Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.
Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.
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DOI：
10.1016/j.xpro.2022.101974
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发表时间：
2023-03-17
期刊：
影响因子：
--
通讯作者：
Schumann, Benjamin
中科院分区：
文献类型：
--
作者：
Calle, Beatriz;Gonzalez-Rodriguez, Edgar;Mahoney, Keira E.;Cioce, Anna;Bineva-Todd, Ganka;Tastan, Omur Y.;Roustan, Chloe;Flynn, Helen;Malaker, Stacy A.;Schumann, Benjamin
尽管已知聚糖与疾病相关，但个体糖基转移酶的生物学功能和底物特异性往往不明确。在这里，我们描述了一种开发GalNAc-T糖基转移酶家族活性的化学、生物正交报告的方案，使用一种称为凹凸孔工程的策略。这允许鉴定单个GalNAc-Ts的蛋白质底物和糖基化位点。尽管需要用工程转移酶和酶转染细胞来进行生物正交底物的生物合成，但该策略补充了分子生物学中的方法。关于该方案使用和执行的完整细节，请参见Schumann等人（2020），Cioce等人（2021）和Cioce等人（2022）GalNAc-T糖基转移酶的碰撞孔报告系统的产生生物合成生物正交底物类似物的人工生物合成途径在活细胞中生物正交标记的单个GalNAc-Ts的蛋白质底物通过ms -糖蛋白组学实现底物鉴定和聚糖位点定位：执行任何实验方案都需要遵守当地机构关于实验室安全和伦理的指导方针。尽管已知聚糖与疾病相关，但个体糖基转移酶的生物学功能和底物特异性往往不明确。在这里，我们描述了一种开发GalNAc-T糖基转移酶家族活性的化学、生物正交报告的方案，使用一种称为凹凸孔工程的策略。这允许鉴定单个GalNAc-Ts的蛋白质底物和糖基化位点。尽管需要用工程转移酶和酶转染细胞来进行生物正交底物的生物合成，但该策略补充了分子生物学中的方法。
Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineered transferases and enzymes for biosynthesis of bioorthogonal substrates, the tactic complements methods in molecular biology. For complete details on the use and execution of this protocol, please refer to Schumann et al. (2020), Cioce et al. (2021), and Cioce et al. (2022) Generation of bump-and-hole reporter systems for GalNAc-T glycosyltransferases Artificial biosynthetic pathway to biosynthesize bioorthogonal substrate analogues Protein substrates of individual GalNAc-Ts bioorthogonally tagged in living cells Substrate identification and glycan site localization achieved by MS-glycoproteomics Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineered transferases and enzymes for biosynthesis of bioorthogonal substrates, the tactic complements methods in molecular biology.
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影响因子：
16.6
作者：
de Las Rivas M;Lira-Navarrete E;Daniel EJP;Compañón I;Coelho H;Diniz A;Jiménez-Barbero J;Peregrina JM;Clausen H;Corzana F;Marcelo F;Jiménez-Osés G;Gerken TA;Hurtado-Guerrero R
通讯作者：
Hurtado-Guerrero R
影响因子：
14.9
作者：
Perez-Riverol Y;Bai J;Bandla C;García-Seisdedos D;Hewapathirana S;Kamatchinathan S;Kundu DJ;Prakash A;Frericks-Zipper A;Eisenacher M;Walzer M;Wang S;Brazma A;Vizcaíno JA
通讯作者：
Vizcaíno JA
影响因子：
16.6
作者：
通讯作者：
--
影响因子：
3.5
作者：
Bennett, EP;Hassan, H;Clausen, H
通讯作者：
Clausen, H
鉴定具有独特催化活性和表达谱的新型人 UDP-GalNAc 转移酶
DOI：
10.1016/j.bbrc.2010.10.084
发表时间：
2010-11-26
影响因子：
3.1
作者：
Peng, Can;Togayachi, Akira;Zhang, Yan
通讯作者：
Zhang, Yan