Characterization of a Novel Alanine-rich Protein Located in Surface Microdomains in Trypanosoma brucei

• Published in: The Journal of biological chemistry Vol. 275; no. 6; pp. 4072 - 4080
• Main Authors: Nolan, Derek P., Jackson, David G., Biggs, Mary J., Brabazon, Elaine D., Pays, Annette, Van Laethem, François, Paturiaux-Hanocq, Françoise, Elliot, John F., Voorheis, H.Paul, Pays, Etienne
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.02.2000; American Society for Biochemistry and Molecular Biology
• Subjects: alanine; Alanine-rich protein; Amino Acid Sequence; amino acid sequences; Animals; bloodstream forms; bloodstream stage alanine rich protein; Cloning, Molecular; complementary DNA; COS Cells; cytochemistry; Escherichia coli; Fluorescent Antibody Technique; genbank/y17281; gene expression; Glycolipids - chemistry; Membrane Proteins - chemistry; messenger RNA; Microscopy, Confocal; Molecular Sequence Data; nucleotide sequences; Polymerase Chain Reaction; polypeptides; proteins; Protozoan Proteins - chemistry; Restriction Mapping; RNA, Messenger - metabolism; Sequence Homology, Amino Acid; transcription (genetics); Trypanosoma brucei; Trypanosoma brucei brucei - chemistry; Trypanosoma congolense
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.6.4072

Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma bruceiresulted in the isolation of a cDNA (∼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 NH2-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 (∼50–70 kDa) significantly higher than predicted from the amino acid sequence (∼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.