Identification of invariant surface glycoproteins in the bloodstream stage of Trypanosoma brucei.
Surface proteins of the mammalian stage of the parasitic protozoan, Trypanosoma brucei, were biotinylated with sulfosuccinimidyl 6-(biotinamido) hexanoate. Since the predominant protein labeled by this reagent is the membrane form of the variant surface glycoprotein (mfVSG), a procedure was developed to convert mfVSG to its soluble form by the endogenous glycosylphosphatidylinositol-specific phospholipase C while retaining other biotinylated surface proteins in a membrane-bound state. From these membranes, three novel glycoproteins of 60, 65, and 75 kDa could be isolated by a combination of Triton X-114 phase separation and precipitations by streptavidin and concanavalin A coupled to solid supports. These polypeptides were detected in trypanosomes expressing different mfVSGs and are thus considered to be invariant. In a variant clone in which the mfVSG is trypsin-sensitive, the invariant surface glycoproteins of 65 and 75 kDa, designated ISG65 and ISG75, respectively, were proteolytically degraded with similar kinetics as the mfVSG. Neither ISG65 nor ISG75 could be detected in procyclic trypanosomes, the stage of the parasite characteristic for the insect midgut. Gene cloning reported in the accompanying paper (Ziegelbauer, K., Multhaup, G., and Overath, P. (1992) J. Biol. Chem. 267, 10797-10803) suggests that ISG65 and ISG75 are transmembrane proteins.
Ziegelbauer
K
K
Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, Tübingen, Federal Republic of Germany.
Overath
P
P
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D003208:Concanavalin A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000032:analysis*; D011233:Precipitin Tests; D015800:Protozoan Proteins / Q000032:analysis*; D019809:Streptavidin; D013388:Succinimides / Q000737:chemistry; D014346:Trypanosoma brucei brucei / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000032:analysis
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D013388:Succinimides; D014643:Variant Surface Glycoproteins, Trypanosoma; C061443:sulfosuccinimidyl 6-(biotinamido)hexanoate; D003208:Concanavalin A; D001710:Biotin; D019809:Streptavidin; D014357:Trypsin