Abstract
The Chinese hamster ovary mutant MI8-5 is known to synthesize Man(9)GlcNAc(2)-P-P-dolichol rather than the fully glucosylated lipid intermediate Glc(3)Man(9)GlcNAc(2)-P-P-dolichol. This nonglucosylated oligosaccharide lipid precursor is used as donor for N-glycosylation. In this paper we demonstrate that a significant part of the glycans bound to the newly synthesized glycoproteins in MI8-5 cells are monoglucosylated. The presence of monoglucosylated glycans on glycoproteins determines their binding to calnexin as part of the quality control machinery. Furthermore, we point out the presence of Glc(1)Man(5)GlcNAc(1) in the cytosol of MI8-5 cells. This indicates that part of the monoglucosylated glycoproteins can be directed toward a deglycosylation process that occurs in the cytosol. Besides studies on glycoprotein degradation based on the disappearance of protein moieties, MI8-5 cells can be used as a tool to elucidate the various step leading to glycoprotein degradation by studying the fate of the glycan moieties.
Highlights
A key reaction of N-glycosylation is the transfer en bloc of a Glc3Man9GlcNAc2 oligosaccharide from a lipid intermediate to an Asn residue in the Asn-Xaa-Ser(Thr) consensus sequence of a nascent protein
The oligosaccharide bound to lipid intermediates in K1–2 and MI8–5 cells was different (Glc3Man9GlcNAc2-PP-Dol for parental cells and Man9GlcNAc2-PP-Dol for mutant cells), the oligomannoside species bound to proteins seems to be similar
The lack of dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyl transferase activity in MI8 –5 cells leads to the synthesis of nonglucosylated Man9GlcNAc2-P-P-dolichol, which is transferred to protein [15]
Summary
A key reaction of N-glycosylation is the transfer en bloc of a Glc3Man9GlcNAc2 oligosaccharide from a lipid intermediate to an Asn residue in the Asn-Xaa-Ser(Thr) consensus sequence of a nascent protein This N-glycosylation process is immediately followed by sequential deglucosylation leading to Man9GlcNAc2 protein. It is worth mentioning that few labs follow the fate of newly synthesized proteins by labeling with [2-3H]mannose In this case, the N-glycosylation process is accompanied by the release of glycans from either lipid intermediates or from newly synthesized glycoproteins (9 –11). Our work demonstrates that the quality control system for newly synthesized glycoproteins is intact in MI8 –5 cells This cell line gives us a biological model to determine the nature of oligosaccharides released from protein independently of what is happening at the reducing end
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