N-arginine dibasic convertase (nardilysin) isoforms are soluble dibasic-specific metalloendopeptidases that localize in the cytoplasm and at the cell surface
N-arginine dibasic convertase (nardilysin) isoforms are soluble dibasic-specific metalloendopeptidases that localize in the cytoplasm and at the cell surface
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DOI：
10.1042/0264-6021:3490587
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发表时间：
2000-07-15
影响因子：
4.1
通讯作者：
Prat, A
中科院分区：
文献类型：
--
作者：
Hospital, V;Chesneau, V;Prat, A
N-精氨酸 (R) 二碱基 (NRD) 转化酶（nardilysin；EC 3.4.24.61）是 M16 家族的金属内肽酶，可在体外特异性切割二碱基基序中精氨酸 N 末端的肽底物。在大鼠睾丸中，该酶位于精子细胞的细胞质内，并与 manchette 和轴丝的微管结合。 NRD1 和 NRD2 转化酶是两种 NRD 转化酶亚型，其不同之处在于活性位点附近是否存在 68 个氨基酸插入（亚型 1）或存在（亚型 2）。在这项研究中，我们通过牛痘病毒感染 BSC40 细胞或转染 COS-7 细胞来过表达两种亚型。部分纯化的酶表现出非常相似的生化和酶学特性。微测序显示 NRD 转化酶经过 N 末端加工。免疫细胞荧光、免疫电子显微镜和亚细胞分级分离研究的结果支持两种肽酶的初级胞质定位。尽管在体外翻译测定中推定的信号肽并未将 NRD 转化酶引导至微粒体，但生物素化实验清楚地表明细胞表面存在两种异构体。总之，虽然大多数已知的碱性残基对的加工事件是通过分泌途径中的前蛋白转化酶实现的，但 NRD 转化酶可能在细胞质和/或细胞表面实现类似的功能。
N-arginine (R) dibasic (NRD) convertase (nardilysin; EC 3.4.24.61), a metalloendopeptidase of the M16 family, specifically cleaves peptide substrates at the N-terminus of arginines in dibasic motifs in vitro. In rat testis, the enzyme localizes within the cytoplasm of spermatids and associates with microtubules of the manchette and axoneme. NRD1 and NRD2 convertases, two NRD convertase isoforms, differ by the absence (isoform 1) or presence (isoform 2) of a 68-amino acid insertion close to the active site. In this study, we overexpressed both isoforms, either by vaccinia virus infection of BSC40 cells or transfection of COS-7 cells. The partially purified enzymes exhibit very similar biochemical and enzymic properties. Microsequencing revealed that NRD convertase is N-terminally processed. Results of immunocytofluorescence, immunoelectron microscopy and subcellular fractionation studies argue in favour of a primary cytosolic localization of both peptidases. Although the putative signal peptide did not direct NRD convertase into microsomes in an in vitro translation assay, biotinylation experiments clearly showed the presence of both isoforms at the cell surface. In conclusion, although most known processing events at pairs of basic residues are achieved by proprotein convertases within the secretory pathway, NRD convertase may fulfil a similar function in the cytoplasm and/or at the cell surface.