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Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma brucei resulted in the isolation of a cDNA ( approximately 1.4 kilobase) which encodes an acidic, alanine-rich polypeptide that is expressed only in bloodstream forms of the parasite and has been termed bloodstream stage alanine-rich protein (BARP). Analysis of the amino acid sequence predicted the presence of a typical NH(2)-terminal leader sequence as well as a COOH-terminal hydrophobic extension with the potential to be replaced by a glycosylphosphatidylinositol anchor. A search of existing protein sequences revealed partial homology between BARP and the major surface antigen of procyclic forms of Trypanosoma congolense. BARP migrated as a complex, heterogeneous series of bands on Western blots with an apparent molecular mass ( approximately 50-70 kDa) significantly higher than predicted from the amino acid sequence ( approximately 26 kDa). Confocal microscopy demonstrated that BARP was present in small discrete spots that were distributed over the entire cellular surface. Detergent extraction experiments revealed that BARP was recovered in the detergent-insoluble, glycolipid-enriched fraction. These data suggested that BARP may be sequestered in lipid rafts.

Type

Journal article

Journal

J Biol Chem

Publication Date

11/02/2000

Volume

275

Pages

4072 - 4080

Keywords

Amino Acid Sequence, Animals, COS Cells, Cloning, Molecular, Escherichia coli, Fluorescent Antibody Technique, Glycolipids, Membrane Proteins, Microscopy, Confocal, Molecular Sequence Data, Polymerase Chain Reaction, Protozoan Proteins, RNA, Messenger, Restriction Mapping, Sequence Homology, Amino Acid, Trypanosoma brucei brucei