The PSA-2 glycoprotein complex of Leishmania major is a glycosylphosphatidylinositol-linked promastigote surface antigen.
Polyclonal rabbit antiserum to the Triton X-114 phase material of Leishmania major, which comprises the surface and internal integral membrane proteins of the parasite, was used to screen a lambda gt11 genomic expression library. A recombinant clone producing a Mr 123,000 beta-galactosidase fusion protein was isolated. Antibodies affinity-purified on this fusion protein recognized a complex of three surface-oriented proteins of promastigotes of L. major of Mr 94,000, 90,000, and 80,000 that we have termed the promastigote surface Ag 2 (PSA-2) complex. The DNA sequence of the insert in this clone predicted the 3' end of an open reading frame encoding a hydrophobic C-terminus. The inferred C-terminal sequence was suggestive of a glycosylphosphatidyl-inositol membrane anchoring mechanism. Phosphatidylinositol-specific phospholipase C treatment of the native PSA-2 proteins caused a shift in their electrophoretic mobility with an apparent reduction in the molecular weight of the PSA-2 complex. After phospholipase C treatment these proteins also displayed the cryptic cross-reacting determinant recognized by antibodies to the Trypanosoma brucei variant surface Ag. Moreover, PSA-2, which previously partitioned in the detergent phase after Triton X-114 phase separation, became water-soluble after phospholipase C treatment. Immunoprecipitation of the PSA-2 proteins with sera directed to lectin-binding proteins indicated that these polypeptides may be differentially glycosylated. Finally, these PSA-2 proteins were recognized by sera from some patients with cutaneous leishmaniasis.
Murray
P J
PJ
Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.
Spithill
T W
TW
Handman
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000032:analysis; D000953:Antigens, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D000954:Antigens, Surface / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000302:isolation & purification; D006017:Glycolipids / Q000378:metabolism*; D007106:Immune Sera / Q000032:analysis; D007895:Leishmania tropica / Q000235:genetics / Q000276:immunology*; D007896:Leishmaniasis / Q000276:immunology; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D004274:DNA, Recombinant; D006017:Glycolipids; D007106:Immune Sera; D010716:Phosphatidylinositols; D015800:Protozoan Proteins