Enzymic characterization in vitro of recombinant proprotein convertase PC4

Proprotein convertase PC4A, a member of the subtilisin/kexin family of seri ne proteases, was obtained in enzymically active form following expression of vaccinia virus recombinant rat (r)PC4A in GH4Cl cells. It displayed maxi mal activity at pH 7.0 and a Ca2+ concentration of 2.0 mM. Using PC4-specif ic antibodies, Western blot analysis of the medium revealed a major band at approximate to 54 kDa, corresponding to the molecular size of mature rPC4A . Among the various peptidyl-[4-methylcoumarin 7-amide (MCA)] substrates te sted, the one that was preferred the most by rPC4A was acetyl (Ac)-Arg-Lys- Lys-Arg-MCA, which is cleaved 9 times faster las judged from V-max/K-m meas urements) than the best furin and PC1 substrate, pGlu-Arg-Thr-Lys-Arg-MCA. Recombinant rPC4A, along with human (h)furin and hPC1, cleaved a 17-amino-a cid synthetic peptide, YQTLRRRVKR down arrow SLVVPTD (where down arrow deno tes site of cleavage, and the important basic residues are shown in bold), encompassing the junction between the putative pro-segment of rPC4A and the active enzyme, suggesting a possible auto-activation of the enzyme. In an effort to identify potential physiological substrates for PC4, studies were performed with pro-[insulin-growth-factor (IGF)]-derived synthetic peptide s, namely Ac-PAKSAR down arrow SVRA (IGF-I66-75) and Ac-PAKSER down arrow D VST (IGF-II63-72), as well as two lysine mutants [(IGF-I(66-75)Lys(70)) and (IGF-II(63-72)Lys(67))]. Unlike PC1 and furin, rPC4A cleaved efficiently b oth IGF-I66-75 and IGF-II63-72, suggesting a possible role of PC4 in the ma turation of IGF-I and -II. In contrast, the peptides with a position 2 (P2) lysine mutation, IGF-I(66-75)Lys(70) and IGF-II(63-72)Lys(67), were cleave d more efficiently by PC1 and furin compared with rPC4A. Furthermore, using synthetic peptides containing the processing sites of pituitary adenylate- cyclase-activating polypeptide (PACAP)-38, we were able to confirm that, of the two testicular enzymes PC4 and PC7, PC4 is the best candidate enzyme f or maturation of PACAP. Our data suggest that rPC4A is a functionally activ e convertase, with a substrate specificity somewhat different from that of other convertases, namely KXXR down arrow (where X denotes any other residu e). As expected, p-chloromercuribenzoic acid and metal chelators such as ED TA, EGTA and trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid inhibi t the proteolytic activity of rPC4A, whereas it is activated by dithiothrei tol. PC4A was also inhibited by transition-metal ions (Cu2+ > Hg2+ > Zn2+ N i2+ > Co2+), as well as by small peptide semicarbazones (SCs), such as Arg- Lys-Lys-Arg-SC (K-i 0.75 mu M) and Arg-Ser-Lys-Arg-SC (K-i 11.4 mu M).