Zacchi Lucia F, Schulz Benjamin L
From the ‡School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, 4072, Australia; §Fundación Instituto Leloir, Avenida Patricias Argentinas 435, Ciudad Autónoma de Buenos Aires, 1405, Argentina.
From the ‡School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, 4072, Australia;
Mol Cell Proteomics. 2016 Jul;15(7):2435-47. doi: 10.1074/mcp.M115.056366. Epub 2016 Apr 19.
Glycan macro- and microheterogeneity have profound impacts on protein folding and function. This heterogeneity can be regulated by physiological or environmental factors. However, unregulated heterogeneity can lead to disease, and mutations in the glycosylation process cause a growing number of Congenital Disorders of Glycosylation. We systematically studied how mutations in the N-glycosylation pathway lead to defects in mature proteins using all viable Saccharomyces cerevisiae strains with deletions in genes encoding Endoplasmic Reticulum lumenal mannosyltransferases (Alg3, Alg9, and Alg12), glucosyltransferases (Alg6, Alg8, and Die2/Alg10), or oligosaccharyltransferase subunits (Ost3, Ost5, and Ost6). To measure the changes in glycan macro- and microheterogeneity in mature proteins caused by these mutations we developed a SWATH-mass spectrometry glycoproteomics workflow. We measured glycan structures and occupancy on mature cell wall glycoproteins, and relative protein abundance, in the different mutants. All mutants showed decreased glycan occupancy and altered cell wall proteomes compared with wild-type cells. Mutations in earlier mannosyltransferase or glucosyltransferase steps of glycan biosynthesis had stronger hypoglycosylation phenotypes, but glucosyltransferase defects were more severe. ER mannosyltransferase mutants displayed substantial global changes in glycan microheterogeneity consistent with truncations in the glycan transferred to protein in these strains. Although ER glucosyltransferase and oligosaccharyltransferase subunit mutants broadly showed no change in glycan structures, ost3Δ cells had shorter glycan structures at some sites, consistent with increased protein quality control mannosidase processing in this severely hypoglycosylating mutant. This method allows facile relative quantitative glycoproteomics, and our results provide insights into global regulation of site-specific glycosylation.
聚糖的宏观和微观异质性对蛋白质折叠和功能有深远影响。这种异质性可由生理或环境因素调节。然而，不受调控的异质性会导致疾病，糖基化过程中的突变会引发越来越多的先天性糖基化障碍。我们使用所有编码内质网腔甘露糖基转移酶（Alg3、Alg9和Alg12）、葡糖基转移酶（Alg6、Alg8和Die2/Alg10）或寡糖基转移酶亚基（Ost3、Ost5和Ost6）的基因缺失的酿酒酵母可行菌株，系统地研究了N-糖基化途径中的突变如何导致成熟蛋白出现缺陷。为了测量这些突变导致的成熟蛋白中聚糖宏观和微观异质性的变化，我们开发了一种SWATH-质谱糖蛋白质组学工作流程。我们测量了不同突变体中成熟细胞壁糖蛋白上的聚糖结构和占有率，以及相对蛋白质丰度。与野生型细胞相比，所有突变体的聚糖占有率均降低，细胞壁蛋白质组也发生了改变。聚糖生物合成早期甘露糖基转移酶或葡糖基转移酶步骤中的突变具有更强的低糖基化表型，但葡糖基转移酶缺陷更为严重。内质网甘露糖基转移酶突变体在聚糖微观异质性方面表现出大量全局性变化，这与这些菌株中转移到蛋白质上的聚糖截短一致。尽管内质网葡糖基转移酶和寡糖基转移酶亚基突变体在聚糖结构上总体无变化，但ost3Δ细胞在某些位点的聚糖结构较短，这与该严重低糖基化突变体中蛋白质质量控制甘露糖苷酶加工增加一致。该方法可实现简便的相对定量糖蛋白质组学，我们的结果为位点特异性糖基化的全局调控提供了见解。
