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

AbstractEndopeptidase-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

Mosaic expression of membrane peptidases by confluent cultures of Caco-2 cells

AbstractThe cell-surface expression of endopeptidase-24.11 (EC 3.4.24.11) on Caco-2 cells cultured to confluency is markedly heterogeneous unlike that of dipeptidylpeptidase IV (EC 3.4.14.5). Here we have investigated the cell-surface expression of three other ectopeptidases: angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase N (EC 3.4.11.2) and aminopeptidase W (EC 3.4.11.16). We show by indirect immunofluorescent staining that these three enzymes are present on the surface of some cells but not on others. However, these enzymes were detected in the majority of detergent-permeabilised Caco-2 cells indicating the presence of intracellular pools of these enzymes. This suggests that there may either be differential regulation of apical transport for these peptidases or that they recycle at different rates