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

The mannosyltransferase that catalyses the transfer of mannosefrom dolichyl-phosphate-mannose (Dol-P-Man) to the hydroxylgroup of serine/threonine residues in the acceptor peptide (Tyr-Asn-Pro-Thr-Ser-Val)was partially purified 150-fold from the microsomal membranefraction of Saccharomyces cerevisiae. The membrane-bound enzymewas solubilized with 0.5% Triton X-100 at a protein:detergentratio of 2: 1, and was then purified by ionexchange chromatographyon DEAE-cellulose, followed by hydroxyapatite column chromatography.The partially purified enzyme had a pH optimum of 7.2 and requiredMg²⁺ at an optimum concentration of 10 mM for activity. Theapparent mol. wt of the enzyme, as estimated by gel filtrationon Sephacryl S-300, was 125 kDa. The activity of the partiallypurified enzyme was greatly stimulated by phosphatidylcholine(PC), while other naturally occurring phosphoglycerides hadno significant effect.The extent of activation of mannosyltransferaseactivity was greatly affected by the number of carbons and thedegree of saturation/unsaturation of the fatty acid substituents,as well as by their position on the glycerol moiety of the PCmolecule. Maximum stimulation of the mannosyltransferase activitywas induced by a PC derivative in which both sn-1 and sn-2 positionson the glycerol moiety were occupied by C_12:0 fatty acids. Ingeneral, mannosyltransferase was found to exhibit greater specificityfor the L--PC derivatives in which the sn-2 position of theglycerol contained a saturated fatty acid. The mannosyltransferaseshowed a greatly reduced K_m value (five times lower) for thehexapeptide substrate in the presence of PC than in its absence,indicating that the stimulation of mannosyltransferase activitywas at least partially due to the increased affinity for theacceptor peptide. Upon ß-elimination of the radiolabelledmannosyl-peptide formed during the incubation of Dol-P-[¹⁴C]Manand unlabelled acceptor peptide with the partially purifiedenzyme, total radioactivity was released as mannose, confirmingthat a single mannose unit was linked to the serine/threonineresidues via an O-glycosidic bond. mannosyltransferase purification Saccharomyces cerevisiae ¹Present address: Department of Biosciences and Biotechnology,University of Roorkee,Roorkee 247 667, India²Present address: Department of Biochemistry and Molecular Biology,University of Arkansas for Medical Sciences, Little Rock, AR72205, USA