Porcine Muscle Prolyl Endopeptidase and Its Endogenous Substrates

Abstract

Prolyl endopeptidase [EC 3.4.21.26] was purified 4,675-fold with a yield of 26.3% from porcine muscle. The purified enzyme was shown to be very similar to the liver enzyme with respect to its molecular weight (72,000-74,000), antigenicity, substrate specificity, and susceptibility to protease inhibitors. Among several bioactive peptides, angiotensins I, II, and III had the lowest Km of 0.6 to 3 microM with the lowest kcat of 0.19 to 0.85 s-1, while thyrotropin-releasing hormone had the highest Km of 98 microM with the highest kcat of 14.4 s-1. Interestingly, mastoparan was hydrolyzed at alanyl bonds, but insulin was only slightly hydrolyzed and glucagon was not hydrolyzed although the latter two peptides contain prolyl and/or alanyl bonds. Muscle prolyl endopeptidase failed to hydrolyze proteins with high molecular weight such as albumin, immunoglobulin G, elastin, collagen, and muscle soluble and insoluble proteins. However, 8 of 14 peptides with molecular weights lower than 3,000, which were isolated from muscle extract, were digested by this enzyme, and they were proved to contain prolyl and/or alanyl residues in their molecules. The data suggest that they are probable endogenous substrates for prolyl endopeptidase.

Peptidomics of the Prolyl Peptidases

“…Pioneering work by Polgár and coworkers had previously revealed a length dependence in Prep substrate selectivity, namely that Prep clearly preferred shorter peptides. Similarly, our experiments demonstrate that Prep favors the shorter peptide, CGRP (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37), as a substrate, with a specificity constant (k cat /K M ) that is 20-fold higher than that for CGRP(1-37) (Fig. 3).…”

“…The cleavage of the neurosecretory protein VGF (490-507) (Vgf(490-507)), Vgf(24-39), preproenkephalin 1 (114-133) (Penk(114-133)), and acetylated heat shock 70-kDa protein 12a(2-23) (Ac-Hspa12a(2-23)) were studied with the recombinant enzyme. Two of these peptides, Vgf (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39) and Penk(114-133), were cleaved at multiple proline sites in the peptide, which was expected for Prep. In contrast, the other two peptides, Vgf(490-507) and Ac-Hspa12a(2-23), showed cleavage patterns that could not be predicted or explained using known models for Prep selectivity.…”

“…The unique biochemical activity of Prep, namely the ability to cleave peptides at proline amino acids, has prompted a great deal of research into the biochemical, cellular, and physiological functions of this protein (9). In vitro assays have identified Prep substrates that range from the tripeptide, thyrotropin-releasing hormone, to a 31 amino acid peptide, beta endorphin (9,34), which led to new hypotheses about the physiological role of Prep, including a role in cognitive function (15)(16)(17)(18).…”

“…We also compared the cleavage of CGRP (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37), an endogenous Prep substrate, and full-length CGRP(1-37) to investigate the role of peptide length and cleavage site context in Prep biochemistry. Pioneering work by Polgár and coworkers had previously revealed a length dependence in Prep substrate selectivity, namely that Prep clearly preferred shorter peptides.…”

Structure and Localization of the Mouse Prolyl Oligopeptidase Gene

“…A very recent study by Fü löp et al (2) has established the unique three-dimensional structure of porcine muscle prolyl oligopeptidase and may have solved the perplexing problem of why the enzyme only hydrolyzes peptides consisting of no more than 30 amino acid residues (3). Prolyl oligopeptidase is found in various organisms, including mammals (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), insects (13), plants (14), mushrooms (15,16), and bacteria (17)(18)(19). Such a wide distribution suggests the general importance of this enzyme.…”