In Campylobacter jejuni, the PglB oligosaccharyltransferase catalyzes the transfer of a heptasaccharide from a lipid donor to asparagine within the D/E-X₁-N-X₂-S/T sequon (X_1,2≠P) or releases this heptasaccharide as free oligosaccharides (fOS). Using available crystal structures and sequence alignments, we identified a DGGK motif near the active site of PglB that is conserved among all Campylobacter species. We demonstrate that amino acid substitutions in the aspartate and lysine residues result in loss of protein glycosylation in the heterologous E. coli system. Similarly, complementation of a C. jejuni pglB knock-out strain with mutated pglB alleles results in reduced levels of N-linked glycoproteins and fOS in the native host. Analysis of the PglB crystal structures from C. lari and the soluble C-terminal domain from C. jejuni suggests a particularly important structural role for the aspartate residue and the two following glycine residues, as well as a more subtle, less defined role for the lysine residue. Limited proteolysis experiments indicate that conformational changes of wildtype PglB that are induced by the binding of the lipid-linked oligosaccharide are altered by changes in the DGGK motif. Related to these findings, certain Campylobacter species possess two PglB orthologues and we demonstrate that only the orthologue containing the DGGK motif is active. Combining the knowledge gained from the PglB structures and mutagenesis studies, we propose a function for the DGGK motif in affecting the binding of the undecaprenyl-pyrophosphate glycan donor substrate that subsequently influences N-glycan and fOS production.

中文翻译：

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保守的DGGK基序对于空肠弯曲杆菌PglB寡糖基转移酶的功能至关重要

在空肠弯曲菌中，PglB寡糖基转移酶催化D / EX ₁ -NX ₂ -S / T后代（X _1,2 ≠P）中七糖从脂质供体向天冬酰胺的转移，或以游离寡糖（fOS）的形式释放该七糖。 ）。使用可用的晶体结构和序列比对，我们确定了在所有弯曲杆菌物种中保守的PglB活性位点附近的DGGK基序。我们证明天冬氨酸和赖氨酸残基中的氨基酸取代导致异源大肠杆菌系统中蛋白质糖基化的损失。类似地，空肠弯曲杆菌pglB敲除菌株与突变的pglB的互补等位基因导致天然宿主中N-连接的糖蛋白和fOS含量降低。分析来自C. lari的PglB晶体结构和来自空肠弯曲杆菌的可溶性C末端结构域，这表明天冬氨酸残基和随后的两个甘氨酸残基具有特别重要的结构作用，而对谷氨酸残基的作用则更微妙，作用不那么明确赖氨酸残基。有限的蛋白水解实验表明，通过脂质连接的寡糖的结合诱导的野生型PglB的构象变化被DGGK基序的变化所改变。与这些发现有关的是某些弯曲杆菌的物种拥有两个PglB直向同源物，我们证明只有包含DGGK基序的直向同源物才有活性。结合从杆菌PgIB结构和诱变研究中获得的知识，我们在影响十一异戊烯焦磷酸聚糖供体底物的结合提出了一种功能的DGGK主题是后来影响ñ -聚糖和低聚果糖生产。