Isolation of glucose-containing high-mannose glycoprotein core oligosaccharides.

Abstract

The total cell wall mannoprotein has been isolated from a mutant of Saccharomyces cerevisiae that fails to remove the glucose units of the dolichol-linked precursor after transfer of the oligosaccharide to asparagine units in the protein. The oligosaccharides released from this mannoprotein by endoglucosaminidase H digestion show 'H NMR signals assignable to three a-linked glucose units at 6 5.52, 5.27, and 5.17, and a comparison with the chemical shifts of reference compounds shows that these signals are consistent with the structure aGlc-i-2aGlc__)i3aGlc__<3aMan-+2. This provides a direct confirmation for the structure previously assigned to the lipid-linked precursor. Analysis of the larger oligosaccharides confirms that the presence of the glucose units does not prevent elongation of the al-6-linked polymannose backbone or addition of al-*3-linked mannose to the core.Glycosylation of asparagine in eukaryotic glycoproteins generally occurs by the transfer of an oligosaccharide with the composition of Glc3Man9GlcNAc2-from the dolichol pyrophosphate oligosaccharide (1). Because of the low concentration of this precursor in cells and the rapid processing of the glycosylated protein, the oligosaccharide is difficult to obtain and its structure was determined by analysis of radiolabeled material isolated from cells incubated individually with radioactive mannose, glucose, N-acetylglucosamine, and galactose. The structure derived by Li et al. (2) is generally accepted, and confirmation has come from other studies (3, 4) and from synthesis of a hexasaccharide with the same presumed partial structure (5).Recently, a new glucosidase-defective mutant of Saccharomyces cerevisiae was obtained that fails to remove the glucose units from the core oligosaccharide after its transfer to protein (6). This mutant makes carboxypeptidase Y that has a slightly reduced mobility on gel electrophoresis and that appears to contain in its oligosaccharide chains all of the glucose present in the lipid-linked precursor. We have now isolated the bulk cell wall mannoprotein from this mutant, recovered the oligosaccharides released by endoglucosaminidase digestion, fractionated them on a sizing column, and analyzed them by proton NMR. The different oligosaccharide fractions show three 1H NMR signals assignable to alinked glucose that are not present in the oligosaccharides from wild-type cells (7,8), and a comparison with the chemical shifts of reference compounds shows that the signals are consistent with the structure assigned to the precursor by Li et al. (2). The NMR spectra also suggest that processing of the core oligosaccharide is otherwise normal because a single al-+2-linked mannose is removed from the aMan-+2 aMan->3aMan >6 side chain, the backbone is extended by addition of mannose in al-*6 linkage, and some al-*3-linked mannose is added to the core. MATERIALS AND METHODSMaterials. The glucosidase-defective strain of S. cerevisiae X2180 (glsI) was originally isolated in combination with the secretion-defective strain sec1...

The Accumulation of Man6GlcNAc2-PP-dolichol in theSaccharomyces cerevisiae Δalg9 Mutant Reveals a Regulatory Role for the Alg3p α1,3-Man Middle-arm Addition in Downstream Oligosaccharide-lipid and Glycoprotein Glycan Processing

“…This assignment is supported by the 2D DQF-COSY cross-peak at 5.25(C1-H)/ 3.54(C2-H) ppm (Fig. 5A), which is in the region expected for this axial ring proton (25,35). The resonance peak indicates that 10% of pool B is the core ⌬alg9 glycan, assigned isomer 7c, which retained the ␣1,3-Glc on transport through the Golgi after leaving the ER (Scheme II).…”

Endoplasmic Reticulum α-Glycosidases of Candida albicans Are Required for N Glycosylation, Cell Wall Integrity, and Normal Host-Fungus Interaction

“…Therefore, the presence of glucose residues on the N-glycan core may inhibit the ability of the CaOch1 ␣1,6-mannosyltransferase to initiate the outer-Nchain elongation. The absence of ␣-glucosidase I activity in S. cerevisiae did not prevent outer-chain formation or the addition of ␣1,3-mannose residues to the core oligosaccharides (75), suggesting that the importance of the N-glycan core glucose residues for subsequent outer-chain elongation may be different in C. albicans and S. cerevisiae. The N-glycosylation defect in Camns1⌬ null mutant was not as severe as in Carot2⌬ and Cacwh41⌬ null mutants, indicating that partial elongation of the N-glycan core occurred.…”

Schizosaccharomyces pombe mutants that are defective in glycoprotein galactosylation.

“…In contrast, invertases from SpS50 and Spl37 both migrated as compact bands near the leading edge of the band for Sp69 invertase and about half the distance traveled by Sac. cerevisiae mnn9 invertase under similar conditions (15). Wild-type Sch.…”