Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia; Institute for Glycomics, Griffith University, Gold Coast Campus, Gold Coast, QLD 4222, Australia.
Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.
Biochem Biophys Res Commun. 2014 Mar 14;445(3):633-8. doi: 10.1016/j.bbrc.2014.02.044. Epub 2014 Feb 22.
The HMWABC system of non-typeable Haemophilus influenzae (NTHi) encodes the HMWA adhesin glycoprotein, which is glycosylated by the HMWC glycosyltransferase. HMWC is a cytoplasmic N-glycosyltransferase, homologues of which are widespread in the Pasteurellaceae. We developed an assay for nonbiased detection of glycoproteins in NTHi based on metabolic engineering of the Leloir pathway and growth in media containing radiolabelled monosaccharides. The only glycoprotein identified in NTHi by this assay was HMWA. However, glycoproteomic analyses ex vivo in Escherichia coli showed that HMWC of NTHi was a general glycosyltransferase capable of glycosylating selected asparagines in proteins other than its HMWA substrate, including Asn78 in E. coli 30S ribosomal protein S5. The equivalent residue in S5 homologues in H. influenzae or other sequenced Pasteurellaceae genomes is not asparagine, and these organisms also showed significantly fewer than expected potential sites of glycosylation in general. Expression of active HMWC in E. coli resulted in growth inhibition compared with expression of inactive enzyme, consistent with glycosylation by HMWC detrimentally affecting the function of some E. coli proteins. Together, this supports the presence of a selective pressure in the Pasteurellaceae against glycosylation sites that would be modified by the general N-glycosyltransferase activity of HMWC.
无乳链球菌（NTHi）的高分子量 ABC 系统编码 HMWA 黏附糖蛋白，该蛋白由高分子量 C 糖基转移酶（HMWC）糖基化。HMWC 是一种细胞质 N-糖基转移酶，其同源物广泛存在于巴斯德氏菌科中。我们基于 Leloir 途径的代谢工程和含有放射性标记单糖的培养基中的生长，开发了一种用于非偏倚检测 NTHi 中糖蛋白的测定法。通过该测定法鉴定出的唯一糖蛋白是 HMWA。然而，在大肠杆菌中进行的糖蛋白质组学分析表明，NTHi 的 HMWC 是一种通用的糖基转移酶，能够糖基化其 HMWA 底物以外的选定蛋白质中的天冬酰胺，包括大肠杆菌 30S 核糖体蛋白 S5 中的 Asn78。在流感嗜血杆菌或其他已测序的巴斯德氏菌基因组中的 S5 同源物中的等效残基不是天冬酰胺，并且这些生物体通常也显示出比预期少得多的潜在糖基化位点。与表达无活性酶相比，在大肠杆菌中表达活性 HMWC 会导致生长抑制，这与 HMWC 的糖基化会损害某些大肠杆菌蛋白的功能一致。总的来说，这支持巴斯德氏菌科中存在一种选择性压力，以防止被 HMWC 的通用 N-糖基转移酶活性修饰的糖基化位点。
