Glycosylation is essential for functional expression of a human brain ecto-apyrase.
Glycosylation is essential for functional expression of a human brain ecto-apyrase.
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糖基化对于人脑外切腺苷三磷酸双磷酸酶的功能表达至关重要。
DOI：
10.1021/bi9821768
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发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
T. Kirley
中科院分区：
文献类型：
--
作者：
T. M. Smith;T. Kirley
N-连接的糖基化的功能和寡聚化的E-型ATP酶的重要性进行了研究，通过使用衣霉素和肽N-糖苷酶F，两种试剂用于防止和去除糖基化，分别。编码人胞外腺苷三磷酸双磷酸酶（HB 6）的cDNA，预测有7个N-连接的糖基化位点，在哺乳动物COS细胞中瞬时表达，所得的膜制剂用肽N-糖苷酶F（PNGase-F）处理。随着时间的推移，PNGase-F导致蛋白质的表观分子量降低（与聚糖去除一致）和酶活性降低。外腺苷三磷酸双磷酸酶也表达衣霉素的存在下，这完全防止N-连接的糖基化，导致在一个nonglycosylated核心蛋白缺乏ATP和ADP水解活性。然而，控制和衣霉素处理的细胞表达的酶相似的水平和定位。有趣的是，这种E型ATP酶的四级结构似乎取决于聚糖链的存在。糖基化的胞外腺苷三磷酸双磷酸酶作为原位同源二聚体存在，如通过洗涤剂溶解的胞外腺苷三磷酸双磷酸酶的尺寸排阻色谱法和膜结合的胞外腺苷三磷酸双磷酸酶的交联所评估的，与酶促去糖基化的胞外腺苷三磷酸双磷酸酶和衣霉素处理的胞外腺苷三磷酸双磷酸酶相反。这些结果表明，糖基化是必要的同源寡聚化和核苷酸水解活性，但不表达和质膜定位的E型ATP酶。用另一种人胞外腺苷三磷酸双磷酸酶CD 39获得了类似的结果，这表明糖基化的重要性可以推广到所有膜结合的E型ATP酶。
The importance of N-linked glycosylation for the function and oligomerization of an E-type ATPase was examined by using tunicamycin and peptide N-glycosidase F, two agents used to prevent and remove glycosylations, respectively. The cDNA encoding a human ecto-apyrase (HB6), predicted to have seven N-linked glycosylation sites, was transiently expressed in mammalian COS cells and the resulting membrane preparations were treated with peptide N-glycosidase F (PNGase-F). PNGase-F caused a decrease in the apparent molecular weight of the protein (consistent with glycan removal) and a decrease in enzymatic activity over time. The ecto-apyrase was also expressed in the presence of tunicamycin, which completely prevented N-linked glycosylation, resulting in a nonglycosylated core protein devoid of ATP and ADP hydrolyzing activity. However, control and tunicamycin-treated cells expressed the enzyme to similar levels and localization. Interestingly, the quaternary structure of this E-type ATPase appears to be dependent upon the presence of glycan chains. The glycosylated ecto-apyrase exists as a homodimer in situ as assessed by both size-exclusion chromatography of detergent-solubilized ecto-apyrase and cross-linking of membrane-bound ecto-apyrase, in contrast to the enzymatically deglycosylated ecto-apyrase and the tunicamycin-treated ecto-apyrase. These results suggest that glycosylation is necessary for homooligomerization and nucleotide hydrolyzing activity, but not for expression and plasma membrane localization of the E-type ATPase. Similar results were obtained with another human ecto-apyrase, CD39, suggesting that the importance of glycosylation may be generalized to all membrane-bound E-type ATPases.