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Abstract：
Regulated shedding of the ectodomain of cell membrane proteins by proteases is a common process that releases the extracellular domain from the cell and activates cell signaling. Ectodomain shedding occurs in the immediate extracellular juxtamembrane region, which is also where O-glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell models and in vivo in mouse Galnt2 knockouts. The study provides compelling evidence for a wider role of site-specific O-glycosylation in ectodomain shedding.
摘要：
通过蛋白酶调节细胞膜蛋白的胞外域的脱落是从细胞释放胞外结构域并激活细胞信号传导的常见过程。外结构域脱落发生在细胞外近膜区域，这也是经常发现O-糖基化的地方，并且已经报道了脱落和O-糖基化之间的串扰的例子。这里,我们系统地研究了由不同的多肽GalNAc-转移酶(GalNAc-T)亚型介导的位点特异性O-糖基化共同调节蛋白酶A-整合素和金属蛋白酶(ADAM)亚家族介导的胞外域脱落的潜力,特别是ADAM17.我们分析了25种已知经历ADAM17脱落的膜蛋白，其中加工位点包括在±4个残基内的Ser/Thr残基，这些残基可以代表O-糖位。我们使用体外GalNAc-T酶和ADAM切割测定来证明至少12种这些蛋白质的脱落可能通过O-糖基化共同调节。以TNF-α为例,我们证实,在等基因细胞模型和小鼠Galnt2敲除体内,ADAM17介导的脱落受GalNAc-T2亚型控制的O-糖基化共同调节.该研究为位点特异性O-糖基化在胞外域脱落中的更广泛作用提供了令人信服的证据。
