Expression and purification of non-glycosylated Trypanosoma brucei transferrin receptor in insect cells.
The transferrin receptor of the parasite Trypanosoma brucei is a heterodimeric protein complex encoded by the 2 expression site-associated genes (ESAGs) 6 and 7. ESAG6 is a heterogeneously glycosylated protein of 50-60kDa modified by a glycosylphosphatidylinositol anchor at the C-terminus, while ESAG7 is a 40-42kDa glycoprotein carrying an unmodified C-terminus. In order to determine whether glycosylation is necessary for dimer formation and ligand binding, the receptor was expressed in insect cells in the presence of tunicamycin. When insect cells were infected with recombinant ESAG6/ESAG7 double expressor baculovirus and grown in the presence of tunicamycin, non-glycosylated forms of ESAG6 and ESAG7 of 46 and 36kDa, respectively, were synthesized. The non-glycosylated ESAG6 and ESAG7 were capable of forming a heterodimer and of binding transferrin. This results shows that glycosylation is not necessary for synthesis of a functional T. brucei transferrin receptor.
Maier
Alexander
A
Department of Parasitology, Ruprecht-Karls-University, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany.
Steverding
Dietmar
D
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D015151:Immunoblotting; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011990:Receptors, Transferrin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
C090143:ESAG 6 protein, Trypanosoma brucei; D006023:Glycoproteins; D015800:Protozoan Proteins; D011990:Receptors, Transferrin; D014415:Tunicamycin; C078011:ESAG 7 protein, Trypanosoma brucei