Requirement of N-glycosylation for the secretion of recombinant extracellular domain of human Fas in HeLa cells.
Apoptosis has been shown to be associated with altered glycosylation patterns and biosynthesis of glycoproteins. A major cell surface receptor involved in the induction of apoptosis is Fas that is activated by binding Fas ligand but can also be activated by binding anti-Fas antibody. In order to determine whether the Fas receptor is glycosylated, the extracellular domain of human Fas (shFas) was expressed as a cleavable fusion protein (shFas-Fc) in HeLa cells. These cells were shown to express activities of glycosyltransferases involved in N- and O-glycan biosynthesis. The secreted shFas-Fc was shown to be a glycoprotein with heterogeneous glycan chains. MALDI mass spectrometry revealed a disperse molecular weight of shFas with an average of 23.4kDa. Western blots of shFas-Fc secreted from tunicamycin treated transfected HeLa cells showed that only N-glycosylated glycoforms were secreted, while the unglycosylated shFas-Fc remained intracellular. The results suggest that both N-glycosylation sites of the extracellular domain of Fas are occupied with large N-glycans that play a role in the expression of the glycoprotein.
Li
Yi
Y
Department of Medicine, Division of Rheumatology, Human Mobility Research Center and The Arthritis Center, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Yang
Xiaojing
X
Nguyen
Alana H T
AH
Brockhausen
Inka
I
D000595:Amino Acid Sequence; D017209:Apoptosis; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D037102:Lectins / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000032:analysis / Q000096:biosynthesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011961:Receptors, Fc / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019014:fas Receptor / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D037102:Lectins; D011134:Polysaccharides; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D019014:fas Receptor; D006026:Glycoside Hydrolases