CHARACTERIZATION OF A NOVEL, STAGE-SPECIFIC, INVARIANT SURFACE PROTEIN IN TRYPANOSOMA-BRUCEI CONTAINING AN INTERNAL, SERINE-RICH, REPETITIVE MOTIF

A new surface membrane protein, invariant surface glycoprotein termed ISG(100), was identified in Trypanosoma brucei, using catalyzed surfac e, radioiodination of intact cells, This integral membrane glycoprotei n was purified by a combination of detergent extraction, lectin-affini ty, and ion exchange chromatography followed by preparative SDS-polyac rylamide gel electrophoresis. The protein was expressed only in bloods tream forms of the parasite, was heavily N-glycosylated, and was prese nt in different clonal variants of the same serodeme as well as in dif ferent serodemes. The gene for this protein was isolated by screening a cDNA expression library with antibodies against the purified protein followed by screening of a genomic library. The nucleotide sequence o f the gene (4050 base pairs) predicted a highly reiterative polypeptid e containing three distinct domains, a unique N-terminal domain of abo ut 10 kDa containing three potential N-glycosylation sites, which was followed by a large internal domain consisting entirely of 72 consecut ive copies of a serine-rich, 17-amino acid motif (similar to 113 kDa) and terminated with an apparent transmembrane spanning region of about 3.3 kDa, The internal repeat region of this gene (3672 base pairs) re presents the largest reiterative coding sequence to be fully character ized in any species of trypanosome. There was no significant homology with other known proteins, and overall the predicted protein was extre mely hydrophobic, Unlike the genes for other surface proteins, the gen e encoding ISG(100) was present as a single copy. Although present in the flagellar pocket, ISG(100) was predominantly associated with compo nents of the pathways for endo/exocytosis, such as intracellular vesic les located in the proximity of the pocket as web a large, electron-lu cent perinuclear digestive vacuole.