Maturation of secreted meprin alpha during biosynthesis: role of the furin site and identification of the COOH-terminal amino acids of the mouse kidney metalloprotease subunit.

The alpha subunit of meprin A is synthesized as a type I integral membrane protein; however, the mature form contains no membrane-spanning region due to COOH-terminal proteolytic cleavage during biosynthesis. Previous studies with transfected mouse meprin alpha subunit cDNA, showed that the inserted (I) domain in the COOH-terminus was essential for proteolytic processing and transport of the subunit out of the endoplasmic reticulum. A furin site in the I domain was implicated as the site of cleavage in the rat meprin alpha subunit, however, this site was shown not to be required in the homologous mouse subunit. The present studies were designed to determine whether there is a species difference in the use of the furin site for processing of the subunit, and whether the different mutations used in the previous studies could account for the different conclusions regarding the importance of the furin site. When the furin sites in mouse and rat cDNAs were mutated, using similar amino acid substitutions, and expressed in human 293 cells, all mutants were secreted, and had comparable activities compared to the wild-types against a protein (azocasein) and peptide (bradykinin analog) substrate. These data revealed no difference between processing of the rat and mouse subunits, and indicated that the furin site is not essential for COOH-terminal processing in either species. Additional transfection investigations with brefeldin A or low temperature confirmed that COOH-terminal processing occurs in the endoplasmic reticulum, further supporting the contention that furin-type enzymes localized to the Golgi apparatus are not responsible for processing this subunit. COOH-terminal amino acid sequence analysis of the mature detergent-purified membrane form of meprin alpha from ICR mouse kidney indicated that the subunit ends at Arg615, which is NH2-terminal to the I domain in the After-MATH domain. Mutation of Arg615 to an Ala did not affect secretion of the protein from 293 cells. The results indicate that the I domain enables or directs the final COOH-terminal processing of meprin alpha to a region NH2-terminal to the I domain, and that there are no essential dibasic, furin, or single base processing sites.