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Molecular cloning of p67, a lysosomal membrane glycoprotein from Trypanosoma brucei

We have previously characterized a highly glycosylated membrane protein (p67) in Trypanosoma brucei spp that is apparently targeted to lysosomes in a developmentally regulated manner. Antibody to native p67 identified a partial cDNA clone from a T. b. rhodesiense expression library and RT-PCR was us...

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Bibliographic Details
Published in:Molecular and biochemical parasitology 1999-01, Vol.98 (1), p.17-28
Main Authors: Kelley, Robert J., Alexander, David L., Cowan, Chris, Balber, Andrew E., Bangs, James D.
Format: Article
Language:English
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Summary:We have previously characterized a highly glycosylated membrane protein (p67) in Trypanosoma brucei spp that is apparently targeted to lysosomes in a developmentally regulated manner. Antibody to native p67 identified a partial cDNA clone from a T. b. rhodesiense expression library and RT-PCR was used to complete the sequence of the cDNA. Equal levels of p67 transcript are detected in both procyclic and bloodstream stages of the life cycle. The 2771 nt cDNA contains a 1980 nt orf encoding a 659 amino acid polypeptide (72,567 Da). Hydropathy analysis predicts a Type I membrane topology ( N to C): an N-terminal signal sequence, a large hydrophilic lumenal domain with 14 N-glycosylation sites, a trans-membrane domain (19 aa), and a short (24 aa) cytoplasmic domain. Peptide microsequencing of purified p67 identified nine residues identical to the deduced amino acid sequence, confirming the identity of the cDNA and defining the signal sequence cleavage site. Antibody to p67 protein produced in E. coli recognizes the same spectrum of native p67 glycoforms as the antibody used to clone the cDNA. All features of the deduced amino acid sequence are consistent with the known properties of the native protein and suggest a structure similar to mammalian LAMPS. The cytoplasmic domain contains two putative di-leucine targeting motifs similar to those involved in lysosomal targeting in vertebrate cells. Our results suggest that a single p67 polypeptide, or a group of highly related polypeptides, is synthesized in both bloodstream and procyclic trypanosomes and that subsequent post-translational processing and lysosomal targeting is subject to stage-specific regulation.
ISSN:0166-6851
1872-9428
DOI:10.1016/S0166-6851(98)00155-8