The catalytic and lectin domains of UDP-GalNAc:: Polypeptide α-N-acetylgalactosaminyltransferase function in concert to direct glycosylation site selection
The catalytic and lectin domains of UDP-GalNAc:: Polypeptide α-N-acetylgalactosaminyltransferase function in concert to direct glycosylation site selection
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DOI：
10.1074/jbc.m803387200
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发表时间：
2008-08-22
影响因子：
4.8
通讯作者：
Tabak, Lawrence A.
中科院分区：
文献类型：
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作者：
Raman, Jayalakshmi;Fritz, Timothy A.;Tabak, Lawrence A.
UDP-GalNAc：多肽α-N-乙酰半乳糖胺转移酶（ppGalNAcTs），启动粘蛋白型O-糖基化的酶家族（EC2.4.1.41），在结构上由催化结构域和凝集素结构域组成。以前的研究表明，凝集素结构域调节糖肽底物的糖基化，并可能成为某些同种型（ppGalNAcT-7和-10）的严格糖肽特异性的基础。使用一组基于粘蛋白MUC 5AC的序列的合成肽和糖肽，我们已经检查了凝集素结构域缺失和肽/糖肽转移酶ppGalNAcT-2（hT 2）和糖肽转移酶ppGal-NAcT- 10（hT 10）的交换构建体的活性和糖基化位点偏好。我们证明，hT 2的凝集素结构域指导糖肽底物的糖基化位点选择。前稳态动力学测量表明，这种效果是由于两种机制，无论是凝集素域辅助底物结合或凝集素域辅助糖基化后的产物释放。我们发现，hT 10的肽底物的糖基化需要结合现有的GalNAc的底物上的催化或凝集素结构域，从而导致其明显的严格的糖肽特异性。这些结果突出了这些ppGalNAcTs使用的两种模式的位点选择的存在：由催化结构域的本地序列识别和先前糖基化连同本地序列结合介导的远端位点的协同识别，分别由凝集素和催化结构域。后一种模式可以促进丝氨酸或苏氨酸残基的糖基化，其发生在单独的催化结构域不能有效糖基化的序列环境中。催化结构域的局部序列识别在hT 2和hT 10之间的不同之处在于hT 10需要预先存在的GalNAc残基，而hT 2不需要。
UDP-GalNAc: polypeptide alpha-N-Acetylgalactosaminyltransferases (ppGalNAcTs), a family (EC 2.4.1.41) of enzymes that initiate mucin-type O-glycosylation, are structurally composed of a catalytic domain and a lectin domain. Previous studies have suggested that the lectin domain modulates the glycosylation of glycopeptide substrates and may underlie the strict glycopeptide specificity of some isoforms (ppGalNAcT-7 and -10). Using a set of synthetic peptides and glycopeptides based upon the sequence of the mucin, MUC5AC, we have examined the activity and glycosylation site preference of lectin domain deletion and exchange constructs of the peptide/glycopeptide transferase ppGalNAcT-2 (hT2) and the glycopeptide transferase ppGal-NAcT- 10 (hT10). We demonstrate that the lectin domain of hT2 directs glycosylation site selection for glycopeptide substrates. Pre-steady-state kinetic measurements show that this effect is attributable to two mechanisms, either lectin domain-aided substrate binding or lectin domain-aided product release following glycosylation. We find that glycosylation of peptide substrates by hT10 requires binding of existing GalNAcs on the substrate to either its catalytic or lectin domain, thereby resulting in its apparent strict glycopeptide specificity. These results highlight the existence of two modes of site selection used by these ppGalNAcTs: local sequence recognition by the catalytic domain and the concerted recognition of distal sites of prior glycosylation together with local sequence binding mediated, respectively, by the lectin and catalytic domains. The latter mode may facilitate the glycosylation of serine or threonine residues, which occur in sequence contexts that would not be efficiently glycosylated by the catalytic domain alone. Local sequence recognition by the catalytic domain differs between hT2 and hT10 in that hT10 requires a pre-existing GalNAc residue while hT2 does not.