Gerken Thomas A, Tep Chhavy, Rarick Jason
W. A. Bernbaum Center for Cystic Fibrosis Research, Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4948, USA.
Biochemistry. 2004 Aug 3;43(30):9888-900. doi: 10.1021/bi049178e.
A large family of uridine 5'-diphosphate (UDP)-alpha-N-acetylgalactosamine (GalNAc):polypeptide N-acetylgalactosaminyl transferases (ppGalNAc Ts) initiates mucin-type O-glycan biosynthesis at serine and threonine. The peptide substrate specificities of individual family members are not well characterized or understood, leaving an inability to rationally predict or comprehend sites of O-glycosylation. Recently, a kinetic modeling approach demonstrated neighboring residue glycosylation as a major factor modulating the O-glycosylation of the porcine submaxillary gland mucin 81 residue tandem repeat by ppGalNAc T1 and T2 [Gerken et al. (2002) J. Biol. Chem. 277, 49850-49862]. To confirm the general applicability of this model and its parameters, the ppGalNAc T1 and T2 glycosylation kinetics of the 80+ residue tandem repeat from the canine submaxillary gland mucin was obtained and characterized. To reproduce the glycosylation patterns of both mucins (comprising 50+ serine/threonine residues), specific effects of neighboring peptide sequence, in addition to the previously described effects of neighboring residue glycosylation, were required of the model. Differences in specificity of the two transferases were defined by their sensitivities to neighboring proline and nonglycosylated hydroxyamino acid residues, from which a ppGalNAc T2 motif was identified. Importantly, the model can approximate the previously reported ppGalNAc T2 glycosylation kinetics of the IgA1 hinge domain peptide [Iwasaki, et al. (2003) J. Biol. Chem. 278, 5613-5621], further validating both the approach and the ppGalNAc T2 positional weighting parameters. The characterization of ppGalNAc transferase specificity by this approach may prove useful for the search for isoform-specific substrates, the creation of isoform-specific inhibitors, and the prediction of mucin-type O-glycosylation sites.
尿苷5'-二磷酸（UDP）-α-N-乙酰半乳糖胺（GalNAc）：多肽N-乙酰半乳糖胺基转移酶（ppGalNAc Ts）的一个大家族在丝氨酸和苏氨酸处启动粘蛋白型O-聚糖生物合成。各个家族成员的肽底物特异性尚未得到很好的表征或理解，因此无法合理预测或理解O-糖基化位点。最近，一种动力学建模方法表明，相邻残基糖基化是调节ppGalNAc T1和T2对猪下颌下腺粘蛋白81个残基串联重复序列进行O-糖基化的主要因素[Gerken等人（2002年）《生物化学杂志》277, 49850 - 49862]。为了证实该模型及其参数的普遍适用性，我们获得并表征了犬下颌下腺粘蛋白80多个残基串联重复序列的ppGalNAc T1和T2糖基化动力学。为了重现两种粘蛋白（包含50多个丝氨酸/苏氨酸残基）的糖基化模式，除了先前描述的相邻残基糖基化效应外，模型还需要考虑相邻肽序列的特定效应。两种转移酶的特异性差异通过它们对相邻脯氨酸和非糖基化羟基氨基酸残基的敏感性来定义，据此确定了一个ppGalNAc T2基序。重要的是，该模型可以近似先前报道的IgA1铰链区肽的ppGalNAc T2糖基化动力学[Iwasaki等人（2003年）《生物化学杂志》278, 5613 - 5621]，进一步验证了该方法和ppGalNAc T2位置加权参数。通过这种方法对ppGalNAc转移酶特异性进行表征，可能有助于寻找同工型特异性底物、开发同工型特异性抑制剂以及预测粘蛋白型O-糖基化位点。
