Immunochemical analysis of the H and M glycoproteins from Histoplasma capsulatum.
The H and M antigens of Histoplasma capsulatum are glycoproteins, and both possess epitopes found on the C antigen, a cross-reactive galactomannan shared by the major genera of systemic dimorphic fungi. We modified the H and M glycoproteins by chemical and enzymatic digestion to determine the relative contributions of the carbohydrate and protein moieties to the immunological reactivities and the apparent molecular weights of these antigens. Endoglycosidases with known action patterns were used to determine the nature of the glycopeptide bonds in the H and M antigens. The effects of these treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lectin binding, and enzyme-linked immunoelectrotransfer blots probed with polyclonal and monoclonal antibodies (MAbs). Oxidation with 100 mM periodate destroyed 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-glycoproteins F (PNGase F), produced pronounced shifts in the M(r) but did not completely 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. The susceptibilities of the H and M glycoproteins to endo-N-acetyl-beta-D-glucosaminidases suggest that their glycosidic moieties are N linked.(ABSTRACT TRUNCATED AT 250 WORDS)
Zancopé-Oliveira
R M
RM
Laboratorio de Micologia Médica, Hospital Evandro Chagas, Fiocruz, Rio de Janeiro, Brazil.
Bragg
S L
SL
Reiss
E
E
Peralta
J M
JM
D000918:Antibody Specificity; D000946:Antigens, Fungal / Q000032:analysis* / Q000378:metabolism; D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D006031:Glycosylation; D006658:Histoplasma / Q000737:chemistry* / Q000276:immunology; D015151:Immunoblotting; D037102:Lectins / Q000378:metabolism; D008934:Mitogens; D008970:Molecular Weight; D010504:Periodic Acid
D000946:Antigens, Fungal; D002411:Cation Exchange Resins; D006023:Glycoproteins; D037102:Lectins; D008934:Mitogens; D010504:Periodic Acid; C009288:metaperiodate; D010450:Endopeptidases