A large family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) initiate mucin type O-glycosylation transferring α-GalNAc from a UDP-GalNAc donor to the hydroxyl groups of Ser and Thr residues of peptides and proteins, thereby defining sites of O-glycosylation. Mutations and differential expression of several GalNAc-Ts are associated with many disease states including cancers. The mechanisms by which these isozymes choose their targets and their roles in disease are not fully understood. We previously showed that the GalNAc-Ts possess common and unique specificities for acceptor type, peptide sequence and prior neighboring, and/or remote substrate GalNAc glycosylation. In the present study, the role of flanking charged residues was investigated using a library of charged peptide substrates containing the central -YAVTPGP- acceptor sequence. Eleven human and one bird GalNAc-T were initially characterized revealing a range of preferences for net positive, net negative, or unique combinations of flanking N- and/or C-terminal charge, correlating to each isozyme's different electrostatic surface potential. It was further found that isoforms with high sequence identity (>70%) within a subfamily can possess vastly different charge specificities. Enzyme kinetics, activities obtained at elevated ionic strength, and molecular dynamics simulations confirm that the GalNAc-Ts differently recognize substrate charge outside the common +/-3 residue binding site. These electrostatic interactions impact how charged peptide substrates bind/orient on the transferase surface, thus modulating their activities. In summary, we show the GalNAc-Ts utilize more extended surfaces than initially thought for binding substrates based on electrostatic, and likely other hydrophobic/hydrophilic interactions, furthering our understanding of how these transferases select their target.

中文翻译：

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多肽 N-乙酰半乳糖胺基转移酶 (GalNAc-T) 同工酶表面电荷控制电荷底物偏好，以调节粘蛋白型 O-糖基化。

一个大家族的多肽 N-乙酰半乳糖胺基转移酶 (GalNAc-Ts) 启动粘蛋白型 O-糖基化，将 α-GalNAc 从 UDP-GalNAc 供体转移至肽和蛋白质的 Ser 和 Thr 残基的羟基，从而定义 O-糖基化位点。几种 GalNAc-T 的突变和差异表达与包括癌症在内的许多疾病状态相关。这些同工酶选择靶标的机制及其在疾病中的作用尚不完全清楚。我们之前表明，GalNAc-T 对受体类型、肽序列和先前邻近和/或远程底物 GalNAc 糖基化具有共同和独特的特异性。在本研究中，使用含有中央-YAVTPGP-受体序列的带电肽底物文库研究了侧翼带电残基的作用。最初对 11 个人和一只鸟 GalNAc-T 进行了表征，揭示了对净正、净负或侧翼 N 和/或 C 端电荷的独特组合的一系列偏好，与每种同工酶的不同静电表面电位相关。进一步发现，亚家族内具有高序列同一性（>70%）的异构体可以具有截然不同的电荷特异性。酶动力学、在升高的离子强度下获得的活性以及分子动力学模拟证实，GalNAc-Ts 不同地识别常见+/-3残基结合位点之外的底物电荷。这些静电相互作用影响带电肽底物在转移酶表面上的结合/定向方式，从而调节它们的活性。总之，我们表明 GalNAc-T 利用比最初想象的更多的扩展表面来基于静电和可能的其他疏水/亲水相互作用结合底物，进一步加深了我们对这些转移酶如何选择其靶标的理解。