Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, causing functional and structural changes in target proteins. Protein deimination causes generation of neo-epitopes, affects gene regulation and also allows for protein moonlighting. Furthermore, PADs have been found to be a phylogenetically conserved regulator for extracellular vesicle (EVs) release. EVs are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins in serum and serum-EVs are described for the first time in camelids, using the llama (Lama glama L. 1758) as a model animal. We report a poly-dispersed population of llama serum EVs, positive for phylogenetically conserved EV-specific markers and characterised by TEM. In serum, 103 deiminated proteins were overall identified, including key immune and metabolic mediators including complement components, immunoglobulin-based nanobodies, adiponectin and heat shock proteins. In serum, 60 deiminated proteins were identified that were not in EVs, and 25 deiminated proteins were found to be unique to EVs, with 43 shared deiminated protein hits between both serum and EVs. Deiminated histone H3, a marker of neutrophil extracellular trap formation, was also detected in llama serum. PAD homologues were identified in llama serum by Western blotting, via cross reaction with human PAD antibodies, and detected at an expected 70 kDa size. This is the first report of deiminated proteins in serum and EVs of a camelid species, highlighting a hitherto unrecognized post-translational modification in key immune and metabolic proteins in camelids, which may be translatable to and inform a range of human metabolic and inflammatory pathologies.

中文翻译：

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骆驼（Lama glama）细胞外囊泡和血清中的脱亚胺蛋白 - 对骆驼免疫的新见解。


肽基精氨酸脱亚胺酶 (PAD) 是系统发育上保守的钙依赖性酶，它在翻译后以不可逆的方式将目标蛋白中的精氨酸转化为瓜氨酸，从而引起目标蛋白的功能和结构变化。蛋白质脱亚胺作用会导致新表位的产生，影响基因调控，并且还允许蛋白质兼职。此外，PAD 被发现是细胞外囊泡 (EV) 释放的系统发育保守调节因子。 EV 存在于大多数体液中，并通过货物蛋白和遗传物质的转移参与细胞通讯。在这项研究中，使用美洲驼 (Lama glama L. 1758) 作为模型动物，首次在骆驼科动物中描述了血清和血清 EV 中的翻译后脱亚胺蛋白。我们报告了美洲驼血清 EV 的多分散群体，其系统发育保守的 EV 特异性标记呈阳性，并通过 TEM 进行了表征。在血清中，总共鉴定出了 103 种脱亚胺蛋白，包括关键的免疫和代谢介质，包括补体成分、基于免疫球蛋白的纳米抗体、脂联素和热休克蛋白。在血清中，鉴定出 60 种 EV 中不存在的脱亚胺化蛋白，并发现 25 种脱亚胺化蛋白是 EV 特有的，其中血清和 EV 之间有 43 个共享的脱亚胺化蛋白。去亚氨基组蛋白 H3（中性粒细胞胞外陷阱形成的标志物）也在美洲驼血清中检测到。通过与人 PAD 抗体的交叉反应，通过蛋白质印迹法在美洲驼血清中鉴定出 PAD 同源物，并以预期的 70 kDa 大小进行检测。 这是骆驼科动物血清和 EV 中脱亚氨基蛋白的首次报告，强调了骆驼科动物关键免疫和代谢蛋白中迄今为止未被认识的翻译后修饰，这些修饰可能可转化为一系列人类代谢和炎症病理学并为其提供信息。