Enzymatic addition of O-GlcNAc to nuclear and cytoplasmic proteins. Identification of a uridine diphospho-N-acetylglucosamine:peptide beta-N-acetylglucosaminyltransferase.
Enzymatic addition of O-GlcNAc to nuclear and cytoplasmic proteins. Identification of a uridine diphospho-N-acetylglucosamine:peptide beta-N-acetylglucosaminyltransferase.
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DOI：
10.1016/s0021-9258(19)39838-2
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发表时间：
1990-02
期刊：
影响因子：
--
通讯作者：
R. S. Haltiwanger;G. D. Holt;G. Hart
中科院分区：
文献类型：
--
作者：
R. S. Haltiwanger;G. D. Holt;G. Hart
以合成肽YSDSPSTST为受体底物，测定了蛋白质中负责O-连接N-乙酰氨基葡萄糖(O-GlcNAc)加成的酶--UDP-N-乙酰氨基葡萄糖：N-乙酰氨基葡萄糖转移酶。该酶的活性与时间、酶和底物浓度呈线性关系。用甘氨酸取代肽中的脯氨酸使其作为底物无效，而将天冬氨酸改变为甘氨酸则没有影响。产物表征表明，糖基化多肽共价结合的单糖为N-乙酰氨基葡萄糖(GlcNAc)，尚未异构化为N-乙酰半乳糖胺。在温和的碱催化下，体外糖基化多肽的β消除定量产生GlcNAcitol，表明GlcNAc是通过O-键连接的。转移酶活性受UDP强烈抑制，但不受GlcNAc和衣霉素的影响。有趣的是，EDTA只略微抑制活性，这表明该酶可能不需要二价阳离子。大部分活性是可溶的，其余的活性在高盐和EDTA提取后从膜上消失。与大多数含有O-GlcNAc的蛋白质的亚细胞定位一致，与半乳糖基转移酶和甘露糖-6-磷酸酶相比，O-GlcNAc的活性似乎驻留在细胞的胞浆部分。
An assay for the enzyme responsible for the addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins, a UDP-N-acetylglucosamine:peptide N-acetylglucosaminyltransferase, is reported using the synthetic peptide YSDSPSTST as the acceptor substrate. The activity is linearly dependent on time, enzyme, and substrate concentration. Replacement of the proline with a glycine in the peptide renders it ineffective as a substrate, whereas changing of the aspartic acid to a glycine has no effect. Product characterization of the glycosylated peptide demonstrates that the monosaccharide covalently attached to the peptide is N-acetylglucosamine (GlcNAc) and has not been epimerized to N-acetylgalactosamine. Mild base-catalyzed beta-elimination of the in vitro glycosylated peptide quantitatively yields GlcNAcitol, indicating that the GlcNAc is attached via an O-linkage. The transferase activity is strongly inhibited by UDP but is unaffected by GlcNAc or tunicamycin. Interestingly, EDTA only slightly inhibits activity, suggesting that the enzyme may not require divalent cations. The majority of the activity is soluble, and the remainder is lost from membranes after extracting with high salt and EDTA. Consistent with the subcellular localization of most proteins bearing O-GlcNAc, the activity appears to reside in the cytosolic portion of the cell when compared to two lumenal marker enzymes, galactosyltransferase and mannose-6-phosphatase.