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In vitro elucidation of substrate specificity and bioassay of proprotein convertase 4 using intramolecularly quenched fluorogenic peptides.

ABSTRACT: The fourth member of Ca2+-dependent mammalian secretory subtilase, PC4 (proprotein convertase 4), is primarily expressed in testicular germ cell and ovarian macrophage. Its role in sperm fertilization and in early embryonic development has been demonstrated earlier through several studies, including those with PC4 null mice. A number of physiological substrates found in reproductive tissues have been postulated or identified for PC4 by various biochemical studies. These include growth factors IGF-1 (insulin-like growth factor-1) and IGF-2, hormonal polypeptide proPACAP (where PACAP stands for pituitary adenylate cyclase-activating polypeptide) and a number of surface proteins of ADAM (ADisintegrin And Metalloproteinase-like) family such as ADAM-1 (fertilin a), ADAM-2 (fertilin b), ADAM-3 (procyritestin) and ADAM-5. To provide further evidence in support of this notion and also to study the substrate specificity and bioassay of PC4, a series of intramolecularly quenched fluorogenic peptides containing the cleavage sites and several mutants were prepared. A comparative kinetic analysis and measurement of Vmax (app)/Km (app) ratio of these fluorogenic substrates against PC4 and PC7 revealed that the mutant variants of h (human) proPACAP and m (mouse) ADAM-5 derived peptides Q-PACAP141-151-mutant [Abz-141RVKNKGRRI150P151SY(NO2)-A-CONH2] (150A151Y replaced by PS) and Q-ADAM-5380-388-mutant [Abz-380E381PKPARRP388RY(NO2)A-CONH2] (381R replaced by P) are most efficiently and selectively cleaved by PC4. Using these two and Q-IGF-263-71 peptides, we showed that the sperm extract of normal adult mice is much higher when compared with that of PC4-null mice. This suggests that these fluorogenic peptides are useful for specific bioassay of PC4 activity. In addition, kinetic studies with various peptidyl-MCA indicate that the hexapeptide Ac-KTKQLR-MCA (where MCA stands for 4-methyl coumaryl-7-amide) is most efficiently and selectively cleaved by PC4 at RMCA, making it another effective agent for bioassay of PC4 activity. The study concludes that the most probable sequence motif for recognition by PC4 is KXKXXR or KXXR, where X is any amino acid other than cysteine and that it prefers proline at P3, P5 and/or P2' positions. It was also revealed that PC4 is a good candidate processing enzyme for growth factors IGF-1 and -2, neuropeptide proPACAP and several ADAM proteins such as ADAM-1, -2, -3 and -5.

SUBMITTER: Basak S

PROVIDER: S-EPMC1224175 | biostudies-other | 2004 Jun

REPOSITORIES: biostudies-other

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Project description: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 Ca(2+) concentration of 2.0 mM. Using PC4-specific antibodies, Western blot analysis of the medium revealed a major band at approximately 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 V(max)/K(m) 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 downward arrowSLVVPTD (where downward arrow 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 downward arrowSVRA (IGF-I(66-75)) and Ac-PAKSER downward arrowDVST (IGF-II(63-72)), 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 both IGF-I(66-75) and IGF-II(63-72), 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-I(66-75)Lys(70) and IGF-II(63-72)Lys(67), 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 downward arrow (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 (Cu(2+)>Hg(2+)>Zn(2+) Ni(2+)>Co(2+)), as well as by small peptide semicarbazones (SCs), such as Arg-Lys-Lys-Arg-SC (K(i) 0.75 microM) and Arg-Ser-Lys-Arg-SC (K(i) 11.4 microM).

Project description:The application of D-stereospecific proteases (DSPs) in resolution of racemic amino acids and in the semisynthesis of proteins has been a successful strategy. The main limitation for a broader application is, however, the accessibility of suitable DSPs covering multiple substrate specificities. To identify DSPs with novel primary substrate preferences, a fast specificity screening method using the easily accessible internally quenched fluorogenic substrate aminobenzoyl-D-arginyl-D-alanyl-p-nitroanilide was developed. By monitoring both UV/vis-absorbance and fluorescence signals at the same time it allows to detect two distinct D-amino acid substrate specificities simultaneously and separately with respect to the individual specificities. In order to identify novel DSP specificities for synthesis applications, DSPs specific for D-arginine were of special interest due to their potential ability as catalysts for substrate mimetics-mediated peptide and protein ligations. D-alanine in the substrate served as positive control and reference based on its known acceptance by numerous DSPs. In silico analysis suggested that DSPs are predominantly present in gram-positive microorganisms, therefore this study focused on the bacilli strains Bacillus thuringiensis and Bacillus subtilis as potential hosts of D-Arg-specific DSPs. While protease activities toward D-alanine were found in both organisms, a novel and so far unknown D-arginine specific DSP was detected within the culture supernatant of B. thuringiensis. Enrichment of this activity via cation exchange and size exclusion chromatography allowed isolation and further characterization of this novel enzyme consisting of a molecular mass of 37.7 kDa and an enzymatic activity of 8.3 U mg-1 for cleaving the D-Arg|D-Ala bond in the detecting substrate. Independent experiments also showed that the identified enzyme shows similarities to the class of penicillin binding proteins. In future applications this enzyme will be a promising starting point for the development of novel strategies for the semisynthesis of all-L-proteins.

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In vitro elucidation of substrate specificity and bioassay of proprotein convertase 4 using intramolecularly quenched fluorogenic peptides.

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