Secretory expression of a cell surface protein phospholipase B1 with altered N-glycan structure in the Human Pathogen Cryptococcus neoformans

다국어 초록 (Multilingual Abstract)

Cryptococcus neoformans is an encapsulated basidiomycete causing severe diseases, mostly meningoencephalitis, in immunocompromised individuals such as AIDS patients. Secreted phospholipase B1 (PLB1) is important for infection of C. neoformans to the host lung. PLB1 are conserved in all serotypes of C. neoformans, with an overall sequence homology of more than 90%. Previous studies have shown that PLB1 is highly modified by N-linked glycosylation which is essential for the three PLB1 enzymatic activities and secretion. Noticeably, the PLB1 proteins for all cryptococcal serotypes are reported to have different molecular mass, reflecting serotype-specific N-glycans assembled on the secreted PLB1 proteins. As an effort to obtain the purified PLB1 proteins with altered N-glycan structure, which would be used to analyze the effect of N-linked glycans on its role as a virulence determinant we constructed His-tagged and glycosylphosphatidylinositol (GPI) anchorless PLB1 that can be secreted due to lack of membrane association. The GPI anchorless PLB1 protein was expressed in C. neoformans H99 wild type (serotypes A), and glycosylation mutant strains such as Cnoch1Δ, and Cnmnn2Δ, which have defects in the outer chine biosynthesis of N-linked glycans. Compared to the size of the wild type PLB1, those of secreted PLB1 in the mutant strains appeared to be reduced but converged to the same molecular mass after N-glycosidase treatment, indicating the size difference is attributed to the altered structure of N- glycan outer chines. Moreover, the results indicate that the outer chain structures of N-glycans do not crucially affect the secretion efficiency of PLB1.