One hundred human proteins have one or more EGF-like domains (EGF-LD) bearing the O-fucosylation consensus motif C²X₄(S/T)C³ but to date, only a few of them have been shown to be O-fucosylated. The protein O-fucosyltransferase (POFUT1) specifically recognizes correctly folded EGF-LD of the human EGF (hEGF) type and transfers fucose on serine or threonine residue within the O-fucosylation motif. Here, we propose a strategy for a rapid screening for ability of any EGF-LD to be O-fucosylated, using copper-catalyzed azide-alkyne cycloaddition (CuAAC). By an oligonucleotide hybridization approach, double-stranded fragments encoding any EGF-LD can be first rapidly cloned into the prokaryotic vector pET-25b to promote its targeting to periplasm and formation of the three conserved disulfide bonds. After protein production and purification, an in vitro POFUT1-mediated O-fucosylation can be performed with azido GDP-fucose. Successful transfer of O-fucose is finally revealed by blotting after CuAAC. In this study, we specially focused on mouse NOTCH1 EGF12 and EGF26, which are both known to be O-fucosylated although having different binding affinities towards POFUT1. Indeed, we clearly showed here that addition of O-fucose by POFUT1 was much more efficient for EGF26 than for EGF12. This experimental approach is rapid and sufficiently sensitive to reveal propensity of any EGF-LD to be O-fucosylated; it is thus useful prior to perform structure–function studies on target proteins containing one or several EGF-LD.

中文翻译：

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体外非细胞方法揭示 EGF 样结构域的 O-岩藻糖基化


一百种人类蛋白质具有一个或多个带有O -岩藻糖基化共有基序 C ² X ₄ ( S / T )C ³的 EGF 样结构域 (EGF-LD)，但迄今为止，其中只有少数已被证明是O -岩藻糖基化。蛋白质O -岩藻糖基转移酶 (POFUT1) 特异性识别正确折叠的人 EGF (hEGF) 类型的 EGF-LD，并将岩藻糖转移到O -岩藻糖基化基序内的丝氨酸或苏氨酸残基上。在这里，我们提出了一种使用铜催化叠氮化物-炔环加成 (CuAAC) 快速筛选任何 EGF-LD 被O -岩藻糖基化的能力的策略。通过寡核苷酸杂交方法，可以首先将编码任意EGF-LD的双链片段快速克隆到原核载体pET-25b中，以促进其靶向周质并形成三个保守的二硫键。蛋白质生产和纯化后，可以使用叠氮基 GDP-岩藻糖进行体外 POFUT1 介导的O-岩藻糖基化。 CuAAC 后的印迹最终显示O-岩藻糖的成功转移。在本研究中，我们特别关注小鼠 NOTCH1 EGF12 和 EGF26，尽管它们对 POFUT1 具有不同的结合亲和力，但已知它们均被O-岩藻糖基化。事实上，我们在这里清楚地表明，通过 POFUT1 添加O-岩藻糖对于 EGF26 比 EGF12 更有效。该实验方法快速且足够灵敏，可揭示任何 EGF-LD 被O-岩藻糖基化的倾向；因此，在对含有一种或多种 EGF-LD 的靶蛋白进行结构-功能研究之前，它非常有用。