Role of peptide sequence and neighboring residue glycosylation on the substrate specificity of the uridine 5′-diphosphate-α-N-acetylgalactosamine:: Polypeptide N-acetylgalactosaminyl transferases T1 and T2:: Kinetic modeling of the porcine and canine submaxillary gland mucin tandem repeats
Role of peptide sequence and neighboring residue glycosylation on the substrate specificity of the uridine 5′-diphosphate-α-N-acetylgalactosamine:: Polypeptide N-acetylgalactosaminyl transferases T1 and T2:: Kinetic modeling of the porcine and canine submaxillary gland mucin tandem repeats
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DOI：
10.1021/bi049178e
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发表时间：
2004-08-03
期刊：
影响因子：
2.9
通讯作者：
Rarick, J
中科院分区：
文献类型：
--
作者：
Gerken, TA;Tep, C;Rarick, J
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）的大家族在丝氨酸和苏氨酸处启动粘蛋白型0-聚糖生物合成。单个家族成员的肽底物特异性没有得到很好的表征或理解，从而无法合理地预测或理解O-糖基化位点。最近，动力学建模方法证明相邻残基糖基化是通过ppGalNAc T1和T2调节猪颌下腺粘蛋白81残基串联重复的O-糖基化的主要因素[Gerken等（2002）J.Biol.Chem.277，49850-49862]。为了确认该模型及其参数的普遍适用性，获得并表征了来自犬颌下腺粘蛋白的80+残基串联重复序列的ppGalNAc T1和T2糖基化动力学。为了再现两种粘蛋白（包含50+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的影响之外，模型还需要相邻肽序列的特定影响。这两种转移酶的特异性的差异被定义为他们的敏感性相邻的脯氨酸和nonglycosylated羟基氨基酸残基，从其中一个ppGalNAc T2基序被确定。重要的是，该模型可以近似先前报道的IgAl铰链结构域肽的ppGalNAc T2糖基化动力学[Iwasaki，et al.（2003）J.Biol.Chem.278，5613-5621]，进一步验证了该方法和ppGalNAc T2位置加权参数。通过这种方法的ppGalNAc转移酶特异性的表征可能证明是有用的异构体特异性底物的搜索，异构体特异性抑制剂的创建，和粘蛋白型O-糖基化位点的预测。
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 IgAl 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.