Calcium-dependent conformation of a mouse macrophage calcium-type lectin. Carbohydrate binding activity is stabilized by an antibody specific for a calcium-dependent epitope.
We established monoclonal antibodies (mAbs) against the mouse macrophage galactose/N-acetylgalactosamine-specific lectin (MMGL) that is a 42-kDa calcium-dependent lectin, using a solid phase carbohydrate binding assay as a novel strategy for screening mAbs. The specificity of six mAbs were investigated by antibody binding to native or recombinant forms (rML) of MMGL, flow cytometry, and immunoprecipitation using a macrophage cell line RAW264.7. Four of these mAbs strongly inhibited the binding of fluorescein 5-isothiocyanate-labeled galactosylated polylysine to immobilized rML, one inhibited moderately, and one did not inhibit binding. The competitive binding study revealed that the binding sites of these four blocking mAbs were closely related to each other but were different from the rest of these mAbs. A non-blocking mAb having a unique binding specificity (LOM-11) exhibited calcium-dependent binding to rML, suggesting that calcium-dependent epitope was not situated in the vicinity of the ligand binding site. Furthermore, pretreatment of rML with the mAb LOM-11 preserved ligand binding activity, especially in a low calcium environment. The four blocking mAbs mentioned above facilitated the binding of the mAb LOM-11 to rML. These results indicate that there is a positive cooperativity between the lectin's ligand binding site and its physically distinct calcium-dependent epitope.
Kimura
T
T
Department of Cancer Biology and Molecular Immunology, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Imai
Y
Y
Irimura
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D001212:Asialoglycoproteins; D002118:Calcium / Q000494:pharmacology*; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005434:Flow Cytometry; D037102:Lectins / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D037181:Lectins, C-Type; D008264:Macrophages / Q000737:chemistry*; D008565:Membrane Proteins; D051379:Mice; D011233:Precipitin Tests; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D001212:Asialoglycoproteins; D002352:Carrier Proteins; C064720:Clec10a protein, mouse; D000939:Epitopes; D037102:Lectins; D037181:Lectins, C-Type; D008565:Membrane Proteins; D002118:Calcium