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 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.
Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA.
R J
RJ
Kelley
D L
DL
Alexander
C
C
Cowan
A E
AE
Balber
J D
JD
Bangs
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D001692:Biological Transport; D002451:Cell Compartmentation; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics* / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D015703:Antigens, CD; D018076:DNA, Complementary; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D021382:Protein Sorting Signals; D015800:Protozoan Proteins