N‐Glycosylation of Yeast Proteins

Abstract

The enzyme transferring the oligosaccharide from D~lPP-(GlcNAc)z(Man)~(Glc)~ to asparagine residues of glycoproteins has been solubilized from yeast membranes by extraction with detergents. Enzyme activity was tested by measuring transfer of the glycosyl moiety from D~IPP-['~C]saccharides to the hexapeptide Tyr-AsnLeu-Thr-Ser-Val. The rate of transfer was linear for 20 min, with about 40 of dolichyl-diphosphate-bound radioactivity transferred to the peptide.The solubilized enzyme has been characterized as follows :1. The enzyme is most efficiently solubilized (60 of the membrane-associated activity) by 0.5 (x Nonidet P40 at a protein/detergent ratio of 2. Octylglucoside solubilizes one third of the activity, but strongly inhibits the reaction if present in the test at a concentration of 1 %.2. Divalent cations are absolutely required. 1 mM Mn2+ is optimal; Mg2+ at a concentration of 10 mM yields only one third the activity observed with Mn2+.3. The enzyme transfers besides dolichyl-diphosphate-bound (GlcNAc)2(Man)9(Glc)3 also (GlcNAc)2(Man)l and ( G I~N A C )~ ; the rate decreases in this order. No transfer is observed from DolPP-(GlcNAc)z(Man)g and from DolPP-GlcNAc.4. The K , value for DolPP-(GlcNAc)2(Man)g(Glc)3 of 0.5 pM does not differ significantly l'rom that for DOIPP-(GICNAC)~ of 1.2 pM. A broad pH-optimum for the reaction with both substrates was found between 6.5 and 7.7. 5. However, a clear difference in K,,, values for the hexapeptide was observed with different dolichol-linked sugar derivatives. With DolPP-(GlcNAc)2 a peptide concentration of 0.6 mM resulted in half-maximal transfer rate, whereas 0.05 mM peptide were sufficient with DolPP-(GlcNAc)2(Man)~(Glc)3 as donor.

Dimeric organization of the yeast oligosaccharyl transferase complex

“…Digitonin has been the detergent of choice of many researchers for the purification of the active OT complex (4,30,31), although others have reported purification of catalytically active OT complexes by use of detergents such as Nikkol and Nonidet P-40 (13,14,32). Although a comparative study of OT activity in various detergents has not been reported yet, digitonin seems to be an ideal detergent for the purification of the OT complex because we found that the digitonin-solubilized microsomal extract retains high OT activity even after storage for several days at 4°C (data not shown).…”

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…One other very important note is that glucose-starved cells produce truncated LLO intermediates, which are less favorable to OST compared to fully glucosylated donors (Gao et al, 2005;Kelleher et al, 2003;Turco et al, 1977). An accumulation of truncated LLO intermediates can result in the addition of premature N-glycans onto the nascent polypeptides at a low rate or leaving Asn residue completely empty (Jackson et al, 1989;Quellhorst et al, 1999;Sharma et al, 1981). Both premature N-glycans and hypoglycosylation may have an effect on the N-glycosylation profile of the final product, which will be investigated in the next experiments in this thesis.…”

“…Studies have showed that a broad spectrum of assembly intermediates (GlcNAc2-PP-Dol to Glc2Man9GlcNAc2-PP-Dol) can be utilized by OST in vitro and in vivo (Jackson et al, 1989;Sharma et al, 1981). However, the presence of glucose units in the completed N-glycosylation precursor, Glc3Man9GlcNAc2-P-P-Dol, is essential for an efficient transfer reaction, and being preferred by more than 20-fold over nonglucosylated oligosaccharide-PP-Dol in cell free assays (Ballou et al, 1986;Parodi, 2000).…”

Unraveling the Mechanism of Protein N-Glycosylation

“…Substrate Recognition and Glycopeptide Turnover at Active Site May Involve Allosteric Regulation It has been demonstrated that oligosaccharide chains of varying length (Glc 0 -2 Man 0 -9 GlcNAc 2 -PP-Dol) can serve as the donor substrate of OT in vivo (47) and in vitro (48). Because OT preferentially transfers the fully assembled donor substrate, Glc 3 Man 9 GlcNAc 2 -PPDol, to the nascent polypeptide chain in vivo, investigation of the kinetic differences between the various sized intermediates as substrates versus the fully assembled dolichol-linked oligosaccharide substrate would be expected to provide insight into the mechanism utilized by OT to monitor the donor substrate specificity.…”