J Thromb Haemost. 2014 May;12(5):670-9. doi: 10.1111/jth.12535.
Acquired deficiency of ADAMTS13 causes a rare and life-threatening disorder called thrombotic thrombocytopenic purpura (TTP). Several studies have shown that aberrant glycosylation can play an important role in the pathogenesis of autoimmune diseases.N-linked glycosylation and putative O-fucosylation sites have been predicted or identified in recombinant ADAMTS13. However, it is not known which of these sites are glycosylated in plasma derived ADAMTS13.
Here we investigated the presence of putative O-fucosylation, C-mannosylation and N-linked glycosylation sites on plasma derived ADAMTS13.
METHODS/RESULTS: Sites of N-linked glycosylation were determined by the use of peptide N-glycosidase-F (PNGase F), which removes the entire carbohydrate from the side chain of asparagines. Nine of the 10 predicted N-linked glycosylation sites were identified in or near the metalloproteinase,spacer, thrombospondin type 1 repeat (TSR1) and the CUB domain of plasma ADAMTS13. Moreover, six putative O-fucosylated sites were identified in the TSR domains of plasma ADAMTS13 by performing searches of the tandem mass spectrometry (MS/MS) data for loss of hexose (162 Da), deoxyhexose (146 Da), or hexose deoxyhexose(308 Da). The use of electron transfer dissociation (ETD) allowed for unambiguous identification of the modified sites. In addition to putative O-fucosylation and N-linked glycosylation, two putative C-mannosylation sites were identified within the TSR1 and TSR4 domains of ADAMTS13.
Our data identify several glycosylation sites on plasma derived ADAMTS13. We anticipate that our findings may be relevant for the initiation of autoimmune reactivity against ADAMTS13 in patients with acquired TTP.
ADAMTS13 的获得性缺乏会导致一种罕见且危及生命的疾病，称为血栓性血小板减少性紫癜（TTP）。几项研究表明，异常糖基化可能在自身免疫性疾病的发病机制中起重要作用。在重组 ADAMTS13 中已经预测或鉴定了 N-连接糖基化和假定的 O-岩藻糖化位点。然而，尚不清楚在血浆衍生的 ADAMTS13 中哪些位点发生了糖基化。
本研究旨在研究血浆衍生的 ADAMTS13 上存在的假定 O-岩藻糖基化、C-甘露糖化和 N-连接糖基化位点。
方法/结果：使用肽 N-糖苷酶 F（PNGase F）确定 N-连接糖基化位点，该酶从天冬酰胺侧链上除去整个碳水化合物。在血浆 ADAMTS13 的金属蛋白酶、间隔区、血小板反应蛋白 1 型重复（TSR1）和 CUB 结构域中或附近鉴定出 10 个预测的 N-连接糖基化位点中的 9 个。此外，通过搜索串联质谱（MS/MS）数据中六碳糖（162 Da）、脱氧六碳糖（146 Da）或六碳糖脱氧六碳糖（308 Da）的丢失，在血浆 ADAMTS13 的 TSR 结构域中鉴定出 6 个假定的 O-岩藻糖基化位点。使用电子转移解离（ETD）可明确鉴定修饰位点。除了假定的 O-岩藻糖基化和 N-连接糖基化之外，还在 ADAMTS13 的 TSR1 和 TSR4 结构域中鉴定出 2 个假定的 C-甘露糖化位点。
本研究确定了血浆衍生的 ADAMTS13 上的几个糖基化位点。我们预计，我们的发现可能与获得性 TTP 患者中针对 ADAMTS13 的自身免疫反应的启动有关。
