Abstract：
Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved post-translational modification (PTM) that fulfills important biological roles during embryonic development. Three non-redundant enzyme families, POMT1/POMT2, TMTC1-4 and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knock-out (KO) of O-Man glycosyltransferase genes. We established a human cell library for analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man glycosylated adhesion molecules and receptors.
摘要：
蛋白质O-连接的甘露糖(O-Man)糖基化是一种进化保守的翻译后修饰(PTM),其在胚胎发育过程中发挥重要的生物学作用。三个非冗余酶家族，POMT1/POMT2，TMTC1-4和TMEM260选择性地协调蛋白质O-Man糖基化在不同类型的跨膜蛋白上的起始，包括α-营养不良聚糖，钙黏着蛋白和丛蛋白受体。然而,缺乏对其底物特异性的系统研究，部分是由于O-Man糖基转移酶在细胞中的普遍表达，这排除了在蛋白质组范围内对途径特异性O-Man糖基化的分析。这里,我们在五种人类细胞系中应用了膜糖蛋白质组学的靶向工作流程，以广泛定位O-Man底物，并通过O-Man糖基转移酶基因的个体和组合敲除（KO）基因解构O-Man起始。我们建立了人类细胞文库，用于通过定量糖蛋白质组学分析单个O-Man起始途径的底物特异性。我们的结果鉴定了180个O-Man糖蛋白，证明了POMT1/POMT2途径的新蛋白质靶标，并表明TMTC1-4和TMEM260途径广泛靶向参与细胞-细胞和细胞-细胞外基质相互作用的质膜蛋白的不同Ig样蛋白质结构域。在Ig样折叠上鉴定O-Man增加了对结构域特异性O-Man糖基化的新兴概念的进一步了解，这为O-Man糖基化粘附分子和受体的功能研究打开了大门。
