The influence of flanking sequence on the O-glycosylation of threonine in vitro.
The influence of flanking sequence on the O-glycosylation of threonine in vitro.
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DOI：
10.1016/s0021-9258(19)73998-2
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发表时间：
1992-12
期刊：
影响因子：
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通讯作者：
B. O'Connell;F. Hagen;L. Tabak
中科院分区：
文献类型：
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作者：
B. O'Connell;F. Hagen;L. Tabak
为了研究侧翼氨基酸序列对单个苏氨酸残基体外O-糖基化的影响，我们研究了一系列52个相关的多肽。底物基于已知在体内糖基化的人von Willebrand因子的序列(-6PHMAQVTVGPGL+5)。母肽的每个残基依次被异亮氨酸、丙氨酸、脯氨酸、谷氨酸或精氨酸取代。用UDP-GalNAc：多肽N-乙酰半乳糖胺转移酶从牛初乳中纯化了15,000倍的多肽N-乙酰半乳糖氨基转移酶，经DEAE-Sephacel、SP-Sephadex、Sephacryl S-300、Affi-Gel Blue和5-Mercuri-UDP-GalNAc硫丙基-Sepharose柱层析，得到了糖基化的多肽。苏氨酸侧翼序列中单个氨基酸的变化可以深刻地改变底物多肽的糖基化。在+3、-3和-2位测试的任何氨基酸的取代显著降低O-糖基化，-1位的带电残基的存在也是如此。肽底物上其他位置的氨基酸取代对GalNAc的掺入影响不大。对已知糖基化苏氨酸和丝氨酸残基两侧序列的统计分析表明，在相同的序列背景下，它们应该以同样的效率被糖基化(O‘Connell等人，1991年)。然而，牛初乳转移酶不能糖基化来自人促红细胞生成素的多肽，该多肽含有体内糖基化的丝氨酸(-5PPDAASAAPLR+5)。当苏氨酸取代该肽(-5PPDAATAAPLR+5)中的丝氨酸时，该底物被证明是GalNAc的良好受体。这些观察表明，虽然侧翼氨基酸序列对于特定羟基氨基酸的O-糖基化很重要，但可能存在离散的苏氨酸和丝氨酸特异性转移酶。
To investigate the influence of flanking amino acid sequence on the O-glycosylation of a single threonine residue in vitro, we have examined a series of 52 related peptides. The substrates were based upon a sequence from human von Willebrand factor which is known to be glycosylated in vivo (-6PHMAQVTVGPGL+5). Each residue of the parent peptide was substituted, in turn, with isoleucine, alanine, proline, glutamic acid, or arginine. Peptides were glycosylated using a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase purified 15,000-fold from bovine colostrum by chromatography on DEAE-Sephacel, SP-Sephadex, Sephacryl S-300, Affi-Gel Blue, and 5-mercuri-UDP-GalNAc thiopropyl-Sepharose. Single amino acid changes in the sequences flanking the threonine could profoundly alter the glycosylation of the substrate peptides. Substitution of any amino acid tested at positions +3, -3, and -2 markedly decreased O-glycosylation, as did the presence of a charged residue at position -1. The substitution of amino acids at the other positions of the peptide substrate had little effect on the incorporation of GalNAc. Statistical analysis of sequences flanking known glycosylated threonine and serine residues suggests that they should be glycosylated with equal efficiency in the same sequence context (O'Connell et al., 1991). However, the bovine colostrum transferase failed to glycosylate a peptide derived from human erythropoietin which contains a serine that is glycosylated in vivo (-5PPDAASAAPLR+5). When a threonine was substituted for the serine in this peptide (-5PPDAATAAPLR+5), the substrate proved to be an excellent acceptor of GalNAc. These observations indicate that although flanking amino acid sequence is important for the O-glycosylation of specific hydroxyamino acids, discrete threonine- and serine-specific transferases may exist.