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

doi:10.1074/jbc.272.46.29212

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

From the Department of Molecular Biology, University of Brussels, 67 rue des Chevaux, B-1640 Rhode St. Genèse, Belgium, the^§Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK, the ^¶Department of Biochemistry, Trinity College, Dublin 2, Ireland, and the ^‖Laboratoire de Chimie Biologique, Universite de Mons-Hainaut, B-7000 Mons, Belgium

Abstract

A new surface membrane protein, invariant surface glycoprotein termed ISG₁₀₀, was identified inTrypanosoma 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 heavilyN-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 (∼113 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 ISG₁₀₀ was present as a single copy. Although present in the flagellar pocket, ISG₁₀₀ 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.

Footnotes

↵* This work was supported by research contracts with the Communauté Française de Belgique, the Interuniversity Poles of Attraction Programme of the Belgian State Prime Minister’s Office—the Federal Office for Scientific, Technical and Cultural Affairs and the European Commission Biotech Programme (DG XII).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) Y14833.
↵‡ Supported by a fellowship from the European Commission (DGXII) Biotech Program. To whom correspondence should be addressed. Tel.: 32-2-6509627; Fax: 32-2-6509625; E-mail: dnolan{at}dbm.ulb.ac.be.
↵1 The abbreviations used are: VSG, variant surface glycoprotein; ISG, invariant surface glycoprotein; PMSF, phenylmethylesulfonyl flouride; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunoadsorbent assay; Tes, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; bp, base pair(s); kb, kilobase pair(s).
- Received August 6, 1997.