A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation
A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation
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DOI：
10.1073/pnas.1511175112
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发表时间：
2015-11-24
影响因子：
11.1
通讯作者：
Schjoldager, Katrine T. -B. G.
中科院分区：
文献类型：
--
作者：
Goth, Christoffer K.;Halim, Adnan;Schjoldager, Katrine T. -B. G.
蛋白水解酶调节细胞膜蛋白胞外区的脱落是从细胞中释放胞外区并激活细胞信号的一个常见过程。胞外结构域脱落发生在即刻的细胞外膜旁区域，这也是O-糖基化经常被发现的地方，已经有报道了脱落和O-糖基化之间的串扰的例子。在这里，我们系统地研究了不同的多肽GalNAc-转移酶(GalNAc-T)亚型介导的位点特异性O-糖基化共同调节由A去整合素和金属蛋白酶(ADAM)亚家族，特别是ADAM17介导的胞外结构域脱落的可能性。我们分析了25个已知经历ADAM17脱落的膜蛋白，其中加工位点包括+/-4个残基中的Ser/Thr残基，这些残基可能代表O-糖基。我们使用体外GalNAc-T酶和ADAM裂解实验证明，至少有12个这些蛋白质的脱落可能受到O-糖基化的共同调节。以肿瘤坏死因子-α为例，我们在体外同基因细胞模型和小鼠Galnt2基因敲除模型中证实了ADAM17介导的脱落是由GalNAc-T2亚型控制的O-糖基化共同调节的。这项研究为位点特异性O-糖基化在胞外结构域脱落中发挥更广泛的作用提供了令人信服的证据。
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-alpha 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.