substantial proportion of which are attached via internal Le(x) or N-acetyllactosamine units to the trimannosyl core. The structures characterized are unique and distinct from those of other previously reported tumor-specific carbohydrate antigens. It is concluded that MBP requires clusters of tandem repeats of the Le(b)/Le(a) epitope for recognition.     

Identification of carbohydrates binding to lectins by using surface plasmon resonance in combination with HPLC profiling

A new, powerful method is presented for screening the binding in real time and taking place under dynamic conditions of oligosaccharides to lectins. The approach combines an SPR biosensor and HPLC profiling with fluorescence detection, and is applicable to complex mixtures of oligosaccharides in terms of ligand-fishing. Labeling the oligosaccharides with 2-aminobenzamide ensures a detection level in the fmol range. In an explorative study the binding of RNase B-derived oligomannose-type N-glycans to biosensor-immobilized concanavalin A (Con A) was examined, and an affinity ranking could be established for Man(5)GlcNAc(2) to Man(9)GlcNAc(2), as monitored by HPLC. In subsequent experiments and using well-defined labeled as well as nonlabeled oligosaccharides, it was found that the fluorescent tag does not interfere with the binding and that the optimum epitope for the interaction with Con A comprises the tetramannoside unit Manalpha2Manalpha6(Manalpha3)Man[D(3)B(A)4'], rather than the generally accepted trimannoside Manalpha6 (Manalpha3)Man [B(A)4' or 4(4')3]. In a similar experimental setup, the interaction of various fucosylated human milk oligosaccharides with the fucose-binding lectin from Lotus tetragonolobus purpureaus was studied, and it appeared that oligosaccharides containing blood group H could selectively be retained and eluted from the lectin-coated surface. Finally, using the same lectin and a mixture of O-glycans derived from bovine submaxillary gland mucin, minor constituents but containing fucose could selectively be picked from the analyte solution as demonstrated by HPLC profiling.     

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.     

The expression of free oligosaccharides in human seminal plasma

Human seminal plasma is a complex mixture of proteins, glycoproteins, peptides, glycopeptides, and prostaglandins secreted by organs of the male reproductive tract. The components of this fluid have been implicated in the suppression of immune response, agonistic effects on sperm-egg binding, and promotion of successful implantation of the human embryo. Fractionation followed by biophysical analyses revealed that free oligosaccharides constitute a major component of the total glycoconjugates within seminal plasma. Significant findings of our analyses include the following: (i) the concentration of free oligosaccharides is 0.3-0.4 mg/ml; (ii) mono- and difucosylated forms of the disaccharide lactose are major components; (iii) many of the remaining oligosaccharides are also rich in fucose and carry Lewis(x) and/or Lewis(y) epitopes; (iv) a subset of the oligosaccharides express the reducing end sequence (GlcNAcbeta1-3/4Glc) not reported in human milk oligosaccharides; (v) oligosaccharides in seminal plasma exclusively express type 2 (Galbeta1-4GlcNAc) but not the type 1 sequences (Galbeta1-3GlcNAc) that predominate in human milk glycans; and (vi) the structural diversity of seminal plasma oligosaccharides is far less than human milk oligosaccharides. The agonistic effect of both fucose and fucosylated glycoconjugates on human sperm-egg binding in vitro suggests that fucosylated oligosaccharides may also promote fertilization in the female reproductive tract.     

Evidence for the presence of complex high-molecular mass N-linked oligosaccharides in intranuclear glycoproteins from HeLa cells.

Nonhistone proteins were extracted in 0.4 M NaCl from membrane-depleted nuclei of HeLa cells grown in the presence or the absence of [5,6-3H]fucose. Control experiments strongly suggest that most extracted proteins were indeed nuclear components. Several proteins, present in the 0.4 M NaCl nuclear extract, with M(r) ranging from 35,000 to 115,000 were identified on Western blots as fucosylated glycoproteins owing to their binding to the fucose-specific lectin, Ulex europeus agglutinin I. Results of experiments involving mild alkaline treatment and peptide N-glycosidase F digestion showed that the carbohydrate moieties of these fucosylated nuclear glycoproteins were N-linked to the polypeptide backbone. Analysis of the N-glycans revealed the presence of two populations of sialylated oligosaccharides on the basis of their relative molecular masses. The sensitivity of the high-M(r) oligosaccharides to endo-beta-galactosidase and their incorporation of [3H]glucosamine suggest that they could contain repeating N-acetyllactosamine units. [3H]Fucose incorporated into nuclei was confined to the nucleoli, as judged by autoradiography of sections cut through cells grown in the presence of [3H]fucose. Electron microscopy autoradiography showed that the fibrillar centers were never labeled, while silver grains were observed on the dense and the granular components of nucleoli. Taking into account of these data most nuclear fucosylated glycoproteins extracted in 0.4 M NaCl might be nucleolar ribonucleoproteins.     

A new Ralstonia solanacearum high-affinity mannose-binding lectin RS-IIL structurally resembling the Pseudomonas aeruginosa fucose-specific lectin PA-IIL

The plant pathogen Ralstonia solanacearum produces two lectins, each with different affinity to fucose. We described previously the properties and sequence of the first lectin, RSL (subunit M(r) 9.9 kDa), which is related to fungal lectins (Sudakevitz, D., Imberty, A., and Gilboa-Garber, N., 2002, J Biochem 132: 353-358). The present communication reports the discovery of the second one, RS-IIL (subunit M(r) 11.6 kDa), a tetrameric lectin, with high sequence similarity to the fucose-binding lectin PA-IIL of Pseudomonas aeruginosa. RS-IIL recognizes fucose but displays much higher affinity to mannose and fructose, which is opposite to the preference spectrum of PA-IIL. Determination of the crystal structure of RS-IIL complexed with a mannose derivative demonstrates a tetrameric structure very similar to the recently solved PA-IIL structure (Mitchell, E., et al., 2002, Nature Struct Biol 9: 918-921). Each monomer contains two close calcium cations that mediate the binding of the monosaccharide and explain the outstandingly high affinity to the monosaccharide ligand. The binding loop of the cations is fully conserved in RS-IIL and PA-IIL, whereas the preference for mannose versus fucose can be attributed to the change of a three-amino-acid sequence in the 'specificity loop'.     

Oligosaccharide composition of an influenza virus hemagglutinin with host-determined binding properties

We have previously reported that the binding properties of the hemagglutinin (HA) of the WSN-F strain of influenza A are affected by the cells in which the virus is grown (Crecelius, D. M., Deom, C. M., and Schulze, I.T. (1984) Virology 139, 164-177); at 37 degrees C chick embryo fibroblast-grown F virus has a greater affinity for host cells than does the same virus grown in Madin-Darby bovine kidney (MDBK) cells. In an attempt to explain this host-determined property, we have characterized the carbohydrate put onto the viral HA by these two cells. Experiments using tunicamycin indicate that the HA made by MDBK cells contains about 4000 daltons of carbohydrate in excess of that on the HA from chick embryo fibroblast. Serial lectin affinity chromatography of the asparagine-linked oligosaccharides on the HA subunits, HA1 and HA2, detected a number of host-dependent differences in the complex oligosaccharides. Both HA1 and HA2 from MDBK cells contained more highly branched (i.e. tri- and tetraantennary) complex oligosaccharides than did the subunits from chick embryo fibroblasts. In addition, the HA subunits from the two sources differed in the amount of galactose-containing "bisected" complex oligosaccharides and in the presence of certain fucosylated triantennary oligosaccharides. Profiles of the asparagine-linked oligosaccharides from the host cells did not show these differences, indicating that the HA subunit profiles were not necessarily representative of the structures found on the cellular glycoproteins. The data support the conclusion that bulky oligosaccharides on the MDBK-HA subunits of WSN-F reduce the affinity of the virus for cellular receptors.     

Ulex europeus type I agglutinin detects carcinoembryonic antigen in extracts of human colorectal carcinoma

Recent interest has focused on fucosylated epitopes expressed on human neoplasms. The plant lectin Ulex europus agglutinin, Type I (UEA) binds fucosylated oligosaccharides, while UEA-reactive substances have a tissue distribution similar to carcinoembryonic antigen (CEA). We sought to determine if UEA reacted with CEA in extracts of fresh primary and metastatic colorectal carcinomas and paired normal tissues. The extracts were electrophoretically transferred to nitrocellulose membranes after the proteins were separated by SDS-PAGE in 10% polyacrylamide gels. The transfer membranes were then stained with peroxidase-conjugated UEA (UEA-P) or antibody to CEA (CEA-P). UEA-P reacted with a 170-190-kDa band in extracts of 22 of 30 primary tumors, 10 of 12 metastases, but only 1 of 5 villous adenomas. UEA-P generally did not react with normal colon or liver extracts. UEA-P also did not bind to 170-190-kDa molecules in Western transfers of a breast carcinoma metastatic to bowel and a focal nodular hyperplasia of liver. CEA-P displayed similar reactivity and detected CEA in a tumor extract negative for UEA. Fucose blocked binding of UEA-P to Western transfers of tumor extracts. CEA-P reacted with a 170-190-kDa substance in tumor extracts eluted with fucose from a column of immobilized UEA. Thus, UEA reacts with fucosylated oligosaccharides on most, but not all, species of CEA and may be a useful adjunct to anti-CEA immunohistochemistry.     

N-linked glycosylation of the liver cancer biomarker GP73

The association between elevated circulating levels of GP73 (and fucosylated GP73 in particular) and hepatocellular carcinoma suggests that a thorough analysis of the extent of GP73 glycosylation is warranted. Detailed analysis of the glycosylation patterns of such low abundance proteins are hampered by technical difficulties. Using conventional lectin affinity chromatography, we have established that three quarters of the GP73 secreted from a cell line derived from HCC is fucosylated. Using mass spectrometry, we have established that at least two of three potential sites of N-linked glycosylation are occupied on most molecules of GP73 secreted from cultured hepatoma cells. Furthermore, the oligosaccharides added to recombinant GP73 resemble those present in the bulk of secreted protein, mostly bi-antennary with core fucose, with a smaller fraction of tri- and tetra-antennary structures. The frequency of fucosylation observed on the recombinant protein agrees well with the pattern of lectin binding of the endogenous secreted protein. Finally, we have developed a method to interrogate the glycans added to either the near full length protein or at a particular sequon, providing proof of concept that a small peptide embedded in a heterologous context can preserve both fucosylation and a high level of branching of oligosaccharides added.     

Microbial recognition of human cell surface glycoconjugates

Infection by pathogens is generally initiated by the specific recognition of host epithelia surfaces and subsequent adhesion is essential for invasion. In their infection strategy, microorganisms often use sugar-binding proteins, that is lectins and adhesins, to recognize and bind to host glycoconjugates where sialylated and fucosylated oligosaccharides are the major targets. The lectin/glycoconjugate interactions are characterized by their high specificity and most of the time by multivalency to generate higher affinity of binding. Recent crystal structures of viral, bacterial, and parasite receptors in complex with human histo-blood group epitopes or sialylated derivatives reveal new folds and novel sugar-binding modes. They illustrate the tight specificity between tissue glycosylation and lectins.     

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.     

Further refinement of the description of the ligand-binding characteristics for the galactoside-binding mistletoe lectin,a plant agglutinin with immunomodulatory potency

The galactoside-binding lectin from mistletoe (Viscum album L.) is a biological response modifier, eliciting e.g. enhanced secretion of cytokines. This immunological activity warrants the further analysis of its ligand-binding properties with special attention paid to blood group epitopes. To avoid the microheterogeneity and complexity of naturally occurring glycoproteins, chemically strictly defined neoglycoconjugates and a panel of synthetic oligosaccharides were employed in solid-phase assays for direct binding and assessment of the relative inhibitory capacity. Since label incorporation into the lectin, although performed under protective conditions, or surface immobilization by adsorption to plastic may affect its affinity characteristics, the extent of neoglycoconjugate binding in the absence of any interfering substance and in the presence of oligosaccharides was determined comparatively with labeled and with immobilized lectin. In principle, these two factors could be excluded to markedly alter binding features. In addition to lactose, the blood group determinants H and B were strongly reactive. A fucose residue can thus especially be accommodated to the binding site when linked to the non-reducing unit. N-Acetyllactosamine was nearly as potent as an inhibitor as lactose. Le^c and the A determinant were notably inferior to the other ABH blood group epitopes. Le^a and Le^x and their sialylated derivatives displayed only very weak binding capacity. Among the two natural isomers of sialyllactose, the α2,6-form displayed a higher level of inhibitory capacity than the α2,3-derivative. Isomeric variants of the Thomsen-Friedenreich antigen, too, reduced lectin binding to the lactose-carrying polymer. Their capacities were surpassed by those of the H and the B determinants and a related form of the latter, the P₁ epitope. An overlap of specificity with the immunomodulatory human galectin-3 is thus measurable for H/B-like structures. The documented differential reactivity of the mistletoe lectin to blood group oligosaccharides may have a bearing on the responsiveness of blood group-positive cell populations. © 1997 John Wiley & Sons, Ltd.     

Exquisite specificity of mitogenic lectin from <Emphasis Type="Italic">Cephalosporium curvulum</Emphasis> to core fucosylated N-glycans

Lectins are carbohydrate binding proteins that are gaining attention as important tools for the identification of specific glycan markers expressed during different stages of the cancer. We earlier reported the purification of a mitogenic lectin from human pathogenic fungus Cephalosporium curvulum (CSL) that has complex sugar specificity when analysed by hapten inhibition assay. In the present study, we report the fine sugar specificity of CSL as determined by glycan array analysis. The results revealed that CSL has exquisite specificity towards core fucosylated N-glycans. Fucosylated trimannosyl core is the basic structure required for the binding of CSL. The presence of fucose in the side chain further enhances the avidity of CSL towards such glycans. The affinity of CSL is drastically reduced towards the non-core fucosylated glycans, in spite of their side chain fucosylation. CSL showed no binding to the tested O-glycans and monosaccharides. These observations suggest the unique specificity of CSL towards core fucosylated N-glycans, which was further validated by binding of CSL to human colon cancer epithelial and hepatocarcinoma cell lines namely HT29 and HepG2, respectively, that are known to express core fucosylated N-glycans, using AOL and LCA as positive controls. LCA and AOL are fucose specific lectins that are currently being used clinically for the diagnosis of hepatocellular carcinomas. Most of the gastrointestinal markers express core fucosylated N-glycans. The high affinity and exclusive specificity of CSL towards α1-6 linkage of core fucosylated glycans compared to other fucose specific lectins, makes it a promising molecule that needs to be further explored for its application in the diagnosis of gastrointestinal cancer.     

Non-reducing terminal fucose within N-linked glycan plays a significant role in the recognition of human milk lactoferrin by the 1CF11 monoclonal antibody

We have recently demonstrated that the 1CF11 monoclonal antibody bound human milk lactoferrin (hLf) through the recognition of two distinct portions of the molecule, namely the N-glycan-relevant and -irrelevant structural elements. In this present study, we prepared four immunoreactive peptide fractions containing N-linked glycan from tryptic digests of reduced and alkylated hLf by using a concanavalin A lectin column and reverse-phase HPLC. Deglycosylation of these fractions and a competitive binding assay using fucosylated oligosaccharides revealed that the non-reducing terminal fucose residue in N-linked glycan(s) played a significant role in recognizing the N-glycan-relevant element in hLf by 1CF11.     

3'-Azidothymidine potently inhibits the biosynthesis of highly branched N-linked oligosaccharides and poly-N-acetyllactosamine chains in cells

Previous studies in our laboratory have characterized 3'-azido-3'-deoxythymidine (AZT) as a potent inhibitor of glycosphingolipid biosynthesis in cultured cells (Steet, R., Alizadeh, M., Melan?on, P., and Kuchta, R. D. (1999) Glycoconj. J. 16, 237-245; Yan, J.-P., Ilsley, D. D., Frohlick, C., Steet, R., Hall, E. T., Kuchta, R. D., and Melan?on, P. (1995) J. Biol. Chem. 270, 22836-22841). Here, we report that AZT treatment of K562 cells results in significant alterations in the profile of N-linked oligosaccharides. Fractionation of [(3)H]mannose-labeled oligosaccharides from AZT-treated K562 cells using lectin affinity chromatography revealed striking changes in the branching and processing of N-linked glycoconjugates. AZT treatment resulted in the production of fewer highly branched complex glycans (60% of control at 20 micrometer AZT) and a significant accumulation of core-fucosylated biantennary oligosaccharides. In addition, extension of branched oligosaccharides with multiple poly-N-acetyllactosamine repeats is nearly abolished by AZT concentrations as low as 2 micrometer. A shift from multiantennary to moderately branched oligosaccharides was also apparent in the melanoma cell line SK-MEL-30 upon AZT treatment. N-Linked glycans from both cell lines exhibited increased affinity for the beta-galactoside-binding lectin RCA-I in the presence of AZT, suggesting that the addition of terminal sialic acid is sensitive to the drug. These results demonstrate the ability of AZT to modulate strongly the processing of asparagine-linked glycoconjugates in whole cells and reveal a novel mechanism by which AZT treatment may cause anemia.     

Detection of a high affinity binding site in recombinant <Emphasis Type="Italic">Aleuria aurantia</Emphasis> lectin

Lectins are carbohydrate binding proteins that are involved in many recognition events at molecular and cellular levels. Lectin-oligosaccharide interactions are generally considered to be of weak affinity, however some mushroom lectins have unusually high binding affinity towards oligosaccharides with K (d) values in the micromolar range. This would make mushroom lectins ideal candidates to study protein-carbohydrate interactions. In the present study we investigated the properties of a recombinant form of the mushroom lectin Aleuria aurantia (AAL). AAL is a fucose-binding lectin composed of two identical 312-amino acid subunits. Each subunit contains five binding sites for fucose. We found that one of the binding sites in rAAL had unusually high affinities towards fucose and fucose-containing oligosaccharides with K (d) values in the nanomolar range. This site could bind to oligosaccharides with fucose linked alpha1-2, alpha1-3 or alpha1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with alpha1-6 linked fucose. This binding site is not detected in native AAL (nAAL) one possible explanation may be that this site is blocked with free fucose in nAAL. Recombinant AAL was produced in E. coli as a His-tagged protein, and purified in a one-step procedure. The resulting protein was analyzed by electrophoresis, enzyme-linked lectin assay and circular dichroism spectroscopy, and compared to nAAL. Binding properties were measured using tryptophan fluorescence and surface plasmon resonance. Removal of the His-tag did not alter the binding properties of recombinant AAL in the enzyme-linked lectin assay. Our study forms a basis for understanding the AAL-oligosaccharide interaction and for using molecular techniques to design lectins with novel specificities and high binding affinities towards oligosaccharides.     

Comparison of the N-Linked Oligosaccharide Structures of the Two Major Human Myelin Glycoproteins MAG and P0: Assessment of the Structures Bearing the Epitope for HNK-1 and Human Monoclonal Immunoglobulin M Found in Demyelinating Neuropathy

The epitope for HNK-1 and patient's monoclonal autoantibodies in demyelinating polyneuropathy associated with immunoglobulin M gammopathy is borne by different types of N-linked oligosaccharide structures in human P0 and myelin-associated glycoprotein (MAG). Fourteen glycopeptide fractions bearing different oligosaccharide structures were obtained from either MAG or P0 glycopeptides by serial lectin affinity chromatography on concanavalin A-Sepharose, Phaseolus vulgaris erythrophytohemagglutinin-agarose, Pisum sativum agglutinin-agarose, and Phaseolus vulgaris leucophytohemagglutinin-agarose. As shown by dot-TLC plate immunostaining, the same MAG and P0 glycopeptide fractions were recognized by HNK-1 and patient's immunoglobulin M, confirming that these antibodies display similar specificities. The antigenic carbohydrate was present in glycopeptide fractions that either interact with Pisum sativum agglutinin-agarose or were bound by Aleuria aurantia agglutinin-digoxigenin, indicating that these structures contained alpha(1-6)fucose residues. This study demonstrates that the L2/HNK-1 epitope is borne mainly or even exclusively by N-linked oligosaccharide structures alpha(1-6)fucosylated in the core.     

A Novel Core Fucose-specific Lectin from the Mushroom Pholiota squarrosa

Fucα1–6 oligosaccharide has a variety of biological functions and serves as a biomarker for hepatocellular carcinoma because of the elevated presence of fucosylated α-fetoprotein (AFP) in this type of cancer. In this study we purified a novel Fucα1–6-specific lectin from the mushroom Pholiota squarrosa by ion-exchange chromatography and affinity chromatography on thyroglobulin-agarose. The purified lectin was designated as PhoSL (P. squarrosa lectin). SDS-PAGE, MALDI-TOF mass spectrometry, and N-terminal amino acid sequencing indicate that PhoSL has a molecular mass of 4.5 kDa and consists of 40 amino acids (NH₂-APVPVTKLVCDGDTYKCTAYLDFGDGRWVAQWDTNVFHTG-OH). Isoelectric focusing of the lectin showed bands near pI 4.0. The lectin activity was stable between pH 2.0 and 11.0 and at temperatures ranging from 0 to 100 °C for incubation times of 30 min. When PhoSL was investigated with frontal affinity chromatography using 132 pyridylaminated oligosaccharides, it was found that the lectin binds only to core α1–6-fucosylated N-glycans and not to other types of fucosylated oligosaccharides, such as α1–2-, α1–3-, and α1–4-fucosylated glycans. Furthermore, PhoSL bound to α1–6-fucosylated AFP but not to non-fucosylated AFP. In addition, PhoSL was able to demonstrate the differential expression of α1–6 fucosylation between primary and metastatic colon cancer tissues. Thus, PhoSL will be a promising tool for analyzing the biological functions of α1–6 fucosylation and evaluating Fucα1–6 oligosaccharides as cancer biomarkers.