Rm. Zancopeoliveira et al., IMMUNOCHEMICAL ANALYSIS OF THE H-GLYCOPROTEIN AND M-GLYCOPROTEIN FROMHISTOPLASMA-CAPSULATUM, Clinical and diagnostic laboratory immunology, 1(5), 1994, pp. 563-568
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.