Large-scale isolation of dolichol-linkedoligosaccharides with homogeneous oligosaccharide structures: determinationof steady-state dolichol-linked oligosaccharide compositions.

The dolichol-linked oligosaccharide donor (Glc3Man9GlcNAc2-PP-Dol)for N-linked glycosylation of proteins is assembled in a seriesof reactions that initiate on the cytoplasmic face of the roughendoplasmic reticulum and terminate within the lumen. The biochemicalanalysis of the oligosaccharyltransferase and the glycosyltransferases thatmediate assembly of dolichol-linked oligosaccharides (OS-PP-Dol)has been hindered by the lack of structurally homogeneous substratepreparations. We have developed an improved method for the preparative-scaleisolation of dolichol-linked oligosaccharides from vertebrate tissues andyeast cells. Preparations that were highly enriched in either Glc3Man9GlcNAc2-PP-Dolor Man9GlcNAc2-PP-Dol were obtained from porcinepancreas and a Man5GlcNAc2-PP-Dol preparationwas obtained from an Δalg3 yeast culture.Chromatography of the OS-PP-Dol preparations on an aminopropyl silicacolumn was used to obtain dolichol-linked oligosaccharides withdefined structures. A single chromatography step could achieve near-baselineresolution of dolichol-linked oligosaccharides that differed byone sugar residue. A sensitive oligosaccharyltransferase endpointassay was used to determine the concentration and composition ofthe OS-PP-Dol preparations. Typical yields of Glc3Man9GlcNAc2-PP-Dol,Man9GlcNAc2-PP-Dol, and Man5GlcNAc2-PP-Dolranged between 5 and 15 nmol per chromatographic run. The homogeneityof these preparations ranged between 85 and 98% with respectto oligosaccharide composition. Purification of dolichol-linkedoligosaccharides from cultures of alg mutant yeast strainsprovides a general method to obtain authentic OS-PP-Dol assemblyintermediates of high purity. The analytical methods described herecan be used to accurately evaluate the steady-state dolichol-linkedoligosaccharide compositions of wild-type and mutant cell lines. [ABSTRACT FROM PUBLISHER]