RAT ENDOPEPTIDASE-24.18 ALPHA-SUBUNIT IS SECRETED INTO THE CULTURE-MEDIUM AS A ZYMOGEN WHEN EXPRESSED BY COS-1 CELLS

Endopeptidase-24.18 (EC 3.4.24.18, E-24.18) is an oligomeric Zn-ectoen zyme. The a and B subunits have been cloned from both rat and mouse ki dneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of th e astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C-terminal hydrophobic domain. In order to veri fy the mode of anchoring of the rat E-24.18 alpha subunit and to test the functionality of the astacin-like domain in the a subunit when exp ressed alone, COS-1 cells were transfected with a cloned cDNA for rat alpha subunit. Despite the presence of its putative transmembrane doma in, the a subunit was not anchored in the plasma membrane but rather s ecreted as a dimer into the culture medium. When the enzymatic activit y of the secreted recombinant protein was tested in the azocasein degr adation assay, the alpha subunit was found to be inactive. Activity co uld, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu-157 in the active site by Val. Take n together our results suggest that the alpha subunit of Endopeptidase -24.18 contains a latent astacin-Iike Zn metallopeptidase activity whi ch could be secreted as a soluble enzyme by kidney and intestine.