Identification and characterization of an acidic major surface glycoprotein from procyclic stage Trypanosoma congolense.
Monoclonal antibodies (mAbs) were derived against the procyclic culture form of Trypanosoma congolense and 14 were selected which bound to the surface of living procyclics in immunofluorescence assays. These antibodies bound to procyclics and epimastigotes of T. congolense (both savannah-type and Kilifi-type) and procyclics of Trypanosoma simiae, but not to procyclics of other species of trypanosomes, to bloodstream forms of several species of trypanosomes or to Leishmania, and were thus life cycle stage- and subgenus-specific. Fluorescence-activated cell sorter analysis with these antibodies showed that the kinetics of expression of the surface antigen during transformation from bloodstream to procyclic forms was similar to that of procyclin or procyclic acidic repetitive protein (PARP) of T. brucei spp. appearing at the cell surface as early as 8 h after initiating transformation. All fourteen antibodies detected broad bands of 40-44 and 28-32 kDa in immunoblot analysis of whole procyclic lysates and were specific for carbohydrate epitopes. The antigen was purified by cation-exchange chromatography and gel electrophoresis, and was shown to be an acidic glycoprotein. Amino acid microanalysis of the purified antigen showed an abundance of glutamic acid/glutamine and alanine. Sequences of peptides produced by cyanogen bromide cleavage matched amino acid sequences predicted by the nucleotide sequence of a gene described in the accompanying paper by Bayne et al. [26]. No sequence similarity to T. brucei procyclin/PARP or to any other protein was found. However, its stage and subgenus specificity, surface disposition, immunodominance, acidity and kinetics of expression during transformation from bloodstream to procyclic forms indicate that the molecule is an analog of procyclin/PARP described in T. brucei spp.
Beecroft
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RP
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Roditi
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Pearson
T W
TW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000032:analysis / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D014348:Trypanosoma congolense / Q000302:isolation & purification / Q000502:physiology*; D014353:Trypanosomiasis, African / Q000097:blood; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; C053277:procyclic acidic repetitive protein, Trypanosoma