MATURATION OF SECRETED MEPRIN-ALPHA DURING BIOSYNTHESIS - ROLE OF THEFURIN SITE AND IDENTIFICATION OF THE COOH-TERMINAL AMINO-ACIDS OF THEMOUSE KIDNEY METALLOPROTEASE SUBUNIT

The a subunit of meprin A is synthesized as a type I integral membrane protein; however, the mature form contains no membrane-spanning regio n due to COOH-terminal proteolytic cleavage during biosynthesis. Previ ous studies with transfected mouse meprin a subunit cDNA, showed that the inserted (I) domain in the COOH-terminus was essential for proteol ytic processing and transport of the subunit out of the endoplasmic re ticulum. A furin site in the I domain was implicated as the site of cl eavage in the rat meprin a subunit, however, this site was shown not t o be required in the homologous mouse subunit. The present studies wer e designed to determine whether there is a species difference in the u se of the furin site for processing of the subunit? and whether the di fferent mutations used in the previous studies could account for the d ifferent conclusions regarding the importance of the furin site. When the furin sites in mouse and rat cDNAs mere mutated, using similar ami no acid substitutions, and expressed in human 293 cells, all mutants w ere secreted, and had comparable activities compared to the wild-types against a protein (azocasein) and peptide (bradykinin analog) substra te. These data revealed no difference between processing of the rat an d mouse subunits, and indicated that the furin site is not essential f or COOH-terminal processing in either species. Additional transfection investigations with brefeldin A or low temperature confirmed that COO H-terminal processing occurs in the endoplasmic reticulum, further sup porting the contention that furin-type enzymes localized to the Golgi apparatus are not responsible for processing this subunit, COOH-termin al amino acid sequence analysis of the mature detergent-purified membr ane form of meprin alpha from ICR mouse kidney indicated that the subu nit 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 en ables or directs the final COOH-terminal processing of meprin a: to a region NH2-terminal to the I domain, and that there are no essential d ibasic, furin, or single base processing sites. (C) 1998 Academic Pres s.