Elucidation of the mechanism enhancing the avidity of lectin with oligosaccharides on the solid phase surface

Shinohara, Yasuro; Hasegawa, Yukio; Kaku, Hanae; Shibuya, Naoto

doi:10.1093/glycob/7.8.1201

Cited by 71 publications

(45 citation statements)

References 35 publications

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“…The peptides were spotted at a low concentration, such that they still generated a measurable binding signal with the lowest possible surface density to minimize potential MEM-265 rebinding. In this system (data not shown), and in previously reported SPR analyses, high ligand surface density aggravates avidity and analyte-rebinding effects (10). Lower surface density is expected to minimize avidity effects caused by binding of bivalent antibody molecules to two immobilized antigen molecules.…”

supporting

confidence: 54%

Induced Fit of an Epitope Peptide to a Monoclonal Antibody Probed with a Novel Parallel Surface Plasmon Resonance Assay

Baggio¹,

Carven

Chiulli³

et al. 2005

Journal of Biological Chemistry

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Class II major histocompatibility complex proteins bind peptides for presentation to T-cells as part of the immune response process. Monoclonal antibody MEM-265 recognizes the peptide-free conformation of the major histocompatibility complex class II protein HLA-DR1 through specific binding to an epitope contained between residues 50 -67 of the ␤-chain. In previous work using alanine scanning (1), we identified residues Leu-53, Asp-57, Tyr-60, Trp-61, Ser-63, and Leu-67 as essential for specific recognition by MEM-265. The spacing of these residues approximates a 3.5-residue repeat, suggesting that MEM-265 may recognize the epitope in an ␣-helical conformation. In the folded, peptide-loaded DR1 structure, the ␤-chain residues 50 -67 contain a kinked ␣-helical segment spanning Glu-52-Ser-63 (2). However, the conformation of this segment in the peptide-free form is unknown. We have used a new surface plasmon resonance approach in a SpotMatrix format to compare the kinetic rates and affinities for 18 alanine scanning mutants comprising epitope residues 50 -67. In addition to the six essential residues described previously, we found two additional residues, Glu-52 and Gln-64, that contribute by enhancing MEM-265 binding. By contrast, mutation of either Gly-54 or Pro-56 to an alanine actually improved binding to MEM-265. In essentially all cases peptide substitutions that either improve or reduce MEM-265 recognition could be traced to differences in the dissociation rate (k off ). The kinetic details of the present study support the presence of a structural component in the antigenic epitope recognized by MEM-265 in the peptide-free form of major histocompatibility complex II DR1 ␤-chain.Research on factors influencing molecular recognition is an area of vibrant activity. The molecular basis of specificity for antigen-antibody recognition can involve numerous factors and is not limited to the primary amino acid sequence of the antigenic epitope. The epitope can have a complex structural component in the context of the entire tertiary structure of the antigen. In such a case, an analogous linear variant of the original antigen, for instance, a short peptide, would not be expected to retain much structural information, particularly in the absence of any disulfide bridges. However, antibodies that can recognize short peptides that appear to adopt a defined conformation have previously been described (3, 4).MEM-265 is a mouse monoclonal antibody that recognizes the empty conformation of the human class II MHC protein HLA-DR1. Although the antibody was raised against the denatured ␤ subunit, it appears to recognize a conformational epitope present in the native ␣-␤ heterodimer, which becomes unavailable upon peptide binding (1). Preliminary characterization has mapped the epitope to residues 50 -67 of the ␤ subunit, with residues Leu-53, Asp-57, Tyr-60, Trp-61, Ser-63, and Leu-67 being essential for binding. To assess the individual contributions of nonessential residues to binding, we used SpotMatrix SPR 1 to obtain a co...

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supporting

confidence: 54%

Induced Fit of an Epitope Peptide to a Monoclonal Antibody Probed with a Novel Parallel Surface Plasmon Resonance Assay

Baggio¹,

Carven

Chiulli³

et al. 2005

Journal of Biological Chemistry

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“…However, since those oligosaccharides were only retarded by affinity chromatography and did not require hapten injection for elution, our results seemed to magnify the differences in relative sugar binding specificities. This magnification of relative binding strength may be explained by an enhanced subunit multivalence effect whereby immobilization of oligosaccharide and free multivalent lectin made the binding conditions nonhomogeneous (8,30). The large differences in binding specificities we observed parallels the exclusivity of their erythro-(E 4 ) and leuko-(L 4 ) agglutinating activities (31).…”

Section: Discussionmentioning

confidence: 75%

The High Specificities of Phaseolus vulgaris Erythro- and Leukoagglutinating Lectins for Bisecting GlcNAc or β1–6-Linked Branch Structures, Respectively, Are Attributable to Loop B

Kaneda¹,

Whittier²,

Yamanaka³

et al. 2002

Journal of Biological Chemistry

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Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgaris erythroagglutinating and leukoagglutinating lectins (E 4 -and L 4 -PHA) are unique in recognizing larger structures. E 4 -and L 4 -PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a ␤1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E 4 -and L 4 -PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E 4 -and L 4 -PHA toward bisecting GlcNAc and ␤1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E 4 -and L 4 -PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a ␤1,6-linked branch.The legume lectins are a family of sugar-binding proteins found mainly in the seeds of plants belonging to the Leguminosae family (1-3). Lectins from leguminous plants constitute a large family of homologous proteins displaying remarkable divergence in their carbohydrate specificity. Elucidation of the mechanism by which these lectins can possess such a broad range of binding specificities while maintaining a strikingly similar threedimensional monomer structure will be key to understanding the essence of carbohydrate-protein interactions.At present, the crystal structures of ϳ20 legume lectins have been solved.1 These various lectin monomers share a common so-called "jellyroll" structure composed primary of a six-and a seven-stranded antiparallel ␤-sheet. Each monomer binds a manganese and a calcium ion that are both essential for carbohydrate binding. Amino acid sequence comparisons, x-ray crystallographic analysis, and mutagenesis studies revealed that the differences in carbohydrate specificity appear to be due primarily to differences in amino acid residues residing in loops adjacent to the carbohydrate binding site.The primary carbohydrate-binding site of legume lectins is a shallow depression on loops associated with the concave face of the seven-stranded curved ␤-sheet (5). It is constructed mainly by residues from four sequentially se...

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“…LbTec2 binds to Sepharose and is specific for 2-O-Mefucose and 3-O-Me-mannose residues. The affinity of LbTec2 for these monosaccharides is very low (millimolar range), but, according to a commonly accepted concept in the lectin field (Shinohara et al 1997), when they are displayed on a surface, the avidity to these carbohydrates could be very high due to the oligomerization of the protein. Finally, PVL, whose primary sequence is not related to AAL or LbTec2, sharing 8 and 11 % sequence identity (33 % similarity), respectively, folds into a monomeric, seven-blade β-propeller with a total of six binding sites for terminal GlcNAc or Neu5Ac residues located at the interfaces between the blades (Audfray et al 2015;Cioci et al 2006;Ueda et al 2002).…”

Section: β-Propeller-type Lectinsmentioning

confidence: 99%

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

Sabotič

Ohm

Künzler

2015

Appl Microbiol Biotechnol

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Fruiting bodies or sporocarps of dikaryotic (ascomycetous and basidiomycetous) fungi, commonly referred to as mushrooms, are often rich in entomotoxic and nematotoxic proteins that include lectins and protease inhibitors. These protein toxins are thought to act as effectors of an innate defense system of mushrooms against animal predators including fungivorous insects and nematodes. In this review, we summarize current knowledge about the structures, target molecules, and regulation of the biosynthesis of the best characterized representatives of these fungal defense proteins, including galectins, beta-trefoil-type lectins, actinoporin-type lectins, beta-propeller-type lectins and beta-trefoil-type chimerolectins, as well as mycospin and mycocypin families of protease inhibitors. We also present an overview of the phylogenetic distribution of these proteins among a selection of fungal genomes and draw some conclusions about their evolution and physiological function. Finally, we present an outlook for future research directions in this field and their potential applications in medicine and crop protection.

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Elucidation of the mechanism enhancing the avidity of lectin with oligosaccharides on the solid phase surface

Cited by 71 publications

References 35 publications

Induced Fit of an Epitope Peptide to a Monoclonal Antibody Probed with a Novel Parallel Surface Plasmon Resonance Assay

Induced Fit of an Epitope Peptide to a Monoclonal Antibody Probed with a Novel Parallel Surface Plasmon Resonance Assay

The High Specificities of Phaseolus vulgaris Erythro- and Leukoagglutinating Lectins for Bisecting GlcNAc or β1–6-Linked Branch Structures, Respectively, Are Attributable to Loop B

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

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