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J Biol Chem, Vol. 274, Issue 42, 29763-29771, October 15, 1999

The Procyclin Repertoire of Trypanosoma brucei
IDENTIFICATION AND STRUCTURAL CHARACTERIZATION OF THE GLU-PRO-RICH POLYPEPTIDES

Alvaro Acosta-SerranoDagger , Robert N. ColeDagger , Angela Mehlertparallel , Mary G.-S. Lee**, Michael A. J. Fergusonparallel , and Paul T. EnglundDagger

From the Dagger  Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the parallel  Department of Biochemistry, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and the ** Department of Pathology, New York University School of Medicine, New York, New York 10016

The surface of the insect stages of the protozoan parasite Trypanosoma brucei is covered by abundant glycosyl phosphatidylinositol (GPI)-anchored glycoproteins known as procyclins. One type of procyclin, the EP isoform, is predicted to have 22-30 Glu-Pro (EP) repeats in its C-terminal domain and is encoded by multiple genes. Because of the similarity of the EP isoform sequences and the heterogeneity of their GPI anchors, it has been impossible to separate and characterize these polypeptides by standard protein fractionation techniques. To facilitate their structural and functional characterization, we used a combination of matrix-assisted laser desorption ionization and electrospray mass spectrometry to analyze the entire procyclin repertoire expressed on the trypanosome cell. This analysis, which required removal of the GPI anchors by aqueous hydrofluoric acid treatment and cleavage at aspartate-proline bonds by mild acid hydrolysis, provided precise information about the glycosylation state and the number of Glu-Pro repeats in these proteins. Using this methodology we detected in a T. brucei clone the glycosylated products of the EP3 gene and two different products of the EP1 gene (EP1-1 and EP1-2). Furthermore, only low amounts of the nonglycosylated products of the GPEET and EP2 genes were detected. Because all procyclin genes are transcribed polycistronically, the latter finding indicates that the expression of the GPEET and EP2 genes is post-transcriptionaly regulated. This is the first time that the whole procyclin repertoire from procyclic trypanosomes has been characterized at the protein level.


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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