Verostek M F, Atkinson P H, Trimble R B
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
J Biol Chem. 1991 Mar 25;266(9):5547-51.
Asparagine-linked oligosaccharides are synthesized by transfer of Glc3Man9GlcNAc2 from dolichol pyrophosphate to nascent polypeptides. Assembly of the precursor proceeds by highly ordered sequential addition of mannose and glucose to form Glc3Man9GlcNAc2-P-P-dolichol. Yeast mutants in asparagine-linked glycosylation (alg), generated by an 3H-Man suicide technique, were assigned to eight complementation groups which define steps in oligosaccharide-lipid synthesis (Huffaker, T.C., and Robbins, P.W. (1982) J. Biol. Chem. 257, 3203-3210). Alg3 invertase oligosaccharides are resistant to endo-beta-N-acetylglucosaminidase H, and the lipid-oligosaccharide pool yields Man5Glc-NAc2, suggesting its structure may be that from mammalian cells lacking Man-P-dolichol (Chapman, A., et al. (1980) J. Biol. Chem. 255, 4441-4446). To test this supposition, the endoplasmic reticulum form of invertase derepressed in alg3,sec18 yeast at 37 degrees C was isolated as a source of oligosaccharides whose processing beyond glucose and/or mannose trimming, if involved, would be prevented. Man8GlcNAc2 and Man5GlcNAc2 were released by peptide-N-glycosidase F from alg3,sec18 invertase in a 1:5 molar ratio. 1H NMR spectroscopy revealed Man8GlcNAc2 to be the alpha 1,2-mannosidase-trimming product described earlier (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666), while Man5GlcNAc2 was Man alpha 1, 2Man alpha 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc beta 1, 4GlcNAc. This provides a structural proof for the lipid-linked Man5GlcNAc2 originally proposed from enzymatic and chemical analyses of the radiolabeled mammalian precursor. Experimental evidence indicates that, unlike the mammalian cell mutants which are unable to synthesize Man-P-dolichol, alg3 yeast accumulate Man5GlcNAc2-P-P-dolichol due to a defective alpha 1,3-mannosyltransferase required for the next step in oligosaccharide-lipid elongation.
天冬酰胺连接的寡糖是通过将Glc3Man9GlcNAc2从焦磷酸多萜醇转移至新生多肽而合成的。前体的组装是通过高度有序地依次添加甘露糖和葡萄糖以形成Glc3Man9GlcNAc2-P-P-多萜醇来进行的。通过3H-甘露糖自杀技术产生的天冬酰胺连接糖基化（alg）酵母突变体被分为八个互补组，这些互补组定义了寡糖-脂质合成中的步骤（哈法克，T.C.，和罗宾斯，P.W.（1982年）《生物化学杂志》257卷，3203 - 3210页）。Alg3转化酶寡糖对内切β-N-乙酰葡糖胺糖苷酶H有抗性，并且脂质-寡糖库产生Man5GlcNAc2，这表明其结构可能是缺乏甘露糖-P-多萜醇的哺乳动物细胞的结构（查普曼，A.等人（1980年）《生物化学杂志》255卷，4441 - 4446页）。为了验证这一推测，在37℃下在alg3、sec18酵母中去阻遏的内质网形式的转化酶被分离出来，作为寡糖的来源，如果涉及到葡萄糖和/或甘露糖修剪之外的加工过程，将会被阻止。Man8GlcNAc2和Man5GlcNAc2以1:5的摩尔比被肽-N-糖苷酶F从alg3、sec18转化酶中释放出来。1H核磁共振光谱显示Man8GlcNAc2是先前描述的α1,2-甘露糖苷酶修剪产物（伯德，J.C.，塔伦蒂诺，A.L.，马利，F.，阿特金森，P.H.，和特林布尔，R.B.（1982年）《生物化学杂志》257卷，14657 - 1,4666页），而Man5GlcNAc2是Manα1,2Manα1,2Manα1,3(Manα1,6)Manβ1,4GlcNAcβ1,4GlcNAc。这为最初从放射性标记的哺乳动物前体的酶促和化学分析中提出的脂质连接的Man5GlcNAc2提供了结构证据。实验证据表明，与无法合成甘露糖-P-多萜醇的哺乳动物细胞突变体不同，alg3酵母由于寡糖-脂质延伸下一步所需的α1,3-甘露糖基转移酶有缺陷而积累Man5GlcNAc2-P-P-多萜醇。
