Enhanced antibody-defucosylation capability of α-L-fucosidase by proximity-based protein fusion.
Enhanced antibody-defucosylation capability of α-L-fucosidase by proximity-based protein fusion.
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DOI：
10.1016/j.bbrc.2023.01.031
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发表时间：
2023-01
影响因子：
3.1
通讯作者：
Shuquan Fan;Wanzhen Li;Kuixing Zhang;Xiangman Zou;Weiwei Shi;Zhi Liu;Caihong Tang;Wei Huang;Feng Tang
中科院分区：
文献类型：
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作者：
Shuquan Fan;Wanzhen Li;Kuixing Zhang;Xiangman Zou;Weiwei Shi;Zhi Liu;Caihong Tang;Wei Huang;Feng Tang
迄今为止，报道的岩藻糖苷酶通常对IgG核心岩藻糖显示出较差的活性，这限制了ENGase催化的糖工程过程的效率。然而，EndoS或EndoS 2对IgG的N-糖基化具有很高的活性和选择性，并且它们的非催化结构域与IgG Fc结构域具有特异性的相互作用，从而产生了很高的活性和选择性。本研究利用AlfC取代EndoS/S2的GH（glycan hydrolysis）催化结构域，构建了一系列含有EndoS/S2非催化结构域的AlfC融合蛋白。我们发现所有这些融合的AlfC对去糖基化的IgG（Fucα1，6 GlcNAc-IgG）显示出显著增强的去岩藻糖基化活性。我们还进行了这些融合酶的动力学研究，我们的结果倾向于告诉EndoS为基础的融合蛋白具有较高的kcat值，而EndoS 2为基础的具有较低的Km值，而不是较高的kcat。本研究为提高AlfC的活性提供了一种有效的途径，并在几分钟内显著缩短了AlfC的去岩藻糖基化过程，这将极大地促进糖工程抗体的发展。
Up to date, the reported fucosidases generally show poor activities toward the IgG core-fucose, which limits the efficiency of ENGase-catalyzed glycoengineering process. However, EndoS or EndoS2 owns excellent activity and great selectivity towards the N-glycosylation of IgGs, and their non-catalytic domains are deduced to have specific interactions to IgG Fc domain that result in the great activity and selectivity. Herein, we constructed a series fusion protein of AlfC (an α-l-fucosidase fromLactobacillus caseiBL23) with EndoS/S2 non-catalytic domain by replacing the catalytic GH (glycan hydrolase) domain of EndoS/S2 with the AlfC. We found that all these fused AlfCs showed significantly enhanced defucosylation activity toward the deglycosylated IgGs (Fucα1,6GlcNAc-IgG). We also performed the kinetic study of these fusion enzymes, and our results tend to tell that the EndoS-based fusion proteins have higherkcatvalues while the EndoS2-based ones possess lowerKmvalues other than higherkcat. Conclusively, our research provides an effective approach to improve the activity of AlfC and remarkably shortened the defucosylation process within several minutes, which will significantly promote the development of glycoengineered antibodies in the future.