A 34- to 38-kilodalton Cryptococcus neoformans glycoprotein produced as an exoantigen bearing a glycosylated species-specific epitope.
Three monoclonal antibodies (MAbs), all of the immunoglobulin G1 subclass, were raised against Cryptococcus neoformans by using the technique of cyclophosphamide ablation of B-cell responses against shared epitopes of the cross-reactive fungus Trichosporon beigelii. MAb 3C2 was reactive against the encapsulated and nonencapsulated isolates of C. neoformans var. neoformans by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot), and in addition to a 34- to 38-kDa determinant, it recognized a series of lower-molecular-weight species. 3C2 also reacted strongly with culture supernatant preparations of C. neoformans var. neoformans by ELISA. 3C2 showed no recognition of either T. beigelii or C. neoformans var. gattii antigens. Enzymatic deglycosylation followed by reaction with 3C2 on Western blots revealed that sialic acid was an integral part of the determinant, together with N-acetylglucosaminyl-asparagine and alpha-mannose. Proteolytic digestion showed that the epitope was pepsin sensitive and that it also contained tryptophan and glycine and/or leucine as determinants of recognition by 3C2. The pI of the glycoprotein was 7.1. Affinity chromatography-purified antigen did not exhibit proteolytic activity on sodium dodecyl sulfate-polyacrylamide substrate gels. Indirect fluorescence antibody tests revealed that 3C2 labelling was confined to the cell membrane and cytoplasm of yeasts. The remaining MAbs, 7H4 and 5G5, recognized both capsulated and nonencapsulated strains of C. neoformans var. neoformans by both ELISA and Western Blot, identifying linear determinants with molecular masses of 36 and 30 kDa. They were unreactive against culture supernatant antigen (exoantigen) from either variant of C. neoformans.
Dermatology Unit, Guy's Hospital, London Bridge, United Kingdom.
A J
AJ
Hamilton
L
L
Jeavons
P
P
Hobby
R J
RJ
Hay
D000818:Animals; D000911:Antibodies, Monoclonal; D000946:Antigens, Fungal / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003429:Cross Reactions; D003453:Cryptococcosis / Q000175:diagnosis; D003455:Cryptococcus neoformans / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry*; D005260:Female; D006023:Glycoproteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D007525:Isoelectric Focusing; D051379:Mice; D008807:Mice, Inbred BALB C; D010447:Peptide Hydrolases / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000946:Antigens, Fungal; D000939:Epitopes; D006023:Glycoproteins; D010447:Peptide Hydrolases