Abstract

Epidermal growth factor-like domains (EGF-LDs) of membrane and secreted proteins can be modified by N-glycans and/or potentially elongated O-linked monosaccharides such as O-glucose (O-Glc) found at two positions (O-Glc 1 and O-Glc2), O-fucose (O-Fuc) and O-N-acetylglucosamine (O-GlcNAc). The presence of three O-linked sugars within the same EGF-LD, such as in EGF-LD 20 of NOTCH1, has rarely been evidenced. We searched in KEGG GENES database to list mouse and human proteins with an EGF-LD sequence including one, two, three or four potential O-glycosylation consensus sites. Among the 129 murine retrieved proteins, most had predicted O-fucosylation and/or O-GlcNAcylation sites. Around 68% of EGF-LDs were subjected to only one O-linked sugar modification and near 5% to three modifications. Among these latter, we focused on the peptidase domain-containing protein associated with muscle regeneration 1 (PAMR1), having only one EGF-LD. To test the ability of this domain to be glycosylated, a correctly folded EGF-LD was produced in Escherichia coli periplasm, purified and subjected to in vitro incubations with the recombinant O-glycosyltransferases POGLUT1, POFUT1 and EOGT, adding O-Glc1, O-Fuc and O-GlcNAc, respectively. Using click chemistry and mass spectrometry, isolated PAMR1 EGF-LD was demonstrated to be modified by the three O-linked sugars. Their presence was individually confirmed on EGF-LD of full-length mouse recombinant PAMR1, with at least some molecules modified by both O-Glc1 and O-Fuc. Overall, these results are consistent with the presence of a triple O-glycosylated EGF-LD in mouse PAMR1.

中文翻译：

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小鼠 PAMR1 的单个 EGF 样结构域被 O-葡萄糖、O-岩藻糖和 O-GlcNAc 修饰。

摘要

膜和分泌蛋白的表皮生长因子样结构域 (EGF-LD) 可以被N-聚糖和/或潜在延长的O-连接单糖修饰，例如在两个位置 ( O -Glc) 发现的O-葡萄糖 ( O - Glc ) 1 和O -Glc2)、O-岩藻糖 ( O -Fuc) 和O - N-乙酰氨基葡萄糖( O -GlcNAc)。三个O的存在同一EGF-LD中的-连接糖，例如在NOTCH1的EGF-LD 20中，很少被证实。我们在 KEGG GENES 数据库中搜索以列出具有 EGF-LD 序列的小鼠和人类蛋白质，包括一个、两个、三个或四个潜在的O -糖基化共有位点。在 129 个小鼠检索到的蛋白质中，大多数预测了O-岩藻糖基化和/或O -GlcNAcylation 位点。大约 68% 的 EGF-LD 仅进行了一次O-连接的糖修饰，近 5% 进行了三次修饰。在后者中，我们专注于与肌肉再生相关的含有肽酶结构域的蛋白质 1 (PAMR1)，它只有一个 EGF-LD。为了测试这个结构域被糖基化的能力，一个正确折叠的 EGF-LD 在大肠杆菌周质，纯化并与重组O-糖基转移酶 POGLUT1、POFUT1 和 EOGT进行体外孵育，分别添加O -Glc1、O -Fuc 和O -GlcNAc。使用点击化学和质谱，分离的 PAMR1 EGF-LD 被证明被三个O-连接糖修饰。它们的存在分别在全长小鼠重组 PAMR1 的 EGF-LD 上得到证实，至少有一些分子被O -Glc1 和O -Fuc 修饰。总体而言，这些结果与小鼠 PAMR1 中存在三重O-糖基化 EGF-LD 一致。