Dotson S B, Rush J S, Ricketts A D, Waechter C J
Department of Immunology, Searle Pharmaceutical Company, Saint Louis, Missouri 63167.
Arch Biochem Biophys. 1995 Feb 1;316(2):773-9. doi: 10.1006/abbi.1995.1103.
Mannosylphosphoryldolichol (Man-P-Dol):protein O-mannosyltransferase (PMT1) was solubilized by extracting a crude microsomal fraction from Saccharomyces cerevisiae with 1.2% Chaps-0.5% desoxycholate and purified 120-fold by standard chromatographic procedures. These very stable, partially purified preparations of PMT1 catalyzed the transfer of mannosyl units from exogenous Man-P-Dol to serine/threonine residues in the synthetic peptide acceptor, Tyr-Asn-Pro-Thr-Ser-Val-NH2, forming O-mannosidic linkages of the alpha-configuration. The specificity of yeast PMT1 was defined with respect to the recognition of the saturated alpha-isoprene unit, the chain length of the dolichyl moiety, and the anomeric configuration of the mannosyl-phosphoryl linkage of the lipophilic mannosyl donor. When Man-P-Dol95 and mannosylphosphorylpolyprenol (Man-P-Poly95), which contains a fully unsaturated polyprenyl chain, were compared as substrates, the initial rate for peptide mannosylation was dramatically higher with Man-P-Dol95 relative to Man-P-Poly95. The chain length of the dolichyl moiety also influenced the mannolipid-enzyme interaction as the partially purified PMT1 had a higher affinity for Man-P-Dol95 than for Man-P-Dol55. When beta-Man-P-Dol95 was compared with chemically synthesized alpha-Man-P-Dol95 as a mannosyl donor, a strict stereo-specificity was observed for the presence of a beta-mannosyl-phosphoryl linkage. In summary, a procedure for isolating a stable, partially purified preparation of PMT1 from S. cerevisiae is described. Enzymological studies with these preparations of PMT1 provide the first evidence that the recognition of the lipophilic mannosyl donor is stereospecific. These results also demonstrate that maximal O-mannosylation of serine/threonine residues in yeast glycoproteins catalyzed by the partially purified preparation of PMT1 requires the presence of a saturated alpha-isoprene unit in the dolichyl moiety of Man-P-Dol.
甘露糖基磷酸多萜醇（Man-P-Dol）：蛋白质O-甘露糖基转移酶（PMT1）通过用1.2%的Chaps-0.5%的脱氧胆酸盐从酿酒酵母中提取粗微粒体部分进行溶解，并通过标准色谱程序纯化了120倍。这些非常稳定的、部分纯化的PMT1制剂催化甘露糖基单元从外源性Man-P-Dol转移到合成肽受体Tyr-Asn-Pro-Thr-Ser-Val-NH2中的丝氨酸/苏氨酸残基上，形成α构型的O-甘露糖苷键。酵母PMT1的特异性在饱和α-异戊二烯单元的识别、多萜醇部分的链长以及亲脂性甘露糖基供体的甘露糖基-磷酸键的异头构型方面得到了明确。当将含有完全不饱和多萜链的Man-P-Dol95和甘露糖基磷酸聚戊烯醇（Man-P-Poly95）作为底物进行比较时，相对于Man-P-Poly95，Man-P-Dol95的肽甘露糖基化初始速率显著更高。多萜醇部分的链长也影响甘露糖脂-酶的相互作用，因为部分纯化的PMT1对Man-P-Dol95的亲和力高于对Man-P-Dol55的亲和力。当将β-Man-P-Dol95与化学合成的α-Man-P-Dol95作为甘露糖基供体进行比较时，观察到对于β-甘露糖基-磷酸键的存在具有严格的立体特异性。总之，描述了一种从酿酒酵母中分离稳定且部分纯化的PMT1制剂的方法。对这些PMT1制剂的酶学研究提供了首个证据，即亲脂性甘露糖基供体的识别是立体特异性的。这些结果还表明，由部分纯化的PMT1制剂催化的酵母糖蛋白中丝氨酸/苏氨酸残基的最大O-甘露糖基化需要在Man-P-Dol的多萜醇部分存在饱和α-异戊二烯单元。
