Cleavage of recombinant proenkephalin and blockade mutants by prohormone convertases 1 and 2: an in vitro specificity study

Abstract

Proenkephalin (PE) derived-peptides are thought to be generated predominantly through endoproteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). In order to compare cleavage site preferences of these convertases, we studied the processing of recombinant wild-type rat PE and of two mutant PEs by recombinant purified mouse PC1 and PC2. Western blot analyses of timed digestions showed that both mouse PC1 and PC2 were able to produce a variety of large and intermediate sized-peptides from wild-type PE as well as from the precursors mutated at initial blockade sites. PC2 exhibited a broader specificity against PE than PC1, generating a much greater number of peptide products. Mass spectrometric identification of cleavage products showed that PC2 appeared to be the principal enzyme involved in the generation of smaller active opioids. Both enzymes were able to cleave various KR-and KK-containing sites, but PC2 was also able to cleave efficiently at an RR-V site and a KK-M site not cleaved by PC1, suggesting the exclusion of large aliphatic residues at the P1¢ position in PC1 cleavage. Alternative cleavage sites were readily chosen by convertases in blockade mutants, confirming in vivo results that cleavages do not follow an obligatory order. Furthermore, glycosylated PE was less efficiently processed by PC2, indicating that glycosylation may serve as a mechanism to hinder processing.

Substrate Cleavage Analysis of Furin and Related Proprotein Convertases

“…Overall, our data correlate well with the results of several laboratories, which extensively studied the individual PCs and their role in the processing of PA83, HA, HIV-1 gp160, MMPs and many other functionally important proteins (9,10,13,15,18,19,24,(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60).…”

Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-Dideoxystreptamine Derivatives

“…However, experimental testing by mutagenesis will be required to confirm direct binding of these molecules to predicted allosteric sites. PC2 stimulation represents a biochemical effect that might have eventual therapeutic relevance in the management of acute and chronic pain, for example by increasing the production of the PC2-synthesized opioid peptides ␤-endorphin (for review, see Mains and Eipper, 2000) and Met-and Leu-enkephalin (Peinado et al, 2003).…”

Spatiotemporal expression of endogenous opioid processing enzymes in mouse uterus at peri-implantation

“…Active opioid peptides are generated by proteolytic cleavage of precursors and PC1 and PC2 mediate this proteolytic cleavage process (Benjannet et al 1991;Zhou et al 1999). PC mainly functions in the posttranslational processing of precursor proteins and is indispensable for the production of active peptides Peinado et al 2003). Moreover, the availability of active opioid peptides also depends on the turnover rate mediated by the degrading enzyme MME (Graf et al 1985).…”

“…The enzymatic activity of PCs can be regulated by ProSAAS (encoded by gene Pcsk1n), a potential inhibitor of PC, which affects PC Cterminal cleavage, a process that is important for its activity in processing the precursors (Cameron et al 2000;Fortenberry et al 2002;Fricker et al 2000;Liu et al 2012;Qian et al 2000). Moreover, the endogenous levels of active opioid peptides also largely depend on the expression and activity of an opioid-degrading enzyme, namely membrane metallo-endopeptidase (MME, also known as NEP; Benjannet et al 1991;Peinado et al 2003;Zhou and Lindberg 1993). Since aberrantly enhanced opioid signaling by morphine adversely affects peri-implantation events (Chen et al 2014;Tang et al 2015), the synthetic enzyme PC and its inhibitor Pcsk1n together with the degrading enzyme MME might coordinately regulate endogenous opioid levels during early pregnancy.…”