Large-scale isolation of dolichol-linked oligosaccharides with homogeneousoligosaccharide structures: determination of steady-state dolichol-linked oligosaccharide compositions

The dolichol-linked oligosaccharide donor (Glc(3)Man(9)GlcNAc(2)-PP-Dol) fo r N-linked glycosylation of proteins is assembled in a series of reactions that initiate on the cytoplasmic face of the rough endoplasmic reticulum an d terminate within the lumen. The biochemical analysis of the oligosacchary ltransferase and the glycosyltransferases that mediate assembly of dolichol -linked oligosaccharides (OS-PP-Dol) has been hindered by the lack of struc turally homogeneous substrate preparations. We have developed an improved m ethod for the preparative-scale isolation of dolichol-linked oligosaccharid es from vertebrate tissues and yeast tells, Preparations that were highly e nriched in either Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9)GlcNAc(2)-PP-Dol we re obtained from porcine pancreas and a Man(5)GlcNAc(2)-PP-Dol preparation was obtained from an Delta alg3 yeast culture. Chromatography of the OS-PP- Dol preparations on an aminopropyl silica column was used to obtain dolicho l-linked oligosaccharides with defined structures, A single chromatography step could achieve near-baseline resolution of dolichol-linked oligosacchar ides that differed by one sugar residue, A sensitive oligosaccharyltransfer ase 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-D ol, 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 ran ged between 85 and 98% with respect to oligosaccharide composition. Purific ation of dolichol-linked oligosaccharides from cultures of alg mutant yeast strains provides a general method to obtain authentic OS-PP-Dol assembly i ntermediates of high purity. The analytical methods described here can be u sed to accurately evaluate the steady-state dolichol-linked oligosaccharide compositions of wild-type and mutant cell lines.