Cipollo John F, Trimble Robert B
Department of Biomedical Sciences, State University of New York at Albany School of Public Health, Albany, NY 12201, USA.
Glycobiology. 2002 Nov;12(11):749-62. doi: 10.1093/glycob/cwf082.
N-glycosylation in nearly all eukaryotes proceeds in the endoplasmic reticulum (ER) by transfer of the precursor Glc(3)Man(9)GlcNAc(2) from dolichyl pyrophosphate (PP-Dol) to consensus Asn residues in nascent proteins. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide lipid properly, and the alg12 mutant accumulates a Man(7)GlcNAc(2)-PP-Dol intermediate. We show that the Man(7)GlcNAc(2) released from alg12Delta-secreted invertase is Manalpha1,2Manalpha1,2Manalpha1,3(Manalpha1,2Manalpha1,3Manalpha1,6)-Manbeta1,4-GlcNAcbeta1-4GlcNAcalpha/beta, confirming that the Man(7)GlcNAc(2) is the product of the middle-arm terminal alpha1,2-mannoslytransferase encoded by the ALG9 gene. Although the ER glucose addition and trimming events are similar in alg12Delta and wild-type cells, the central-arm alpha1,2-linked Man residue normally removed in the ER by Mns1p persists in the alg12Delta background. This confirms in vivo earlier in vitro experiments showing that the upper-arm Manalpha1,2Manalpha1,6-disaccharide moiety, missing in alg12Delta Man(7)GlcNAc(2), is recognized and required by Mns1p for optimum mannosidase activity. The presence of this Man influences downstream glycan processing by reducing the efficiency of Ochlp, the cis-Golgi alpha1,6-mannosyltransferase responsible for initiating outer-chain mannan synthesis, leading to hypoglycosylation of external invertase and vacuolar protease A.
在几乎所有真核生物中，N-糖基化在内质网（ER）中进行，通过将前体Glc(3)Man(9)GlcNAc(2)从焦磷酸多萜醇（PP-Dol）转移到新生蛋白质中的共有Asn残基上。酿酒酵母的alg（天冬酰胺连接的糖基化）突变体无法正确合成寡糖脂，alg12突变体积累了Man(7)GlcNAc(2)-PP-Dol中间体。我们发现，从alg12Δ分泌的转化酶释放的Man(7)GlcNAc(2)是Manα1,2Manα1,2Manα1,3(Manα1,2Manα1,3Manα1,6)-Manβ1,4-GlcNAcβ1-4GlcNAcα/β，证实Man(7)GlcNAc(2)是由ALG9基因编码的中臂末端α1,2-甘露糖基转移酶的产物。尽管alg12Δ和野生型细胞中的内质网葡萄糖添加和修剪事件相似，但通常在内质网中由Mns1p去除的中心臂α1,2-连接的Man残基在alg12Δ背景中持续存在。这证实了体内早期的体外实验，表明alg12Δ Man(7)GlcNAc(2)中缺失的上臂Manα1,2Manα1,6-二糖部分被Mns1p识别并为最佳甘露糖苷酶活性所必需。这种Man的存在通过降低Ochlp（负责启动外链甘露聚糖合成的顺式高尔基体α1,6-甘露糖基转移酶）的效率来影响下游聚糖加工，导致外部转化酶和液泡蛋白酶A的低糖基化。
