Cloning of a novel surface antigen from the insect stages of Trypanosoma brucei by expression in COS cells.
Trypanosoma brucei cDNA libraries constructed in the vector pCDM8 were screened selectively for insect (procyclic) stage surface antigen cDNAs by transient expression in mammalian COS-7 fibroblasts and "panning" with a rabbit polyclonal antiserum. This strategy yielded two surface antigen cDNAs termed PSSA-1 and PSSA-2. The PSSA-1 cDNA encoded an isotype of procyclin, the major phosphatidylinositol-linked stage-specific glycoprotein antigen of the tsetse fly infective forms of T. brucei. The PSSA-2 cDNA encoded a new and previously unidentified stage-specific surface antigen with the features of a typical transmembrane glycoprotein but with an unusual cytoplasmic tail composed of a proline-rich tandem repeat. Fluorescent antibody staining of PSSA-1 transfected COS cells with a panel of procyclin-specific monoclonal antibodies confirmed that the protein was located on the outer surface of the plasma membrane. Furthermore, the antigen on COS cells was insensitive to treatment with phosphatidylinositol-specific phospholipase C suggesting that the inositol of the glycosylinositol phospholipid-lipid anchor contained the same fatty acyl modification reported recently for the procyclin molecule in cultured procyclic trypanosomes. In contrast the PSSA-2 antigen on COS cells was stained very weakly by whole parasite antisera. Northern blot hybridization revealed that the PSSA-2 antigen was encoded by a single 1.7-kilobase transcript which was present in parasites from the insect procyclic stage of the life cycle but not from the animal bloodstream stage. Southern blot hybridization analysis of DNA from procyclic stage trypanosomes indicated that the gene for PSSA-2 may be present in more than one copy in procyclic trypanosomes.
Jackson
D G
DG
Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, England.
Smith
D K
DK
Luo
C
C
Elliott
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan / Q000235:genetics*; D000954:Antigens, Surface / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D005455:Fluorescent Antibody Technique; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D015800:Protozoan Proteins; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000276:immunology*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; C053277:procyclic acidic repetitive protein, Trypanosoma; D004247:DNA