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

Abstract

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 (ϳ1.4 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 (ϳ50 -70 kDa) significantly higher than predicted from the amino acid sequence (ϳ26 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.

The Ancient Small GTPase Rab21 Functions in Intermediate Endocytic Steps in Trypanosomes

“…For immunofluorescence microscopy, BSF or PCF cells were pelleted by centrifugation at 800 ϫ g for 10 min at 4°C. Cells were gently resuspended and washed once with icecold Voorheis's modified PBS (vPBS) (39). BSF cells were fixed for 10 min and PCF cells for 60 min on ice using 3% paraformaldehyde and vPBS.…”

Secretory Pathway of Trypanosomatid Parasites

“…In this respect, a number of recent studies have shown that trypanosomatid GPI proteins become associated with detergent-resistant membranes (DRMs) during transit to the cell surface (73,238,280). DRMs have been found in all eukaryotes that have been investigated and are thought to correspond to dynamic micro-or macrodomains in intracellular and plasma membranes that are present largely in a liquid-ordered, rather than a liquid-disordered (fluid), state (46,47).…”

“…The GPI anchors and N-linked glycans of VSG are variably modified with glycan side chains that further contribute to the function of the VSG as a macromolecular diffusion barrier (100,210,380). While VSG is evenly distributed over the plasma membrane (cell body, flagellum, and flagellar pocket), other GPI proteins on the surface of T. brucei BF can have a punctate distribution or be entirely restricted to the flagellar pocket (183,238). The surface coat of T. brucei BF also contains a number of type 1 integral membrane proteins (the so-called invariant surface glycoproteins) (378) that likewise may be distributed over the entire cell body or sequestered within the flagellar pocket (Table 1).…”

Procyclin Null Mutants ofTrypanosoma bruceiExpress Free Glycosylphosphatidylinositols on Their Surface

“…In the mammal, the parasite evades the immune system by periodically replacing the existing VSG coat by a different variant, a phenomenon known as antigenic variation (Cross, 1996). The VSG coat forms a physical barrier that prevents the binding of antibodies to invariant molecules that are embedded in the plasma membrane Salmon et al, 1994;Nolan et al, 2000). When bloodstream forms are ingested by the tsetse fly, they differentiate to procyclic forms in the insect midgut.…”