Combined proteomic and biochemical analyses redefine the consensus sequence requirement for epidermal growth factor-like domain hydroxylation.
Combined proteomic and biochemical analyses redefine the consensus sequence requirement for epidermal growth factor-like domain hydroxylation.
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DOI：
10.1016/j.jbc.2022.102129
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发表时间：
2022-08
影响因子：
4.8
通讯作者：
Schofield, Christopher J.
中科院分区：
文献类型：
--
作者：
Brewitz, Lennart;Onisko, Bruce C.;Schofield, Christopher J.
表皮生长因子样结构域（EGFD）在细胞间信号传导中具有重要功能。分泌的和细胞表面的人EGFD都经历广泛的修饰，包括由2-酮戊二酸加氧酶天冬氨酸/天冬酰胺-β-羟化酶（AspH）催化的天冬氨酸和天冬酰胺残基C3-羟基化。虽然遗传学研究表明AspH在人类生物学中很重要，但由于对其底物的不完全了解，对其生理作用的研究受到限制。在这里，我们重新定义的AspH催化EGFD羟基化的共识序列要求的基础上结合分析的蛋白质组学质谱数据和质谱为基础的分析与分离的AspH和肽底物。我们提供的细胞和生化证据表明，EGFD羟基化的首选网站是嵌入在一个二硫键桥接的大环形成的10个氨基酸残基。这一定义使以前未分配的羟基化位点的鉴定人纤蛋白作为AspH底物的三个EGFD。一个非EGFD含有蛋白，淋巴细胞抗原-6/纤溶酶原激活剂尿激酶受体结构域含有蛋白6 B（LYPD 6 B）被证明是一个底物分离的AspH，但我们没有观察到LYPD 6 B在细胞中羟基化的证据。AspH催化的纤维蛋白的羟基化是特别感兴趣的，因为它们在细胞外基质动力学中起着重要作用。总之，这些结果导致修改的共识底物的要求，为AspH和扩大的范围内观察到的和潜在的AspH催化的羟基化在细胞中，这将使未来的研究AspH的生物学作用。
Epidermal growth factor-like domains (EGFDs) have important functions in cell–cell signaling. Both secreted and cell surface human EGFDs are subject to extensive modifications, including aspartate and asparagine residue C3-hydroxylations catalyzed by the 2-oxoglutarate oxygenase aspartate/asparagine-β-hydroxylase (AspH). Although genetic studies show AspH is important in human biology, studies on its physiological roles have been limited by incomplete knowledge of its substrates. Here, we redefine the consensus sequence requirements for AspH-catalyzed EGFD hydroxylation based on combined analysis of proteomic mass spectrometric data and mass spectrometry–based assays with isolated AspH and peptide substrates. We provide cellular and biochemical evidence that the preferred site of EGFD hydroxylation is embedded within a disulfide-bridged macrocycle formed of 10 amino acid residues. This definition enabled the identification of previously unassigned hydroxylation sites in three EGFDs of human fibulins as AspH substrates. A non-EGFD containing protein, lymphocyte antigen-6/plasminogen activator urokinase receptor domain containing protein 6B (LYPD6B) was shown to be a substrate for isolated AspH, but we did not observe evidence for LYPD6B hydroxylation in cells. AspH-catalyzed hydroxylation of fibulins is of particular interest given their important roles in extracellular matrix dynamics. In conclusion, these results lead to a revision of the consensus substrate requirements for AspH and expand the range of observed and potential AspH-catalyzed hydroxylation in cells, which will enable future study of the biological roles of AspH.
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影响因子：
8.4
作者：
Brewitz L;Nakashima Y;Schofield CJ
通讯作者：
Schofield CJ
影响因子：
14.9
作者：
Benita Y;Kikuchi H;Smith AD;Zhang MQ;Chung DC;Xavier RJ
通讯作者：
Xavier RJ
影响因子：
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作者：
Coleman, Mathew L.;McDonough, Michael A.;Schofield, Christopher J.
通讯作者：
Schofield, Christopher J.
影响因子：
2
作者：
Chung, Brian Hon-Yin;Lam, Stephen Tak-Sum;Lau, Yu-Lung
通讯作者：
Lau, Yu-Lung
影响因子：
4.8
作者：
Dinchuk, JE;Focht, RJ;Friedman, PA
通讯作者：
Friedman, PA