The glycan processing and site occupancy of recombinant Thy-1 is markedly affected by the presence of a glycosylphosphatidylinositol anchor.
Thy-1 is a cell surface glycoprotein containing three N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The effect of the anchor on its N-linked glyco-sylation was investigated by comparing the glycosylation of soluble recombinant Thy-1 (sThy-1) with that of recombinant GPI anchored Thy-1, both expressed in Chinese hamster ovary cells. The sThy-1 was produced in a variety of isoforms including some which lacked carbohydrate on all three sequons whereas the GPI anchored form appeared fully glycosylated like native Thy-1. This was surprising as the attachment of N-linked sugars occurs cotranslationally and it was not expected that the presence of a C-terminal GPI anchor signal sequence would affect sequon occupancy. Furthermore sThy-1 lacking glycosylation could be produced with the inhibitor tunicamycin but in contrast cell surface expression of unglycosylated GPI anchored Thy-1 could not be obtained. The GPI anchored form appeared less processed with almost 4-fold more oligo-mannose oligosaccharides than in sThy-1 and also with less sialylated and core fucosylated biantennary glycans. Possible mechanisms whereby the anchor or the anchor signal sequence, control site occupancy and maturation are discussed.
Devasahayam
M
M
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, England.
Catalino
P D
PD
Rudd
P M
PM
Dwek
R A
RA
Barclay
A N
AN
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D005643:Fucose / Q000032:analysis; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D008358:Mannose / Q000032:analysis; D019158:N-Acetylneuraminic Acid / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D018800:Thy-1 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D017261:Glycosylphosphatidylinositols; D011994:Recombinant Proteins; D018800:Thy-1 Antigens; D014415:Tunicamycin; D005643:Fucose; D019158:N-Acetylneuraminic Acid; D008358:Mannose