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Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4
ACS Central Science ( IF 12.7 ) Pub Date : 2018-09-14 00:00:00 , DOI: 10.1021/acscentsci.8b00488 Matilde de las Rivas 1 , Earnest James Paul Daniel 2 , Helena Coelho 3, 4, 5 , Erandi Lira-Navarrete 6 , Lluis Raich 7 , Ismael Compañón 8 , Ana Diniz 3 , Laura Lagartera 9 , Jesús Jiménez-Barbero 4, 5, 10 , Henrik Clausen 6 , Carme Rovira 7, 11 , Filipa Marcelo 3 , Francisco Corzana 8 , Thomas A. Gerken 2 , Ramon Hurtado-Guerrero 1, 12
ACS Central Science ( IF 12.7 ) Pub Date : 2018-09-14 00:00:00 , DOI: 10.1021/acscentsci.8b00488 Matilde de las Rivas 1 , Earnest James Paul Daniel 2 , Helena Coelho 3, 4, 5 , Erandi Lira-Navarrete 6 , Lluis Raich 7 , Ismael Compañón 8 , Ana Diniz 3 , Laura Lagartera 9 , Jesús Jiménez-Barbero 4, 5, 10 , Henrik Clausen 6 , Carme Rovira 7, 11 , Filipa Marcelo 3 , Francisco Corzana 8 , Thomas A. Gerken 2 , Ramon Hurtado-Guerrero 1, 12
Affiliation
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Mucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (containing two α-GalNAc glycosylated Thr (T*), the PXP motif and a “naked” Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.
中文翻译：
GalNAc-T4催化域介导的短程糖基化偏好的结构和机理研究
粘蛋白O型-糖基化由多肽GalNAc-转移酶（GalNAc-Ts）家族起始，其是II型跨膜蛋白，其包含通过柔性接头连接的高尔基体腔催化和凝集素结构域。包括GalNAc-T4在内的几种GalNAc-Ts均显示长程和短程先验糖基化特异性，分别受其凝集素和催化结构域支配。尽管凝集素结构域依赖性糖基化的机理是众所周知的，但是糖肽的催化结构域依赖性糖基化的分子基础尚不清楚。在这里，我们报告与二糖肽GAT * GAGAGAGT * TPGPG结合的GalNAc-T4的晶体结构（包含两个α-GalNAc糖基化Thr（T *），PXP基序和一个“裸” Thr受体位点），描述了其催化结构域糖肽GalNAc结合位点。对野生型和GalNAc结合位点突变酶的动力学研究表明，凝集素结构域GalNAc结合活性比催化结构域GalNAc结合活性更重要，并且这些活性可以独立消除。令人惊讶地，发现从凝集素结构域突出的柔性环对于催化结构域的最佳活性是必不可少的。这项工作为GalNAc-T的短程糖基化偏好提供了第一个结构基础。
更新日期：2018-09-14
中文翻译：
GalNAc-T4催化域介导的短程糖基化偏好的结构和机理研究
粘蛋白O型-糖基化由多肽GalNAc-转移酶（GalNAc-Ts）家族起始，其是II型跨膜蛋白，其包含通过柔性接头连接的高尔基体腔催化和凝集素结构域。包括GalNAc-T4在内的几种GalNAc-Ts均显示长程和短程先验糖基化特异性，分别受其凝集素和催化结构域支配。尽管凝集素结构域依赖性糖基化的机理是众所周知的，但是糖肽的催化结构域依赖性糖基化的分子基础尚不清楚。在这里，我们报告与二糖肽GAT * GAGAGAGT * TPGPG结合的GalNAc-T4的晶体结构（包含两个α-GalNAc糖基化Thr（T *），PXP基序和一个“裸” Thr受体位点），描述了其催化结构域糖肽GalNAc结合位点。对野生型和GalNAc结合位点突变酶的动力学研究表明，凝集素结构域GalNAc结合活性比催化结构域GalNAc结合活性更重要，并且这些活性可以独立消除。令人惊讶地，发现从凝集素结构域突出的柔性环对于催化结构域的最佳活性是必不可少的。这项工作为GalNAc-T的短程糖基化偏好提供了第一个结构基础。
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