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Abstract：
Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits.
摘要：
寡糖糖基转移酶（OST）催化N-连接蛋白糖基化的中心步骤，预组装的寡糖从其脂质载体转移到分泌蛋白的天冬酰胺残基上。来自酿酒酵母的原型异八聚体OST复合物以两种同工型存在，其中包含Ost3p或Ost6p，两个非催化亚基。这两种OST复合物在体内具有不同的蛋白质底物特异性。然而,它们的详细生化机制和不同特异性的基础尚不清楚。从仅表达一种同种型的基因工程菌株中纯化两种OST复合物。使用短肽和不同合成脂质连接寡糖(LLO)底物的定量体外糖基化测定表征动力学性质和底物特异性。我们发现，接近糖基化序列的肽序列会影响肽的亲和力和转换率。脂质部分的长度影响LLO亲和力，而脂质双键立体化学对LLO周转率的影响更大。两种OST复合物对肽和LLO底物两者具有相似的亲和力,但显示出显著不同的转换率。这些数据为Ost3p和Ost6p亚基的功能分析提供了基础。
