Molecular characterization of a mannoprotein with homology to chitin deacetylases that stimulates T cell responses to Cryptococcus neoformans

The fungus Cryptococcus neoformans is a major cause of morbidity and mortal ity in patients with impaired CD4(+) T cell function, particularly those wi th AIDS. To identify cryptococcal antigens that could serve as vaccine cand idates by stimulating T cell responses, C. neoformans-reactive CD4(+) T cel l hybridomas were generated by immunization of C57BL/6 mice and fusion of s plenocytes with thymoma cells. The antigen that stimulated one of the hybri domas, designated P1D6, to produce IL-2 was purified to homogeneity by sequ ential anion exchange chromatography, hydrophobic interaction chromatograph y, and SDS/PAGE. Based on its apparent molecular mass of 98 kDa and mannosy lation, the antigen of interest was named MP98. MP98 was N terminal-sequenc ed, and the gene encoding the protein was cloned and sequenced. Recombinant MP98, expressed in Saccharomyces cerevisiae, stimulated P1D6 to produce IL -2. Analysis of the derived 458-aa sequence of MP98 reveals an N-terminal c leavable signal sequence, a polysaccharide deacetylase domain found in fung al chitin deacetylases, and a serine/threonine-rich C-terminal region. Over all, there were 103 serine/threonine residues serving as potential O-linked glycosylation sites as well as 12 possible N-linked glycosylation sites. T hus, a C neoformans mannoprotein has been characterized that stimulates T c ell responses and has molecular properties of a chitin deacetylase.