Affiliation:
1. Molecular Neuroendocrinology Laboratory, Clinical Research Institute of Montréal, Montréal, Qué, Canada H2W 1R7
2. Protein Chemistry Center, Loeb Health Research Institute, Ottawa Hospital Medical Hospital, 725 Parkdale Avenue, Ottawa, ON, Canada K1Y 4K9
3. Biochemical Neuroendocrinology Laboratory, Clinical Research Institute of Montréal, Montréal, Qué, Canada H2W 1R7
4. Structure and Metabolism of Neuropeptides Laboratory, Clinical Research Institute of Montréal, Montréal, Qué, Canada H2W 1R7
Abstract
Proprotein convertase PC4A, a member of the subtilisin/kexin family of serine proteases, was obtained in enzymically active form following expression of vaccinia virus recombinant rat (r)PC4A in GH4C1 cells. It displayed maximal activity at pH 7.0 and a Ca2+ concentration of 2.0 mM. Using PC4-specific antibodies, Western blot analysis of the medium revealed a major band at ≈ 54 kDa, corresponding to the molecular size of mature rPC4A. Among the various peptidyl-[4-methylcoumarin 7-amide (MCA)] substrates tested, the one that was preferred the most by rPC4A was acetyl (Ac)-Arg-Lys-Lys-Arg-MCA, which is cleaved 9 times faster (as judged from Vmax/Km measurements) 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-acid synthetic peptide, YQTLRRRVKR↓ SLVVPTD (where ↓ denotes 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 peptides, namely Ac-PAKSAR↓ SVRA (IGF-I66-75) and Ac-PAKSER↓ DVST (IGF-II63-72), as well as two lysine mutants [(IGF-I66-75Lys70) and (IGF-II63-72Lys67)]. Unlike PC1 and furin, rPC4A cleaved efficiently both IGF-I66-75 and IGF-II63-72, suggesting a possible role of PC4 in the maturation of IGF-I and -II. In contrast, the peptides with a position 2 (P2) lysine mutation, IGF-I66-75Lys70 and IGF-II63-72Lys67, were cleaved 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 for maturation of PACAP. Our data suggest that rPC4A is a functionally active convertase, with a substrate specificity somewhat different from that of other convertases, namely KXXR↓ (where X denotes any other residue). As expected, p-chloromercuribenzoic acid and metal chelators such as EDTA, EGTA and trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid inhibit the proteolytic activity of rPC4A, whereas it is activated by dithiothreitol. PC4A was also inhibited by transition-metal ions (Cu2+>Hg2+>Zn2+ Ni2+>Co2+), as well as by small peptide semicarbazones (SCs), such as Arg-Lys-Lys-Arg-SC (Ki 0.75 μM) and Arg-Ser-Lys-Arg-SC (Ki 11.4 μM).
Subject
Cell Biology,Molecular Biology,Biochemistry