Purification and biochemical characterization of a 65-kilodalton mannoprotein (MP65), a main target of anti-Candida cell-mediated immune responses in humans.
A 65 kDa-constituent (MP65) of a whole-cell mannoprotein (MP) fraction of Candida albicans was purified by immunoaffinity chromatography with monoclonal antibodies directed against periodate-insensitive, protease-sensitive MP epitopes, putatively polypeptide in nature. These antibodies were obtained by immunization of mice with concanavalin A bead-coupled, low-glycosylated MP from hyphal cells of C. albicans grown in the presence of a subinhibitory dose of tunicamycin. The immunoaffinity-purified MP65 molecule had a pI of 4.1 and a protein/polysaccharide ratio of 1.8:1. It was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, or N-glycoffanases but still reactive with concanavalin A. The polysaccharide moiety of MP65 was composed exclusively of mannose and glucose at a ratio of 12.7 to 1. The protein moiety showed numerous potential O-glycosidic linkage sites as suggested by the high proportion of serine and threonine (together accounting for more than 20% of the total amino acid composition) and susceptibility to diluted alkali. This treatment and digestion with alpha-mannosidase caused a reduction in the MP65 molecular mass to around 54 kDa. The N-terminal sequence of MP65 protein moiety was rich in alanine and valine (7 of 13 amino acids) and did not show any significant homology with deposited sequences in data banks. Purified MP65, at doses of a few nanograms, induced extensive T-cell proliferation of human peripheral blood mononuclear cells. This proliferation was specifically inhibited, in a dose-response fashion, by the antigen-binding fragment of the monoclonal antibody used for immunoaffinity purification. Overall, these results highlight biochemical and molecular details of MP65, a main target of human T-cell response to C.albicans.
Gomez
M J
MJ
Department of Bacteriology, Istituto Superiore di Sanità, Rome, Italy.
Torosantucci
A
A
Arancia
S
S
Maras
B
B
Parisi
L
L
Cassone
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D000946:Antigens, Fungal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001665:Binding Sites; D002176:Candida albicans / Q000235:genetics / Q000276:immunology*; D002846:Chromatography, Affinity; D005656:Fungal Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D006801:Humans; D007111:Immunity, Cellular; D007120:Immunochemistry; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013601:T-Lymphocytes / Q000276:immunology
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000946:Antigens, Fungal; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; C025363:mannoproteins