Large-scale isolation of dolichol-linked oligosaccharides with homogeneous oligosaccharide structures: determination of steady-state dolichol-linked oligosaccharide compositions.
The dolichol-linked oligosaccharide donor (Glc(3)Man(9)GlcNAc(2)-PP-Dol) for N-linked glycosylation of proteins is assembled in a series of reactions that initiate on the cytoplasmic face of the rough endoplasmic reticulum and terminate within the lumen. The biochemical analysis of the oligosaccharyltransferase and the glycosyltransferases that mediate assembly of dolichol-linked oligosaccharides (OS-PP-Dol) has been hindered by the lack of structurally homogeneous substrate preparations. We have developed an improved method for the preparative-scale isolation of dolichol-linked oligosaccharides from vertebrate tissues and yeast cells. Preparations that were highly enriched in either Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9)GlcNAc(2)-PP-Dol were obtained from porcine pancreas and a Man(5)GlcNAc(2)-PP-Dol preparation was obtained from an alg3 yeast culture. Chromatography of the OS-PP-Dol preparations on an aminopropyl silica column was used to obtain dolichol-linked oligosaccharides with defined structures. A single chromatography step could achieve near-baseline resolution of dolichol-linked oligosaccharides that differed by one sugar residue. A sensitive oligosaccharyltransferase endpoint assay was used to determine the concentration and composition of the OS-PP-Dol preparations. Typical yields of Glc(3)Man(9)GlcNAc(2)-PP-Dol, Man(9)GlcNAc(2)-PP-Dol, and Man(5)GlcNAc(2)-PP-Dol ranged between 5 and 15 nmol per chromatographic run. The homogeneity of these preparations ranged between 85 and 98% with respect to oligosaccharide composition. Purification of dolichol-linked oligosaccharides from cultures of alg mutant yeast strains provides a general method to obtain authentic OS-PP-Dol assembly intermediates of high purity. The analytical methods described here can be used to accurately evaluate the steady-state dolichol-linked oligosaccharide compositions of wild-type and mutant cell lines.
Kelleher
D J
DJ
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655-0103, USA.
Karaoglu
D
D
Gilmore
R
R
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003208:Concanavalin A / Q000378:metabolism; D004286:Dolichols / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics; D017353:Gene Deletion; D008364:Mannosyltransferases; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010179:Pancreas / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017385:Sequence Homology; D013552:Swine
D004286:Dolichols; D005656:Fungal Proteins; D008565:Membrane Proteins; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D003208:Concanavalin A; C104466:ALG3 protein, S cerevisiae; D008364:Mannosyltransferases