Asparagine 81, an invariant glycosylation site near apyrase conserved region 1, is essential for full enzymatic activity of ecto-nucleoside triphosphate diphosphohydrolase 3.
Asparagine 81, an invariant glycosylation site near apyrase conserved region 1, is essential for full enzymatic activity of ecto-nucleoside triphosphate diphosphohydrolase 3.
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天冬酰胺 81 是腺苷三磷酸双磷酸酶保守区 1 附近的一个不变糖基化位点，对于外核苷三磷酸二磷酸水解酶 3 的完整酶活性至关重要。
DOI：
10.1016/s0003-9861(03)00084-5
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发表时间：
2003
影响因子：
3.9
通讯作者：
Kirley,TerenceL
中科院分区：
文献类型：
--
作者：
Murphy,DeirdreM;Kirley,TerenceL
N-连接的糖基化对于膜结合的胞外三磷酸核苷二磷酸水解酶（eNTPDases）的功能、细胞定位和寡聚化是重要的。NTPDase 3是一种典型的细胞膜相关eNTPDase，与其他两种细胞表面膜NTPDase（NTPDase 1和2）同样相关且具有酶中间体。NTPDase 3的蛋白质序列含有位于胞外结构域的7个推定的N-糖基化位点。只有一个这些推定的糖基化位点，天冬酰胺81在NTPDase 3，这是位于附近的腺苷三磷酸双磷酸酶保守区1（ACR 1），是不变的，在所有的细胞表面膜eNTPDases。使用定点诱变，构建突变体以消除NTPD酶3中高度保守的N-糖基化位点。结果表明，该位置的糖基化对于完全酶活性是必不可少的，突变体ATP酶活性的降低程度大于腺苷三磷酸酶活性。该位点的酶促去糖基化被证明是通过用肽N-糖苷酶-F处理使野生型酶失活的原因。此外，该保守位点的糖基化对于用凝集素伴刀豆球蛋白A处理后核苷酸酶活性的稳定/刺激是必需的。然而，缺乏糖基化在这个网站上并没有导致在三级或四级结构的大的变化，所测量的汽巴蓝结合，化学交联，和天然凝胶电泳分析。由于该N-糖基化位点在细胞膜eNTPD酶中是不变的，因此假定该残基在ACR 1附近的糖基化对于细胞膜NTPD酶的完全酶活性是至关重要的。
N-linked glycosylation is important for the function, cellular localization, and oligomerization of membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases). NTPDase3 is a prototypical cell membrane-associated eNTPDase, which is equally related and enzymatically intermediate to the other two cell surface membrane NTPDases (NTPDase1 and 2). The protein sequence of NTPDase3 contains seven putative N-glycosylation sites located in the ecto-domain. Only one of these putative glycosylation sites, asparagine 81 in NTPDase3, which is located near apyrase conserved region 1 (ACR1), is invariant in all the cell surface membrane eNTPDases. Using site-directed mutagenesis, mutants were constructed to eliminate this highly conserved N-glycosylation site in NTPDase3. The results indicate that glycosylation at this position is essential for full enzymatic activity, with mutant ATPase activity decreased more than ADPase activity. Enzymatic deglycosylation of this site is shown to be responsible for the inactivation of the wild-type enzyme by treatment with peptide N-glycosidase-F. In addition, glycosylation of this conserved site is necessary for the stabilization/stimulation of nucleotidase activity upon treatment with the lectin concanavalin A. However, lack of glycosylation at this site did not result in large changes in tertiary or quaternary structure, as measured by Cibacron blue binding, chemical cross-linking, and native gel electrophoretic analysis. Since this N-glycosylation site is invariant in cell membrane eNTPDases, it is postulated that glycosylation of this residue near ACR1 is crucial for full enzymatic activity of the cell membrane NTPDases.
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