Identification of membrane-associated proteins in Trypanosoma brucei encoding an internal, EARLRAEE amino acid repeat.

Abstract

Differential screening of expression libraries with nonimmune and day 4-6 immune serum from naive rats infected with the protozoan Trypanosoma lewisi was used to identify potential cell surface protein coding genes. Several T. lewisi cDNAs that resulted were partially characterized and used to clone the homologues from Trypanosoma brucei. The nucleotide sequence of the cDNAs encoding the Tb-29 genes of the parasitic protozoan T. brucei obtained by this method (3557 nucleotides for Tb-291 and 8729 nucleotides for Tb-292) encoded predicted open reading frames of 1070 and 2550 amino acids, respectively. The Tb-29 proteins encoded a large domain with an octapeptide (EARLRAEE) repeat (79 repeats in Tb-291 and 60 in Tb-292), which shared significant similarity with the octapeptide repeat of the S-antigen of isolate NF7 of Plasmodium falciparum. The predicted amino acid sequence of the Tb-292 protein encoded potential transmembrane domains (eight in total). Indirect immunofluorescence using confocal image analysis and immunoelectron microscopy located the EARLRAEE proteins to a membranous network. The Tb-29 proteins were most abundantly distributed to the area surrounding the nucleus, the region between the nucleus and the flagellar pocket, and the region immediately underneath the flagellar pocket membrane. The subcellular distribution of Tb-29 proteins suggests that these proteins may provide a constituent associated with the cell's vesicular transport system.