CYSTEINE MUTATIONS IN THE MAM DOMAIN RESULT IN MONOMERIC MEPRIN AND ALTER STABILITY AND ACTIVITY OF THE PROTEINASE

Meprins are oligomeric, glycosylated cell surface or secreted metalloe ndopeptidases that are composed of multidomain disulfide-linked subuni ts. To investigate whether subunit oligomerization is critical for int racellular transport or for the enzymatic and/or physical properties o f the proteinase, specific cysteine residues were mutated, and the mut ants were expressed in 293 cells. Mutation of mouse meprin alpha Cys-3 20 to Ala in the MAM domain (an extracellular domain found in meprin, (A) under bar-5 protein, and receptor protein-tyrosine phosphatase <(m u)under bar>) resulted in expression of a monomeric form of meprin, as determined by SDS-polyacrylamide gel electrophoresis and nondenaturin g gel electrophoresis. The monomeric subunits were considerably more v ulnerable to proteolytic degradation and heat inactivation in vitro co mpared with the oligomeric form of the enzyme. Proteolytic activity of the monomeric meprin using a bradykinin analog or aminobenzoyl-Ala-Al a Phe-p-nitroanilide as substrate was similar to that of disulfide-lin ked oligomeric meprin; however, activity against azocasein was markedl y decreased. Mutation of another cysteine residue in the MAM domain (C 289A), predicted to be involved in intrasubunit disulfide bridging, re sulted in disulfide-linked oligomers and monomers. These results indic ated that this mutant was capable of forming intersubunit disulfide bo nds but less efficiently than wild-type meprin subunits. Mutant C289A also retained activity toward peptides but not the protein substrate a nd was more vulnerable to proteolytic degradation and heat inactivatio n compared with the wild-type enzyme. Both Cys mutants were expressed and secreted into the medium at levels comparable with the wild type a nd had slightly altered glycosylation. This work indicates that 1) Cys -320 of mouse meprin alpha is most likely responsible for the covalent interactions of the subunits; 2) covalent dimerization of subunits is not essential for efficient biosynthesis, trafficking, or posttransla tional processing of the secreted protease; and 3) mutations in the MA M domain affect noncovalent interactions of the subunits and the stabi lity and activity of the protease domain, indicating that domain-domai n interactions are critical for structure and function of the enzyme.