PURIFICATION AND CHARACTERIZATION OF THE CANDIDATE PROHORMONE-PROCESSING ENZYME SPC3 PRODUCED IN A MOUSE L-CELL LINE

SPC3 is a member of a growing family of mammalian subtilisin-like seri ne proteases which play a probable role in proprotein maturation. In t his study we have prepared a mouse L cell line stably expressing rat S PC3 cDNA and characterized the recombinant SPC3 protein secreted into the medium. Three molecular forms of recombinant SPC3 were identified with molecular masses of 86, 75, and 64 kDa. NH2-terminal sequence ana lysis indicated that all three forms were cleaved following the sequen ce -Arg107-Arg-Lys-Arg110, indicating removal of an SPC3 prosequence. All three molecular forms showed a 3-4-kDa decrease in molecular mass following incubation with endoglycosidase F. Two SPC3 carboxyl-termina l-directed antisera recognized only the 86-kDa molecular form of recom binant SPC3, demonstrating that COOH-terminal truncation of SPC3 prote in is responsible for the different molecular mass forms. Recombinant SPC3 had a pH optimum of 6.0 and was stimulated by calcium, with maxim um activity at 10 mM. Recombinant SPC3 was inhibited most effectively by the thiol-reactive reagent p-hydroxymecuribenzoate and the heavy me tal chelators EDTA and EGTA. Recombinant SPC3 was also inhibited by al pha1-antitrypsin Pittsburgh as well as wild type alpha1-antitrypsin an d antithrombin III. The inhibitor specificities revealed using these h igh molecular mass serpins differ from those reported for other member s of the subtilisin-like serine protease family and may be able to be exploited to distinguish between closely related members of this new e nzyme superfamily. Studies of cleavage specificity using tri- and tetr apeptidyl coumarins that contained pairs of basic residues indicated t hat tetrapeptide substrates that contained an S4 Arg residue as part o f an -Arg-X-Lys/Arg-Arg motif were the most effective synthetic peptid e substrates. Recombinant SPC3 also cleaved human proalbumin following the Arg-Gly-Val-Phe-Arg-Arg prosequence. Circulating human proalbumin variants that contained a mutation at either of the basic amino acids adjacent to the cleavage site were not cleaved by recombinant SPC3. R ecombinant SPC3 was also able to cleave after a single arginine residu e in chicken proalbumin following the Arg-Asn-Leu-Gln-Arg-Phe-Ala-Arg prosequence. These results define the primary structure requirements f or cleavage by recombinant SPC3 and remain consistent with a role for SPC3 in proprotein/prohormone maturation.