Structure of the N-linked oligosaccharide of the main diagnostic antigen of the pathogenic fungus Paracoccidioides brasiliensis

The major diagnostic antigen of Paracoccinioides brasiliensis is the exocellularly secreted 43,000 Da glycoprotein (gp43) which contains a single N-linked oligosaccharide chain, This oligosaccharide, although poorly immunogenic in man, is responsible for the cross-reactivity of the gp43 with sera from patients with histoplasmosis, and may have a role in fungal virulence, It contains a neutral high-mannose core (Man₇GlcNAc₂) to which a (1?6)-linked a-D-Manp chain of variable length, substituted at the 2-O positions by single a-D-Manp residues, is attached, A terminal unit of ß-D-galactofuranose is (1?6)-linked to one of the (1?2)-linked mannosyl residues, either in the C or in the A arm of the oligosaccharide, The heterogeneity of the oligosaccharide is determined by the different sizes of the A arm and the sites of insertion of the ß-galactofuranosyl unit. The complete structure was determined hy methylation analysis, ¹H-NMR, mass spectrometry, acetolysis and mannosidase degradation, Electrospray mass spectrometry showed that the oligosaccharide comprises several subtypes ranging from Hex₁₈GlcNAc₂ to Hex₁₀GlcNAc₂ which accounts for the diffuse migration of the gp43 in polyacrylamide gels, The average size of the most frequent subtype is Hex_13.6GlcNAc₂. Dilute acid treatment to remove ß-D-Galf reduced the molecular masses of the majority of the subtypes by a single sugar unit.