Bloodstream forms [BF] of pathogenic African trypanosomes maintain a discrete surface membrane domain where receptor-mediated endocytosis, secretion, membrane turnover, and junction formation occur. This domain includes the membranes of the flagellum, the flagellar pocket [FP] and the flagellar adhesion zone [FAZ]. The variant surface glycoprotein [VSG] coat that protects most of the parasite surface is less coherent in the FP-FAZ region. Consequently, targets for immunological intervention in trypanosomiasis may be exposed in the FP-FAZ domain. Hgp-1 is one of several surface glycoproteins that carry N-linked, ricin-binding oligosaccharides and that are localized exclusively in FP-FAZ membranes. The monoclonal antibody CB1 detects glycosylated forms of Hgp-1 in BF of all Trypanosoma brucei subspecies, but no Hgp-1 is detectable in procyclic forms. Hgp-1 preparations purified by CB1 affinity chromotagraphy contain two core proteins, p57 and p42, that are very heavily but heterogeneously N-glycosylated. Because Hgp-1 is invariant, conserved, and developmentally regulated, we propose that it is involved in one of the important functions of the FAZ-FP membrane domain. We propose to study the biosynthesis, intracellular transport, and turnover of Hgp-1 in order to understand how trypanosomes maintain this critical membrane region. First, we will use pulse/chase biosynthesis experiments, limited protease digestion studies, and microsequencing to determine if p42 is derived from p57 by proteolysis or if these two polypeptides are distinct gene products. Next, we will use immunoelectron microscopy to localize Hgp-1, p57, p42, and VSG within cells and to determine if intracellular sorting contributes to establishing the FP-FAZ membrane domain. Third, surface labeling and pulse/chase studies with various inhibitors will indicate if Hgp-1 is processed in unique ways that reflect or potentiate differential transport of Hgp-1 to the FP-FAZ domain or stability of this protein with this domain. Finally, we will clone and sequence p57 and, if necessary, p46 genes from expression libraries. The DNA sequences may indicate function of these proteins.
| Kelley, R J; Brickman, M J; Balber, A E (1995) Processing and transport of a lysosomal membrane glycoprotein is developmentally regulated in African trypanosomes. Mol Biochem Parasitol 74:167-78 |
