IMMUNOCHEMICAL ANALYSIS OF THE H-GLYCOPROTEIN AND M-GLYCOPROTEIN FROMHISTOPLASMA-CAPSULATUM

The H and M antigens of Histoplasma capsulatum are glycoproteins, and both possess epitopes found on the C antigen, a cross-reactive galacto mannan shared by the major genera of systemic dimorphic fungi. We modi fied the H and M glycoproteins by chemical and enzymatic digestion to determine the relative contributions of the carbohydrate and protein m oieties to the immunological reactivities and the apparent molecular w eights of these antigens. Endoglycosidases with known action patterns were used to determine the nature of the glycopeptide bonds in the H a nd M antigens. The effects of these treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lectin binding, a nd enzyme-linked immunoelectrotransfer blots probed with polyclonal an d monoclonal antibodies (MAbs). Oxidation with 100 mM periodate destro yed the common fungal epitope recognized by MAb CA1-CB4 and nearly all of the concanavalin A-binding sites on both the H and M antigens; it also caused the molecular mass of the M antigen to shift from 94 to 88 kDa. Treatment of samples with O-glycanase had little, if any, effect on the H and M glycoproteins. On the other hand, treatments with endo -beta-N-acetylglucosaminidase H, and particularly peptide N-glycosidas e F (PNGase F), produced pronounced shifts in the M(r) but did not com pletely eliminate concanavalin A- or MAb CA1-CB4-binding sites. PNGase F treatment caused the molecular mass of the H antigen to shift from 116 to 94 kDa and that of the M antigen to shift from 94 to 74 kDa. Th e susceptibilities of the H and M glycoproteins to endo-N-acetyl-beta- D-glucosaminidases suggest that their glycosidic moieties are N linked . The glycosidic moieties are cross-reactive in enzyme-linked immunoel ectrotransfer blots, so their presence is an impediment to achieving d iagnostic specificity. Periodate oxidation appears to remove cross-rea ctive carbohydrate moieties while retaining protein integrity and anti genicity.