Specificity of Prohormone Convertase 2 on Proenkephalin and Proenkephalin-related Substrates

Abstract

In the central and peripheral nervous systems, the neuropeptide precursor proenkephalin must be endoproteolytically cleaved by enzymes known as prohormone convertases 1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. In this study, we have investigated the specificity of recombinant mouse PC2 for proenkephalin-related internally quenched ( ؊1 (<Glu-Arg-Thr-Lys-Arg-MCA; where <Glu is pyroglutamic acid). Substrates containing only a single basic residue were not appreciably hydrolyzed, and substrates lacking a P4 Arg exhibited k cat of less than 0.05 s ؊1 . Substitution of ornithine for Lys at the P4 position did not significantly affect the k cat but increased the K m 2-fold. Data from both sets of fluorogenic substrates supported the contribution of a P4 Arg to PC2 preference. Analysis of proenkephalin reaction products using immunoblotting and gel permeation chromatography demonstrated that PC2 can directly cleave proenkephalin and that the generation of small opioid peptides from intermediates is mediated almost entirely by PC2 rather than by PC1. These results are in accord with the analysis of PC2 knock-out brains, in which the amounts of three mature enkephalins were depleted by more than three-quarters.Prohormones and proneuropeptides are synthesized as inactive large precursors that are proteolytically cleaved during intracellular transport to generate active peptide forms for extracellular release (1, 2). Pairs of basic amino acid residues such as Lys-Arg and Arg-Arg-and to a certain extent Lys-Lys and Arg-Lys-have been recognized as consensus sites of proteolytic cleavage (1, 2). Primary and secondary structures in proproteins are thought to be important for enzyme recognition and selectivity of cleavage (1-3). The involvement of prohormone convertases 1 and 2 (PC1 1 and PC2), serine proteinases expressed in neuroendocrine tissues, in precursor cleavage has been widely accepted (1, 2) although other enzymes have recently also been reported to be involved in opioid peptide precursor cleavage (4).Previous reports from our laboratory using cell-based systems have provided support for the idea that PC1 is the chief enzyme concerned with the generation of intermediate-sized peptides from proenkephalin (PE) and that PC2 is mostly responsible for the production of small, bioactive opioid peptides (5, 6). The profile of peptides generated in PE-expressing AtT-20 cells (7), which contain high quantities of endogenous PC1 (8), consists mainly of 3-18-kDa enkephalin-containing peptides, although some mature enkephalins such as Met-enkArg-Phe and Met-enk are also present (7). In contrast, the major PE products in PC2-containing AtT-20/PE cells, as well as in PC2-expressing Rin-PE cells, are the fully processed opioid peptides Met-enk-Arg-Phe, Met-enk-Arg-Gly-Leu, Metenk, and Leu-enk (6, 7). In agreement with the notion that PC2 expression is correlated with more complete processing of PE, antisense experiments have shown that PC2 is largely responsible for the processing of PE into smaller opioid peptide...

Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter

“…PE processing in brain also involves the subtilisin-like PC2 protease, because mice that contain inactive PC2 possess partial reduction of ME in brain (30,31). The reduction of ME by approximately one-half in cathepsin L KO mice and the partial reduction of brain enkephalin in PC2-deficient mice indicate that ME production in brain involves cathepsin L as well as PC2.…”

Mutations in the Catalytic Domain of Prohormone Convertase 2 Result in Decreased Binding to 7B2 and Loss of Inhibition with 7B2 C-terminal Peptide

“…Ac-LLRVKR-NH 2 (51) was kindly provided by J. Appel and R. A. Houghten (Torrey Pines Institute for Molecular Studies, San Diego, CA). The internally quenched fluorogenic substrate Abz-VPEMEKRYGGFMQ-EDDnp (20) was kindly provided by L. Juliano (Department of Biophysics, Escola Paulista de Medicina, San Paolo, Brazil).…”

“…Abz fluorescence was recorded with a Fluoroscan Ascent fluorometer (Labsystems) using excitation and emission wavelengths of 320 and 420 nm, respectively. k cat /K m values were calculated based on the curves obtained, as described previously (20).…”

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

“…These enzymes are thought to cleave peptide precursors preferentially at multibasic cleavage sites Steiner, 1998). The selectivity and efficiency of cleavage appear to be governed by the extent of processing of PCs and by the level of endogenous inhibitors (Jutras et al, 1997;Lusson et al, 1997;Benjannet et al, 1998;Johanning et al, 1998). An endoprotease designated dynorphin converting enzyme (DCE) cleaves neuropeptides such as dynorphins at monobasic sites (Berman et al, 1995;Devi, 1998).…”