Protein glycosylation is critical to the quaternary structure and collagen-binding activity of the extracellular matrix protein adhesin A (EmaA) associated with Aggregatibacter actinomycetemcomitans. The glycosylation of this large, trimeric autotransporter adhesin is postulated to be mediated by WaaL, an enzyme with the canonical function to ligate the O-polysaccharide (O-PS) antigen with a terminal sugar of the lipid A-core oligosaccharide of lipopolysaccharide (LPS). In this study, we have determined that the Escherichia coli waaL ortholog (rflA) does not restore collagen binding of a waaL mutant strain of A. actinomycetemcomitans but does restore O-PS ligase activity following transformation of a plasmid expressing waaL. Therefore, a heterologous E. coli expression system was developed constituted of two independently replicating plasmids expressing either waaL or emaA of A. actinomycetemcomitans to directly demonstrate the necessity of ligase activity for EmaA collagen binding. Proper expression of the protein encoded by each plasmid was characterized, and the individually transformed strains did not promote collagen binding. However, coexpression of the two plasmids resulted in a strain with a significant increase in collagen binding activity and a change in the biochemical properties of the protein. These results provide additional data supporting the novel hypothesis that the WaaL ligase of A. actinomycetemcomitans shares a dual role as a ligase in LPS biosynthesis and is required for collagen binding activity of EmaA.

中文翻译：

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放线菌 O 抗原 WaaL 连接酶的双重功能

蛋白质糖基化对于与放线菌聚集菌相关的细胞外基质蛋白粘附素 A (EmaA) 的四级结构和胶原蛋白结合活性至关重要。这种大的三聚体自转运蛋白粘附素的糖基化被认为是由 WaaL 介导的，WaaL 是一种酶，具有将 O-多糖 (O-PS) 抗原与脂多糖 (LPS) 脂质 A 核心寡糖的末端糖连接的典型功能。 ）。在这项研究中，我们确定大肠杆菌 waaL直系同源物 ( rflA ) 不会恢复A. actinomycetemcomitans waaL突变菌株的胶原蛋白结合，但在表达waaL的质粒转化后确实恢复了 O-PS 连接酶活性。因此，开发了由两个独立复制的质粒组成的异源大肠杆菌表达系统，表达A. actinomycetemcomitans的waaL或emaA，以直接证明连接酶活性对于EmaA胶原蛋白结合的必要性。表征了每个质粒编码的蛋白质的正确表达，并且单独转化的菌株不促进胶原蛋白结合。然而，两种质粒的共表达导致菌株的胶原蛋白结合活性显着增加，并且蛋白质的生化特性发生变化。这些结果提供了支持新假设的额外数据，即A. actinomycetemcomitans的 WaaL 连接酶在 LPS 生物合成中具有作为连接酶的双重作用，并且是 EmaA 的胶原蛋白结合活性所必需的。