The polypeptide N-acetylgalactosaminyl transferase (GalNAc-T) enzyme family initiates O-linked mucin-type glycosylation. The family constitutes 20 isoenzymes in humans. GalNAc-Ts exhibit both redundancy and finely tuned specificity for a wide range of peptide substrates. In this work, we deciphered the sequence and structural motifs that determine the peptide substrate preferences for the GalNAc-T2 isoform. Our approach involved sampling and characterization of peptide–enzyme conformations obtained from Rosetta Monte Carlo-minimization-based flexible docking. We computationally scanned 19 amino acid residues at positions −1 and +1 of an eight-residue peptide substrate, which comprised a dataset of 361 (19 × 19) peptides with previously characterized experimental GalNAc-T2 glycosylation efficiencies. The calculations recapitulated experimental specificity data, successfully discriminating between glycosylatable and nonglycosylatable peptides with a probability of 96.5% (ROC-AUC score), a balanced accuracy of 85.5%, and a false positive rate of 7.3%. The glycosylatable peptide substrates viz. peptides with proline, serine, threonine, and alanine at the −1 position of the peptide preferentially exhibited cognate sequon-like conformations. The preference for specific residues at the −1 position of the peptide was regulated by enzyme residues R362, K363, Q364, H365, and W331, which modulate the pocket size and specific enzyme–peptide interactions. For the +1 position of the peptide, enzyme residues K281 and K363 formed gating interactions with aromatics and glutamines at the +1 position of the peptide, leading to modes of peptide binding suboptimal for catalysis. Overall, our work revealed enzyme features that lead to the finely tuned specificity observed for a broad range of peptide substrates for the GalNAc-T2 enzyme. We anticipate that the key sequence and structural motifs can be extended to analyze specificities of other isoforms of the GalNAc-T family and can be used to guide design of variants with tailored specificity.

中文翻译：

---

糖基转移酶 GalNAc-T2 肽底物特异性的结构基础

多肽N-乙酰半乳糖胺基转移酶 (GalNAc-T) 酶家族启动O-连接的粘蛋白型糖基化。该家族在人类中构成 20 种同工酶。GalNAc-Ts 对广泛的肽底物表现出冗余和微调的特异性。在这项工作中，我们破译了确定 GalNAc-T2 亚型的肽底物偏好的序列和结构基序。我们的方法涉及对从基于 Rosetta Monte Carlo 最小化的灵活对接获得的肽酶构象进行采样和表征。我们计算扫描了八残基肽底物 -1 和 +1 位的 19 个氨基酸残基，该底物包含 361 个（19 × 19）肽的数据集，具有先前表征的实验性 GalNAc-T2 糖基化效率。计算概括了实验特异性数据，成功区分糖基化和非糖基化肽的概率为 96.5%（ROC-AUC 评分），平衡准确率为 85.5%，假阳性率为 7.3%。糖基化肽底物即。在肽的-1位置具有脯氨酸、丝氨酸、苏氨酸和丙氨酸的肽优先表现出同源序列样构象。对肽 -1 位特定残基的偏好受酶残基 R362、K363、Q364、H365 和 W331 的调节，这些残基调节口袋大小和特定的酶-肽相互作用。对于肽的 +1 位置，酶残基 K281 和 K363 在肽的 +1 位置与芳香族化合物和谷氨酰胺形成门控相互作用，导致肽结合模式对催化而言不是最佳的。全面的，我们的工作揭示了酶的特征，这些特征导致对 GalNAc-T2 酶的广泛肽底物观察到的精细调整的特异性。我们预计关键序列和结构基序可以扩展以分析 GalNAc-T 家族其他同种型的特异性，并可用于指导具有定制特异性的变体设计。