Rat endopeptidase‐24.18 α subunit is secreted into the culture medium as a zymogen when expressed by COS‐1 cells

FEBS Letters

Simon, Valérie²  Boileau, Guy²  Milhiet, Pierre-Emmanuel²  Crine, Philippe²  Kenny, A.John¹  Corbeil, Denis²  Ingram, Jean¹ 

[1] Department of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK;Département de biochimie. Faculté de médecine, Université de Montréal, C.P. 6128, Succ. A., Montréal, H3C 3J7, Canada

关键词: Meprin;    Astacin;    Ectoenzyme;    Zymogen;    E-24.18;    endopeptidase-24.18;    RER;    rough endoplasmic reticulum;    PNGase F;    peptide N-glycosidase F;    endo H;    endo-β-N-acetylglucosaminidase H;    SDS-PAGE;    sodium dodecyi sulfate-polyacrylamide gel electrophoresis;    PBS;    phosphate-buffered saline;    BBM;    brush border membrane;   

DOI  :  10.1016/0014-5793(93)80420-Y

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Endopeptidase-24.18 (EC 3.4.24.18, E-24.18) is an oligomeric Zn-ectoenzyme. The α and β submits have been cloned from both rat and mouse kidneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of the astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C-terminal hydrophobic domain. In order to verify the mode of anchoring of the rat E-24.18 α subunit and to test the functionality of the astacin-like domain in the α subunit when expressed alone, COS-1 cells were transfected with a cloned cDNA for rat α subunit. Despite the presence of its putative transmembrane domain, the α subunit was not anchored in the plasma membrane but rather secreted as a dimer into the culture medium. When the enzymatic activity of the secreted recombinant protein was tested in the azocasein degradation assay, the α subunit was found to be inactive. Activity could, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu-157 in the active site by Val. Taken together our results suggest that the α subunit of Endopeptidase-24.18 contains a latent astacin-like Zn metallopeptidase activity which could be secreted as a soluble enzyme by kidney and intestine.

【 授权许可】

Unknown