Enzymic characterization of immunopurified prohormone convertase 2: Potent inhibition by a 7B2 peptide fragment

Abstract

Prohormone convertase (PCs) are thought to mediate the controlled proteolysis of prohormones and neuropeptide precursors. While recombinant PC1 and furin are currently available, thus far it has not been possible to produce recombinant PC2. We have used conditioned medium obtained from the mouse insulinoma cell line beta TC3 to generate a working preparation of enzymatically active PC2 through immunopurification. Immunopurified PC2 cleaved the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC in a time- and calcium-dependent manner. It was half-maximally stimulated at 75 microM Ca2+, had an optimum pH of 5, and exhibited PCMS and EDTA sensitivity similar to that reported for furin and PC1. The tight-binding inhibitor 27 kDa 7B2 was used to calculate the Kd for this inhibitor and the active enzyme concentration. The Kd was 7.3 +/- 1.7 nM, and the turnover rate of PC2 was 5.2 molecules substrate per enzyme molecule per minute. The specific activity was 4.9 nmol/micrograms/h (assuming a molecular mass for PC2 of 64 kDa). The enzyme preparation was able to cleave recombinant proenkephalin at at least four of the expected paired basic sites in the absence, but not in the presence, of 27 kDa 7B2. Since 21 kDa 7B2 is functionally inactive as a proteinase inhibitor, we examined the inhibitory activity of the carboxy-terminal portion of 27 kDa 7B2 (7B2 CT-peptide). Synthetic peptides were used to demonstrate that the 7B2 CT-peptide (a) represents a potent inhibitor of PC2 (Ki = 57 nM), (b) can block the conversion of proPC2 to PC2, and (c) can block the PC2-mediated conversion of proenkephalin to smaller peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional Characterization of ProSAAS

“…His-tagged proSAAS- (1-225) is, however, not nearly as potent as the LLRVKR hexapeptide (K i ϭ 5 nM), a relative order that is consistent with recent studies comparing GST-proSAAS-(1-225) and other synthetic peptides (19). The reduced potency of proSAAS-(1-225) against PC1 compared with the hexapeptide differs considerably from the situation with 7B2 and PC2, because full-length 7B2 is 10-fold more potent against PC2 than is the 7B2 CT peptide (13,10). Additionally, sequences shorter than 7B2 CT-(1-18) do not represent inhibitors of PC2 (35), indicating a smaller effective binding site for natural inhibitors for PC1 as opposed to PC2.…”

“…In fact, recent studies show that 7B2 represents a complex bifunctional binding protein with two domains encoding opposing functions. The N-terminal domain is responsible for the production of activatable pro-PC2 (11, 12) whereas the C-terminal domain (CT peptide) represents a potent PC2 inhibitor (13,14). The mechanism by which the N-terminal domain of 7B2 is able to effect the productive maturation of pro-PC2 to an active enzyme species is not yet clear, but it is thought to involve Golgi-mediated cellular processes rather than a direct effect on activation per se (reviewed in Ref.…”

The proprotein convertase PC2 is involved in the maturation of prosomatostatin to somatostatin-14 but not in the somatostatin deficit in Alzheimer's disease

“…However, if proPC2 has not encountered 7B2, catalytically active PC2 cannot be generated (Zhu, and Lindberg, 1995;Muller et al, 1997). Moreover, uncleaved 7B2 as well as the CT peptide have been demonstrated to act as nanomolar inhibitors of PC2 (Martens et al, 1994;Lindberg et al, 1995).…”

Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

“…We find that DCE can efficiently process peptides that contain LysLys at the P1 and P2 positions and an Arg at the P4 position; furin and PC1 cannot efficiently process at this site (Jean et al, 1995). Studies with purified PC2 have shown that a Lys-Lys site within the proenkephalin precursor is processed by this enzyme; the efficiency of processing is substantially lower than the efficiency of processing at Lys-Arg sites (Lindberg et al, 1995).…”