Partial purification of a mannosyltransferase involved in the O-mannosylation of glycoproteins from Saccharomyces cerevisiae

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Abstract
The mannosyltransferase that catalyses the transfer of mannose from dolichyl-phosphate-mannose (Dol-P-Man) to the hydroxyl group of serine/threonine residues in the acceptor peptide (Tyr-Asn-Pro-Thr-Ser-Val) was partially purified ∼150-fold from the microsomal membrane fraction of Saccharomyces cerevisiae. The membrane-bound enzyme was solubilized with 0.5% Triton X-100 at a protein:detergent ratio of 2: 1, and was then purified by ionexchange chromatography on DEAE-cellulose, followed by hydroxyapatite column chromatography. The partially purified enzyme had a pH optimum of 7.2 and required Mg2+ at an optimum concentration of 10 mM for activity. The apparent mol. wt of the enzyme, as estimated by gel filtration on Sephacryl S-300, was ∼125 kDa. The activity of the partially purified enzyme was greatly stimulated by phosphatidylcholine (PC), while other naturally occurring phosphoglycerides had no significant effect.The extent of activation of mannosyltransferase activity was greatly affected by the number of carbons and the degree of saturation/unsaturation of the fatty acid substituents, as well as by their position on the glycerol moiety of the PC molecule. Maximum stimulation of the mannosyltransferase activity was induced by a PC derivative in which both sn-1 and sn-2 positions on the glycerol moiety were occupied by C12:0 fatty acids. In general, mannosyltransferase was found to exhibit greater specificity for the L-α-PC derivatives in which the sn-2 position of the glycerol contained a saturated fatty acid. The mannosyltransferase showed a greatly reduced Km value (five times lower) for the hexapeptide substrate in the presence of PC than in its absence, indicating that the stimulation of mannosyltransferase activity was at least partially due to the increased affinity for the acceptor peptide. Upon β-elimination of the radiolabelled mannosyl-peptide formed during the incubation of Dol-P-[14C]Man and unlabelled acceptor peptide with the partially purified enzyme, total radioactivity was released as mannose, confirming that a single mannose unit was linked to the serine/threonine residues via an O-glycosidic bond.

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