The specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase as inferred from a database of in vivo substrates and from the in vitro glycosylation of proteins and peptides.
The specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase as inferred from a database of in vivo substrates and from the in vitro glycosylation of proteins and peptides.
复制标题
DOI：
10.1016/s0021-9258(18)82168-8
复制
发表时间：
1993-05
期刊：
影响因子：
--
通讯作者：
Å. Elhammer;R. Poorman;E. Brown;L. Maggiora;J. G. Hoogerheide;F. Kézdy
中科院分区：
文献类型：
--
作者：
Å. Elhammer;R. Poorman;E. Brown;L. Maggiora;J. G. Hoogerheide;F. Kézdy
UDP-GalNAc的受体底物特异性：多肽N-乙酰氨基半乳糖转移酶（GalNAc-转移酶）从国家生物医学研究基金会蛋白质数据库中提取的196个O-糖基化位点周围的氨基酸序列推断。当根据累积酶特异性模型（Poorman，R.A.，Tomasselli，A.G.，Heinrikson，R.L.，和Kézdy，F.J.（1991）J.Biol.Chem.266，14554 - 14561），发现这些数据与酶活性位点一致，所述酶活性位点与底物的8-氨基酸长片段相互作用，跨越反应性丝氨酸或苏氨酸之前的3个氨基酸残基和之后的4个氨基酸残基。该模型假定8个氨基酸部分与其各自的结合位点（指定为亚位点P3至P0和P1 '至P4'）的独立相互作用。高选择性表达在所有亚位点对丝氨酸，苏氨酸和脯氨酸。通过体外牛初乳GalNAc-转移酶催化的糖基化未糖基化蛋白质（含有预测的O-糖基化位点）和设计为GalNAc受体的合成肽证实了推断的特异性。在合成肽中，牛初乳GalNAc-转移酶使苏氨酸糖基化的速度比丝氨酸快约35倍。我们的研究结果表明，酶的特异性是不依赖于任何特定的二级结构的基板，但相反，它是由受体肽段中的氨基酸，以及由该区段的可访问性。牛初乳GalNAc-转移酶似乎也能够在体内催化苏氨酸和丝氨酸残基的糖基化。
The acceptor substrate specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) was inferred from the amino acid sequences surrounding 196 O-glycosylation sites extracted from the National Biomedical Research Foundation Protein Database. When analyzed according to the cumulative enzyme specificity model (Poorman, R.A., Tomasselli, A.G., Heinrikson, R.L., and Kézdy, F.J. (1991) J. Biol. Chem. 266, 14554-14561) these data were found to be consistent with an enzymatic active site which interacts with an 8-amino-acid long segment of the substrate, spanning 3 amino acid residues preceding and 4 amino acid residues following the reactive serine or threonine. The model postulates independent interactions of the 8 amino acid moieties with their respective binding sites, designated as subsites P3 through P0 and P1‘ to P4‘. High selectivity is expressed at all subsites toward serine, threonine, and proline. The inferred specificity was confirmed by in vitro bovine colostrum GalNAc-transferase-catalyzed glycosylation of unglycosylated proteins containing predicted sites for O-glycosylation and synthetic peptides designed to be GalNAc acceptors. In synthetic peptides the bovine colostrum GalNAc-transferase glycosylates threonine about 35 times faster than serine. Our results suggest that the specificity of the enzyme is not dependent on any particular secondary structure of the substrate but, rather, it is determined by the amino acids in the acceptor peptide segment as well as by the accessibility of this segment. It also appears likely that bovine colostrum GalNAc-transferase is able to catalyze in vivo the glycosylation of both threonine and serine residues.