Purification and characterization of a second immunoreactive mannoprotein from Cryptococcus neoformans that stimulates T-Cell responses.
Although T-cell responses are known to be critical for effective host defenses against the fungal pathogen Cryptococcus neoformans, the antigens that stimulate protective responses are poorly characterized but are thought to be comprised, at least in part, of mannoproteins. Recently, we created a panel of murine CD4(+)-T-cell hybridomas that react with C. neoformans antigens. A mannoprotein antigen, MP98, that stimulated one of the hybridomas was purified, and the gene encoding MP98 was cloned. In the present study, the cryptococcal antigen, MP88, that stimulated a second T-cell hybridoma, X5A3, to secrete interleukin-2 was characterized. MP88 was purified from supernatants of glass bead-disrupted C. neoformans by anion-exchange and hydrophobic interaction chromatography. A single band with an apparent molecular mass of 88 kDa was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to partial internal amino acid sequencing. The gene encoding MP88 was cloned and sequenced. MP88 features a C-terminal serine/threonine-rich region, which presumably serves as a site for extensive O glycosylation, followed by a putative glycosylphosphatidylinositol anchor site. A search of C. neoformans genomic databases revealed that MP88 shares this feature with at least 11 other genes, including MP98. The mannoprotein nature of MP88 was established based upon the capacity of (i) the mannoprotein fraction of C. neoformans supernatants to stimulate X5A3 and (ii) mannosylated ligands to competitively inhibit this stimulation. Thus, a second cryptococcal mannoprotein has been identified which stimulates T-cell responses and is a vaccine candidate.
Huang
Chao
C
Evans Memorial Department of Clinical Research, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Nong
Shu-Hua
SH
Mansour
Michael K
MK
Specht
Charles A
CA
Levitz
Stuart M
SM
D000595:Amino Acid Sequence; D000818:Animals; D000946:Antigens, Fungal / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D003001:Cloning, Molecular; D003453:Cryptococcosis / Q000276:immunology / Q000517:prevention & control; D003455:Cryptococcus neoformans / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D005657:Fungal Vaccines / Q000302:isolation & purification / Q000494:pharmacology; D015870:Gene Expression; D005800:Genes, Fungal; D006825:Hybridomas / Q000276:immunology; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data
D000946:Antigens, Fungal; D004271:DNA, Fungal; D005656:Fungal Proteins; D005657:Fungal Vaccines; D008562:Membrane Glycoproteins; C025363:mannoproteins