Agaricus bisporus lectin binds mainly O-glycans but also N-glycans of human IgA subclasses

The primary interaction between purified Agaricus bisporus lectin (ABL) and human IgA subclasses was studied by ABL-affinity chromatography, dot blot assay and competitive enzyme-lectin assay, considering that ABL could be an alternative tool for detection of IgA1 O-glycans. Both secretory IgA subclasses bound to ABL-Sepharose and the IgA2 subclass (which contains only N-glycans) was recovered with a high degree of purity when NH4OH was used as eluent. ABL-Ig interaction was also observed by dot blot assays using ABL-peroxidase against monoclonal IgA1 k Pan, IgA2m(1)k Gir, IgA2m(2)k Bel, secretory IgA2 and normal IgG (also contains only N-glycans). When these immunoglobulins were enzymatically treated with peptide N-glycosidase F (N-glycan hydrolysis), the ABL-IgA2 and -IgG interaction did not occur while IgA1 maintained a high degree of interaction with ABL. Also, the ABL-IgA interaction was observed by competitive enzyme-lectin assay, and when IgA1 subclass was treated with endo-α-N-acetylgalactosaminidase for O-glycans hydrolysis, the reactivity with ABL was very low. We conclude that the complementary use of ABL and peptide N-glycosidase F could be a useful tool to assess the O-glycosylation state of human IgA1 subclass, which is of relevant importance in the effector functions of immunoglobulins. Abbreviations: ABL, Agaricus bisporus lectin; α1 and α2, heavy chains from human IgA1 and IgA2; C1-3α, constant domains (1–3) of heavy chains from human IgA; ECL, Erythrina cristagalli lectin; EEO, electroendoosmosis; EIA, enzyme immunoassay; ELA, enzyme lectin assay; Ig, immunoglobulin; HRP, horseradish peroxidase; ID50, 50% inhibitory dose; Ka, affinity constant; O.D., optical density; PBS, phosphate buffered saline; PBS-t, phosphate buffered saline with Tween 20; PNGase F, peptide N-glycosidase F; RID, radial immunodiffusion; SD, standard deviation; TBS, Tris-HCL buffered saline; TBS-t, Tris-HCI buffered saline with Tween 20; T-disaccharide, Thomsen-Friedenreich disaccharide