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 mortality in patients with impaired CD4(+) T cell function, particularly those with AIDS. To identify cryptococcal antigens that could serve as vaccine candidates by stimulating T cell responses, C. neoformans-reactive CD4(+) T cell hybridomas were generated by immunization of C57BL/6 mice and fusion of splenocytes with thymoma cells. The antigen that stimulated one of the hybridomas, designated P1D6, to produce IL-2 was purified to homogeneity by sequential anion exchange chromatography, hydrophobic interaction chromatography, and SDS/PAGE. Based on its apparent molecular mass of 98 kDa and mannosylation, the antigen of interest was named MP98. MP98 was N terminal-sequenced, 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 cleavable signal sequence, a polysaccharide deacetylase domain found in fungal chitin deacetylases, and a serine/threonine-rich C-terminal region. Overall, there were 103 serine/threonine residues serving as potential O-linked glycosylation sites as well as 12 possible N-linked glycosylation sites. Thus, a C. neoformans mannoprotein has been characterized that stimulates T cell responses and has molecular properties of a chitin deacetylase.
Levitz
S M
SM
Evans Memorial Department of Clinical Research and Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA. slevitz@bu.edu
Nong
S
S
Mansour
M K
MK
Huang
C
C
Specht
C A
CA
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000946:Antigens, Fungal / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003453:Cryptococcosis / Q000276:immunology; D003455:Cryptococcus neoformans / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D017931:DNA Primers / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D005657:Fungal Vaccines / Q000302:isolation & purification; D006825:Hybridomas / Q000276:immunology; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000276:immunology*
D000946:Antigens, Fungal; D017931:DNA Primers; D005656:Fungal Proteins; D005657:Fungal Vaccines; D008562:Membrane Glycoproteins; C025363:mannoproteins; D000581:Amidohydrolases; C019866:chitin deacetylase