Isolation and cell-free synthesis of variant surface glycoproteins from Trypanosoma congolense.
Two Trypanosoma congolense stocks, 1/148 FLY and TREU 921, were cloned in A/J strain mice immunosuppressed with cyclophosphamide. The cloned populations, AmNat 1.1 and AmNat 3.1, each characterized by a different variant antigen type, were checked for homogeneity by the indirect fluorescent antibody test using 6-day antisera developed in rabbits. The variant surface glycoproteins (VSGs) from both AmNat clones were purified to homogeneity. Electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gradient gels revealed that the apparent Mr values of the two VSGs were 51 700 (AmNat 1.1) and 49 900 (AmNat 3.1). Monospecific antisera prepared in rabbits to each VSG were used to confirm the homogeneity of the clones by the indirect fluorescent antibody test. The VSGs were susceptible to endoglycosidase H digestion, indicating the presence of high-mannose type oligosaccharides in these glycoproteins. The apparent Mr values of the endoglycosidase H-digested VSGs were 48 800 and 46 900 for AmNat 1.1 and 3.1, respectively. Poly(A+)-enriched RNA isolated from each clone was assayed for template activity using a mRNA-dependent rabbit reticulocyte lysate for in vitro protein synthesis. Radioactively labeled polypeptides were initially characterized by SDS-polyacrylamide gradient gel electrophoresis and visualized by fluorography. VSG-specific translation products were immunoprecipitated with IgGs isolated from the homologous monospecific antisera and analyzed on SDS-polyacrylamide gradient gels. The apparent Mr values for the AmNat 1.1 and 3.1 precursor VSGs synthesized in vitro were 39 000 and 43 000, respectively.
G A
GA
Cook
B M
BM
Honigberg
R A
RA
Zimmermann
D000818:Animals; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D006026:Glycoside Hydrolases / Q000378:metabolism; D008358:Mannose / Q000032:analysis; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger; D014348:Trypanosoma congolense / Q000235:genetics / Q000378:metabolism*; D014643:Variant Surface Glycoproteins, Trypanosoma
D000939:Epitopes; D006023:Glycoproteins; D012333:RNA, Messenger; D014643:Variant Surface Glycoproteins, Trypanosoma; D011061:Poly A; D012313:RNA; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008358:Mannose