Characterization of a Novel, Stage-specific, Invariant Surface Protein in Trypanosoma brucei Containing an Internal, Serine-rich, Repetitive Motif

Abstract

A new surface membrane protein, invariant surface glycoprotein termed ISG 100 , was identified in Trypanosoma brucei, using catalyzed surface, radioiodination of intact cells. This integral membrane glycoprotein was purified by a combination of detergent extraction, lectin-affinity, and ion-exchange chromatography followed by preparative SDS-polyacrylamide gel electrophoresis. The protein was expressed only in bloodstream forms of the parasite, was heavily N-glycosylated, and was present in different clonal variants of the same serodeme as well as in different serodemes. The gene for this protein was isolated by screening a cDNA expression library with antibodies against the purified protein followed by screening of a genomic library. The nucleotide sequence of the gene (4050 base pairs) predicted a highly reiterative polypeptide containing three distinct domains, a unique N-terminal domain of about 10 kDa containing three potential N-glycosylation sites, which was followed by a large internal domain consisting entirely of 72 consecutive copies of a serine-rich, 17-amino acid motif (ϳ113 kDa) and terminated with an apparent transmembrane spanning region of about 3.3 kDa. The internal repeat region of this gene (3672 base pairs) represents the largest reiterative coding sequence to be fully characterized in any species of trypanosome. There was no significant homology with other known proteins, and overall the predicted protein was extremely hydrophobic. Unlike the genes for other surface proteins, the gene encoding ISG 100 was present as a single copy. Although present in the flagellar pocket, ISG 100 was predominantly associated with components of the pathways for endo/exocytosis, such as intracellular vesicles located in the proximity of the pocket as well a large, electron-lucent perinuclear digestive vacuole.The African trypanosomes are a group of unicellular eukaryotes responsible for sleeping sickness in man and related diseases in animals. The life cycle of these parasites, which alternates between the mammalian host and the tsetse fly vector, is characterized by the stage-specific expression of two major surface glycoproteins, namely the variant surface glycoprotein (VSG) 1 and procyclin during the bloodstream and insect mid-gut stages, respectively. These two proteins cover the entire surface of the trypanosome and are by far the most abundant of the surface proteins during these stages of the life cycle. Although there is a considerable body of data in the literature concerning the structure, function, and differential expression of VSG and procyclin (1-3), only relatively recently (for review, see Overath et al. (4) has attention focused on other surface proteins located either beneath the surface coats of VSG/procyclin or within the the flagellar pocket, a specialized invagination of the plasma membrane, which is involved in the pathways for endo-and exocytosis in these cells. There are several reasons why interest in these proteins is increasing. First, some of these proteins must be involved in ...

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

“…After these additions the sample was incubated at 75°C for 3 min, allowed to cool, and then 1/5 of the volume (of the sample) of 6 ϫ deglycosylation buffer (0.3 M Na 2 HPO 4 , pH 7.6, 60 mM EDTA, N-octyl glucoside (6%, w/v), and ␤-mercaptoethanol (6%, v/v))was added plus N-glycosidase F (5 units/ ml, final) and incubated for 20 h at 37°C. These conditions have been shown to result in the complete removal of N-linked carbohydrate from several surface proteins from T. brucei (21,22).…”

“…The column was washed with 5 volumes of buffer before the application of a pH gradient (pH 6.5-4.0). Fractions were collected and samples analyzed for their content of radioactivity as well as for the presence of BARP by ELISA as described previously for ISG 100 (22). Fractions containing BARP, as judged by ELISA, or the peak of the 125 I label were subjected to Western blot analysis using antibodies against VSG or BARP, and those fractions containing the heterogeneous material corresponding to BARP were pooled.…”

“…These pooled fractions were concentrated, dialyzed, and the imidazole buffer replaced with Tes buffer by ultrafiltration using Centricon 10 units (Amicon). Samples of these pooled fractions were treated with N-glycosidase F essentially as described previously (21,22). Briefly, SDS and ␤-mercaptoethanol were added to the to give a final concentration of 0.05% (w/v) and 19 mM, respectively.…”

“…Gels, Immunoblotting and ELISA-SDS-PAGE, autoradiography, and Western blots were performed as described previously (21,22).…”

Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei

“…The few surviving cells failed to divide unless tetracycline was reintroduced at day 10 ( Fig. 5C) or until day [16][17][18][19][20], when the cultures spontaneously started to grow once more at normal rates (Fig. 5B).…”