Abstract
BackgroundBiosynthesis of the dolichol linked oligosaccharide (DLO) required for protein N-glycosylation starts on the cytoplasmic face of the ER to give Man5GlcNAc2-PP-dolichol, which then flips into the ER for further glycosylation yielding mature DLO (Glc3Man9GlcNAc2-PP-dolichol). After transfer of Glc3Man9GlcNAc2 onto protein, dolichol-PP is recycled to dolichol-P and reused for DLO biosynthesis. Because de novo dolichol synthesis is slow, dolichol recycling is rate limiting for protein glycosylation. Immature DLO intermediates may also be recycled by pyrophosphatase-mediated cleavage to yield dolichol-P and phosphorylated oligosaccharides (fOSGN2-P). Here, we examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I) in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation.Methods and Principal FindingsIn EBV-transformed lymphoblastoid cells from CDG I patients and normal subjects a correlation exists between the quantities of metabolically radiolabeled fOSGN2-P and truncated DLO intermediates only when these two classes of compounds possess 7 or less hexose residues. Larger fOSGN2-P were difficult to detect despite an abundance of more fully mannosylated and glucosylated DLO. When CDG Ig cells, which accumulate Man7GlcNAc2-PP-dolichol, are permeabilised so that vesicular transport and protein synthesis are abolished, the DLO pool required for Man7GlcNAc2-P generation could be depleted by adding exogenous glycosylation acceptor peptide. Under conditions where a glycotripeptide and neutral free oligosaccharides remain predominantly in the lumen of the ER, Man7GlcNAc2-P appears in the cytosol without detectable generation of ER luminal Man7GlcNAc2-P.Conclusions and SignificanceThe DLO pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that pyrophosphatase-mediated cleavage of DLO intermediates yields recyclable dolichol-P. The kinetics of cytosolic fOSGN2-P generation from a luminally-generated DLO intermediate demonstrate the presence of a previously undetected ER-to-cytosol translocation process for either fOSGN2-P or DLO.
Highlights
The majority of secretory and cell surface glycoproteins are Nglycosylated by the co, or post-translational addition of the oligosaccharide, Glc3Man9GlcNAc2, that is transferred from the mature dolichol-linked oligosaccharide (DLO), Glc3Man9Glc-NAc2-PP-dolichol, onto nascent polypeptides in the lumen of the endoplasmic reticulum (ER) by oligosaccharyltransferase (OST, see Fig. 1)
The dolichol linked oligosaccharide (DLO) pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that pyrophosphatase-mediated cleavage of DLO intermediates yields recyclable dolichol-P
The release of fOSGN2 [13] and fOSGN2-P [36] from DLO during glycoprotein biosynthesis is thought to occur as a consequence of mechanisms that regulate DLO availability for protein glycosylation [21]
Summary
The majority of secretory and cell surface glycoproteins are Nglycosylated by the co-, or post-translational addition of the oligosaccharide, Glc3Man9GlcNAc2, that is transferred from the mature dolichol-linked oligosaccharide (DLO), Glc3Man9Glc-NAc2-PP-dolichol, onto nascent polypeptides in the lumen of the endoplasmic reticulum (ER) by oligosaccharyltransferase (OST, see Fig. 1). After flipping into the lumen of the ER [6,7,8], the growing DLO is completed by DPM- and DPG-requiring glycosyltransferases, whose active sites are thought to face the lumen of the ER [9,10], to yield the mature DLO The ensemble of these reactions constitutes the dolichol cycle and its interruption leads to hypoglycosylation of glycoproteins in yeast [11] and mammalian cells [12]. We examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I) in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation
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