Characterization of two azurphil granule proteases with active-site homology to neutrophil elastase.
Characterization of two azurphil granule proteases with active-site homology to neutrophil elastase.
复制标题
与中性粒细胞弹性蛋白酶具有活性位点同源性的两种天青颗粒蛋白酶的表征。
DOI：
10.1016/s0021-9258(19)39936-3
复制
发表时间：
1990
期刊：
影响因子：
--
通讯作者：
R W Scott
中科院分区：
文献类型：
--
作者：
C. Wilde;J. L. Snable;J. Griffith;R W Scott
许多与肺气肿和其他炎症性疾病相关的组织损伤归因于中性粒细胞弹性蛋白酶的蛋白水解活性，中性粒细胞弹性蛋白酶是中性粒细胞颗粒的主要成分。最近，另外两个与弹性酶具有nh2末端序列同源的氮性粒细胞颗粒蛋白被分离出来(Gabay， J. E., Scott， R. W., Campanelli， D., Griffith， J., Wilde， C., Marra， M. N., Seeger， M., and Nathan， C. F. (1989) Proc. Natl。学会科学。美国法典第86号，5610-5614号)和指定的azurophil颗粒蛋白7 （AGP7）和azuroidin。azuroidin和AGP7是azurophil颗粒的重要蛋白质成分，通过反相高效液相色谱分析判断，它们共同占酸可提取蛋白质的约15%。AGP7在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上以分子量为28-34 kDa的4种不同糖型迁移，而偶氮灭虫素在分子量为28-30 kDa的3种糖型为主。用[3H]氟磷酸二异丙基处理完整的氮唑菌颗粒可标记弹性蛋白酶、组织蛋白酶G和AGP7，而氮唑菌素未被标记。3h标记AGP7的色氨酸图谱使我们能够识别和测序活性位点多肽，该多肽与弹性酶在20多个残基中具有70%的同一性。通过色氨酸片段的序列分析，鉴定了嘧虫啶的活性位点肽，其与弹性酶活性位点的同源性为65%。令人惊讶的是，唑虫啶的催化丝氨酸被甘氨酸取代，这解释了它无法用[3H]氟磷酸二异丙基标记。因此，我们已经确定了两个与中性粒细胞弹性酶密切相关的中性粒细胞蛋白，其中一个由于催化丝氨酸的突变而明显失去了其蛋白水解活性。
Much of the tissue damage associated with emphysema and other inflammatory diseases has been attributed to the proteolytic activity of neutrophil elastase, a major component of the azurophil granule. Recently, two additional azurophil granule proteins with NH2-terminal sequence homology to elastase were isolated (Gabay, J. E., Scott, R. W., Campanelli, D., Griffith, J., Wilde, C., Marra, M. N., Seeger, M., and Nathan, C. F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5610-5614) and designated azurophil granule protein 7 (AGP7) and azurocidin. Azurocidin and AGP7 represent significant protein components of the azurophil granule, together comprising approximately 15% of the acid-extractable protein as judged by reverse-phase high performance liquid chromatography analysis. AGP7 migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as four distinct glycoforms of molecular mass 28-34 kDa, whereas azurocidin exhibits three predominant bands with molecular mass of 28-30 kDa. Treatment of intact azurophil granules with [3H]diisopropyl fluorophosphate resulted in labeling of elastase, cathepsin G, and AGP7, whereas azurocidin was not labeled. Tryptic mapping of 3H-labeled AGP7 allowed us to identify and sequence the active-site polypeptide that has 70% identity to elastase over 20 residues. The active site peptide of azurocidin was also identified by sequence analysis of tryptic fragments and showed 65% identity to the active site of elastase. Surprisingly, the catalytic serine of azurocidin is replaced by glycine, explaining its inability to label with [3H]diisopropyl fluorophosphate. Thus, we have identified two azurophil proteins closely related to neutrophil elastase, one of which has apparently lost its proteolytic activity due to mutation of the catalytic serine.