Pyroglutamyl peptidase: an overview of the three known enzymatic forms

Biochimica et Biophysica Acta 1429 (1998) 1^17

Review

Pyroglutamyl peptidase: an overview of the three known enzymatic forms Philip M. Cummins *, Brendan O'Connor School of Biological Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland Received 9 July 1998; received in revised form 19 October 1998; accepted 19 October 1998

Abstract Pyroglutamyl peptidase can be classified as an omega peptidase which hydrolytically removes the amino terminal pyroglutamate (pGlu) residue from specific pyroglutamyl substrates. To date, three distinct forms of this enzyme have been identified in mammalian tissues. Type I is typically a cytosolic, cysteine peptidase displaying a broad pyroglutamyl substrate specificity and low molecular mass. Type II has been shown to be a membrane anchored metalloenzyme of high molecular mass with a narrow substrate specificity restricted to the hypothalamic releasing factor, thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2 ). A third pyroglutamyl peptidase activity has also been observed in mammalian serum which displays biochemical characteristics remarkably similar to those of tissue Type II, namely a high molecular mass, sensitivity to metal chelating agents, and a narrow substrate specificity also restricted to TRH. This serum activity has subsequently been designated `thyroliberinase'. This review surveys the biochemical, enzymatic, and structural properties of this interesting and unique class of peptidases. It also addresses the putative physiological roles which have been ascribed to these enzymes. Pyroglutamyl peptidase activities isolated and characterized from bacterial sources are also reviewed and compared with their mammalian counterparts. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Pyroglutamyl; Brain; Neuropeptide; Mammalian; Thyrotropin-releasing hormone; Peptidase

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

2.

Pyroglutamic acid: physiological signi¢cance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: LNA, L-naphthylamide; cDNA, complimentary DNA; CPHNA, N-[1(R,S)-carboxy-2-phenylethyl]-N-imidazole benzyl-histidyl L-naphthylamide; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; Km , Michaelis^Menten constant; Ki , inhibition constant; LHRH, luteinizing hormone-releasing hormone; Mca, 7-amino-4-methyl coumarin; mRNA, messenger RNA; ORF, open reading frame; PACAP, pituitary adenylate cyclase activator peptide 38; PCMB, p-chloromercuribenzoate; pcp, pyrrolidone carboxylyl peptidase; PCR, polymerase chain reaction; pGlu, pyroglutamate; PP, pyroglutamyl peptidase; pNA, p-nitroanalide; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis ; T3 , triiodothyronine; TRH, thyrotropin-releasing hormone; TSH, thyroidstimulating hormone; Z, N-benzyloxycarbonyl * Corresponding author. Present address: Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1065, New York, NY 10029, USA. Fax: +1 (212) 996-9785; E-mail: [email protected] 0167-4838 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 2 4 8 - 9

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P.M. Cummins, B. O'Connor / Biochimica et Biophysica Acta 1429 (1998) 1^17 3.

Pyroglutamyl peptidase: biochemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Pyroglutamyl peptidase type I (PP I, EC 3.4.19.3, MEROPS classi¢cation: C15 [180]) 3.2. Pyroglutamyl peptidase type II (PP II, EC 3.4.19.6, MEROPS Classi¢cation: M1 [181]) 3.3. Serum thyroliberinase (EC 3.4.19.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4 4 6 8

4.

Pyroglutamyl peptidase: molecular properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Bacterial pyroglutamyl peptidase: molecular characterization . . . . . . . . . . . . . . . . . . . 4.2. Mammalian pyroglutamyl peptidases: molecular characterization . . . . . . . . . . . . . . . .

9 9 10

5.

Pyroglutamyl peptidase: inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Inhibitors of pyroglutamyl peptidase I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Inhibitors of pyroglutamyl peptidase II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11 11 13

6.

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Neuropeptides released in autocrine, paracrine, and endocrine systems allow cells to communicate [1]. Intercellular communication is essential to integrate the basic processes of life such as metabolic activity, cell di¡erentiation, and growth. Structurally, neuropeptides consist of polypeptide chains of up to 40 amino acid residues in length with speci¢c modi¢cations to individual residues which govern the distinctive biological activity of each particular neuropeptide. These include modi¢cation of the amino terminus (cyclization of glutamine to pGlu, acetylation) and the carboxy terminus (amidation). These structural modi¢cations determine the highly speci¢c biological activities of neuropeptides, such as their ability to recognize and bind to cell surface receptors and their relative stability towards inactivation by neuropeptidases. Pyroglutamyl peptidase (PP) can be classi¢ed as an omega peptidase (EC 3.4.19.-) which hydrolytically removes the pyroglutamate (pGlu) residue from the amino terminus of pGlu peptides and proteins [2], with an apparent speci¢city for L-pGlu-L-amino acid optical isomers [3]. PP was ¢rst described in a strain of Pseudomonas £uorescens by Doolittle and Armentrout [4], who subsequently termed the activity pyrrolidonyl peptidase. It has since been observed in the tissues of mammals, birds, ¢sh, plants, and bacteria [5,6] and has been referred to by several other names (pyrrolidonecarboxylate peptidase, 5-oxoprolyl peptidase, pyrrolidonecarboxylyl peptidase, pyro-

glutamate aminopeptidase, pyroglutamyl peptide hydrolase, PYRase, and pyroglutamyl aminopeptidase). Numerous workers have investigated the biochemical and enzymatic properties of this interesting enzyme and have speculated on its possible role(s) in neuroendocrine regulation and metabolism in mammalian systems. More recently, genes encoding PP in both bacterial and mammalian systems have been cloned and sequenced, subsequently enabling the expression and detailed structural analysis of recombinant enzymes. Considering the high level of interest in pyroglutamyl peptidases, and their likely participation in neuroendocrine events, we have endeavoured to examine the extensive volume of literature currently available on this class of enzymes and to report on aspects of their biochemical and enzymatic properties, putative physiological role(s), tissue/species distribution, molecular characterization, and selective inhibitors. 2. Pyroglutamic acid: physiological signi¢cance pGlu, also known as 5-oxo-L-proline or pyrrolidone carboxylic acid, was ¢rst described by Haitinger as a glutamate derivative that lacked a molecule of water [7]. Although much evidence exists for the nonenzymatic formation of pGlu from glutamine, glutamate, and various esters and diesters of glutamate [8^10], the enzymatic formation of this compound is well established, suggesting important physiological function(s). Akita et al. [11] for example, have

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Table 1 Some peptides and proteins with an amino terminal pGlu residuea Protein/peptide

Sequence

Reference

TRH TRH-like peptide (prostate) LHRH Neurotensin Eledoisin Gastrin Anorexigenic peptide Colon mitosis-inhibitory peptide Peptide-inhibiting epidermal mitosis Vasoactive polypeptide Fibrinopeptide B Eisenine

pGlu-His-Pro-NH2 pGlu-Glu-Pro-NH2 pGlu-His-Trp-SerpGlu-Leu-Tyr-GlupGlu-Pro-Ser-LyspGlu-Gly-Pro-TrppGlu-His-Gly pGlu-Glu-His-Gly(OH) pGlu-Glu-Asp-CyspGlu-Val-Pro-GlnpGlu-Gly-Val-AsppGlu-Glu-Ala(OH)

[13,14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]

a

For further examples, the reader is directed to Awade et al. [89].

previously described an L-glutamyl cyclotransferase activity in P. cruciviae which is responsible for the conversion of L-glutamate to L-pGlu. The mechanism by which pGlu occurs at the amino terminus of proteins and peptides is still not fully clear. Most current evidence would seem to indicate that, in mammalian tissues, this results from the post-translational cyclization of an amino terminal glutamyl or glutaminyl residue. Fischer and Speiss [12] for example, have previously identi¢ed a mammalian glutaminyl cyclase capable of converting glutaminyl peptides into pGlu peptides. There is no evidence as yet to support the amino terminal addition of a pGlu residue. Many proteins and bioactive peptides exhibit an amino terminal pGlu residue (Table 1 [13^25]), subsequently minimizing their susceptibility to degradation by aminopeptidases. Interestingly, this residue is also a frequent determinant of overall peptide function. For example, Hinkle and Tashijian [26] have demonstrated that any structural substitution in the pGlu lactam ring of the hypothalamic releasing factor, TRH (Fig. 1), signi¢cantly decreases both hormone potency and receptor binding ability. More recently, Perlman et al. [27] have demonstrated that TRH binds to its receptor via an interaction between the ring carbonyl of the TRH pGlu moiety with Tyr106 of the TRH receptor. The functions of pGlu as a free acid are less clear, although its pharmacological properties have been well described. pGlu has been shown to prevent scopolamine induced amnesia

in the rat [28] and to improve learning and age-associated memory loss [29]. In addition, elevated plasma levels of free pGlu have been demonstrated in patients su¡ering from Huntington's disease [30] and pyroglutamic acidemia [31].

Fig. 1. Chemical structures of TRH and its primary metabolite, cyclo(His-Pro).

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3. Pyroglutamyl peptidase: biochemical properties 3.1. Pyroglutamyl peptidase type I (PP I, EC 3.4.19.3, MEROPS classi¢cation: C15 [180]) Workers have described the puri¢cation (partial or otherwise) of PP I from human cerebral cortex, kidney, and skeletal muscle [32^34], bovine whole brain [35], rat liver [36,37], bovine pituitary [38], guinea pig brain [39], and rat brain [40]. PP I activity has also been reported in hamster hypothalamus [41], mouse brain [42], cat brain cortex [43], rat retina and hypothalamus [44], rat adenohypophysis [45], rat pancreas [46], and various rat systemic organs [47]. Non-mammalian animal sources such as avian, ¢sh, and amphibian tissues have also been reported to contain PP I activity [6,48^50]. Mammalian PP I is typically a monomeric enzyme with a low relative molecular mass, a soluble or cytosolic location, and a pH optimum between 6.5 and 8.5 [32,35,38]. Molecular mass estimates range from 22 kDa (human kidney and skeletal muscle [33,34]), 23 kDa (human brain [32]) and 24 kDa (bovine and guinea pig brain [35,39]) to 60 kDa (rat brain [40]). PP I also displays a strict requirement for a thiol-reducing agent, such as DTT or L-mercaptoethanol. Consequently, several reports have demonstrated the extremely inhibitory nature of sulfhydryl-blocking reagents such as N-ethylmaleimide, PCMB, and 2-iodoacetamide towards PP I [35,39,45]. Serine protease inhibitors typically have no e¡ect on this enzyme. With regard to the e¡ects of thiol-reducing agents on PP I activity as a function of tissue speci¢city, Prasad has demonstrated, using rat tissues, that enzyme activity could be either activated or inactivated by DTT depending on the tissue from which the enzyme was isolated [51]. A distinctive biochemical feature of PP I is its broad pyroglutamyl substrate speci¢city, the rate of hydrolysis of any given substrate usually depending on the nature of the amino acid residue (or other group) immediately carboxyl to the pGlu residue. Substrates include biologically active peptides such as TRH, acid TRH (pGlu-His-Pro), luteinizing hormone-releasing hormone (LHRH), neurotensin, bombesin, and anorexigenic peptide, and synthetic substrates such as pGlu-Mca, pGlu-pNa, pGluLNA, pGlu-Ala, and pGlu-Val [35,39,45,48]. Interestingly, pGlu-Pro bonds are not normally hydro-

lyzed by mammalian PP I [38,39]. One notable exception in this instance is a PP activity isolated from Klebsiella cloacae which has been shown to be capable of splitting pGlu-proline [52]. Despite its broad substrate speci¢city, however, PP I is highly speci¢c for amino terminal pGlu residues. A study by Capecchi and Loudon [53] reports that minor alterations to the pGlu residue of a given substrate, such as the introduction of a second ureido nitrogen into the pyroglutamyl ring or increasing the ring size from 5 to 6 members, almost completely eliminates the ability of PP I to cleave the adjacent peptide bond. More recently, researchers have exploited this pGlu specificity as a means of developing potentially useful `prodrugs' which are resistant to PP I attack. pGlu derivatized prodrugs have been found to be completely resistant to PP I hydrolysis by simply substituting the N-H group on the pGlu ring for an N-x group where x is an acyl group (N-CO-R1 ), Mannich base (N-CH2 N-R2 -R3 ), glyoxylic acid adduct (NCH(OH)-COO-R4 ), or one of several other derivatives also investigated [54^56]. The physiological role of PP I currently remains unclear. An early study by Albert and Szewczuk [48] suggested that PP I may participate in the absorption of peptides and proteins from the mammalian alimentary tract. This view is supported by the occurrence of the enzyme in both the small intestine [57] and the intestinal mucous membrane and duodenum, in addition to its broad substrate speci¢city [48]. Based on its relatively ubiquitous distribution in such functionally dissimilar tissues as skeletal muscle, brain, and kidney, and through comparison with other soluble aminopeptidases, workers have further proposed that PP I may contribute to the intracellular catabolism of peptides to free amino acids which are then reincorporated into biosynthetic pathways [32^34,58]. Thus, PP I may, at least in part, be involved in regulating the cellular pool of free pGlu. In this regard, it is noteworthy that free pGlu is known to have pharmacological properties [28,29], whilst elevated levels of free pGlu have been demonstrated in certain disease states [30,31]. Research into the causes/symptoms of various neurodegenerative disorders by Falkous and coworkers also supports the notion of PP I involvement in protein turnover. This group has recently observed signi¢cantly increased levels of PP I (relative to several other cyto-

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solic and lysosomal proteases/peptidases) in the spinal cord of patients su¡ering from motor neuron disease, a disorder manifesting itself in deposits of ubiqutinated proteins in degenerating spinal cord motor neurons [59]. In a parallel study, these workers also demonstrate that PP I from human cerebral cortex is extremely susceptible to inhibition by potentially neurotoxic metal ions (Cu2‡ , Cd2‡ , Zn2‡ , and Pb2‡ ) [60]. The widely accepted inability of soluble peptidases to obtain an extracellular location has cast some doubt on a physiologically signi¢cant role for PP I in neuropeptide metabolism [61^66]. Workers have subsequently hypothesized that PP I, working in combination with other cytosolic enzymes, may represent a mechanism for returning pGlu terminating neuropeptides released from damaged or aging vesicles back to the cellular amino acid pool [67], or, in cases where secretion from neuropeptide-synthesizing cells is suppressed, cytosolic degradation of neuropeptides might conceivably represent a security mechanism to ensure the degradation of neuropeptides which are produced in excess [61]. Consistent with this hypothesis is the ¢nding by Faivre-Bauman and coworkers [68] that addition of speci¢c inhibitors of PP I and prolyl endopeptidase (EC 3.4.21.26) to TRH-synthesizing hypothalamic cells in primary culture results in a signi¢cant increase in both their TRH content and in the amount of TRH being released from these cells under basal or K‡ -stimulated conditions. Therefore, as neuropeptides do not generally have access to the cytosolic compartment, it would be of great interest to identify mechanisms which may possibly exist to bring them into contact with intracellular peptidases. Noteworthy in this regard are `non-classical' release mechanisms which obviate a path through the endoplasmic reticulum and golgi apparatus, allowing synthesis of proteins lacking a signal peptide within the cytosol, followed by their exit from the cell in mature form [69]. Publications in recent years have reported the apparent release of numerous soluble proteins [70^72], all lacking readily identi¢able secretory sequences and hydrophobic transmembrane domains, lending weight to the possibility that intracellular peptidases may act at extracellular locations. The author has also recently observed release of endopeptidase EC 3.4.24.15 (EP24.15), a soluble, 77-kDa, zinc metal-

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loenzyme, from both anterior pituitary gonadotroph and corticotroph cell lines [73]. Interestingly, secreted EP24.15 was seen to display a slightly lower molecular mass (V2 kDa) than the cytosolic form (P.M. Cummins, unpublished observation), suggesting either an as yet unde¢ned processing step necessary for secretion, or that the secreted enzyme is the product of genetic splice variance. At this point, one is reminded that pyroglutamyl peptidase activity was ¢rst observed in a bacterium [4]. PP has since been puri¢ed and characterized from a number of bacterial sources such as P. £uorescens [74,75], K. cloacae [52], Bacillus subtilis [5,76,77], B. amyloliquifaciens [78^81], Streptococcus pyogenes [6,82,83], Enterococcus faecium [84], and Staphylococcus aureus [85]. Interestingly, bacterial PPs display many of the biochemical characteristics common to mammalian PP I. With the exception of the K. cloacae enzyme which is associated with a particulate fraction, all of the bacterial PPs examined to date have been shown to be soluble [77,78,82], to display a broad pGlu substrate speci¢city, and to exhibit a strict requirement for a highly reduced environment [75,79,80]. In contrast to the monomeric nature of native mammalian PP I, however, bacterial PPs invariably exist as multimers. Molecular mass determinations for the bacterial enzyme under denaturing conditions indicate an average subunit mass of 25 kDa which is almost identical to that of the native mammalian enzyme. Native determinations, on the other hand, indicate more variability in size and show an average molecular mass from 40 to 90 kDa. Tsuru and coworkers [78,79] have proposed that the B. amyloliquifaciens PP, with a native molecular mass of 72 kDa and a subunit molecular mass of 24 kDa, probably functions as a trimer. More recently, however, Yoshimoto et al. [81] have cloned the gene for the B. amyloliquifaciens enzyme and, following its expression in Escherichia coli, have shown that the recombinant enzyme appears to exist as a dimer, suggesting di¡erences between the recombinant and wild type forms of the enzyme. Other studies indicate that the recombinant PPs from B. subtilis [77,86] and S. pyogenes [82] are probably tetramers, whilst the recombinant PP from P. £uorescens [86] appears to be dimeric in nature. Although one cannot discount the possibility that these multimers are artefactual in nature due to the in vitro

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oxidation of highly reactive SH groups, multimerization has recently been identi¢ed as a novel mechanism for regulating active site accessibility, and hence enzyme activity, in soluble, thiol-sensitive, mammalian peptidases [87]. Consequently, multimerization may serve a similar function with respect to PP in bacteria. Like its mammalian counterpart, a de¢nitive role for PP in bacteria has yet to be identi¢ed. In bacteria, PP and other peptidases are thought to be involved in protein maturation, protein degradation, and the utilization of peptides as nutrients [88]. Therefore, one might expect that bacterial PPs are involved in intracellular protein metabolism. Awade et al. [89] have also suggested that PP may be involved in detoxi¢cation, since the accumulation of peptides with an amino terminal pGlu may abnormally acidify the bacterial cell cytoplasm. Such proposed roles for bacterial PPs are weakened, however, by the observation that this enzyme is noticeably absent from numerous bacterial strains. In this regard, it would be of interest to determine whether those bacterial strains that lack PP activity exhibit 5-oxoprolinase activity, an enzyme which can decyclize pGlu. 3.2. Pyroglutamyl peptidase type II (PP II, EC 3.4.19.6, MEROPS Classi¢cation: M1 [181]) Initial indications that a proportion of total mammalian brain PP activity might possibly be membrane associated stemmed from the work of several researchers [90^94]. Browne and coworkers [95] were the ¢rst to propose that there were indeed two distinct PP activities in mammalian brain. This research group observed in guinea pig whole brain the previously characterized soluble enzyme, PP I, which required DTT and EDTA for the expression of optimal activity, as well as a membrane anchored activity, designated PP II, which was inhibited by these reagents. These observations were later con¢rmed by O'Connor and O'Cuinn [96] who localized this activity to a synaptosomal membrane preparation of guinea pig brain, from which it could be solubilized by papain treatment. Particulate PP activities have also been demonstrated in rat brain [97,98] and pituitary [99] as well as in rabbit [100], porcine [101], and quite recently, bovine brain [102,103]. Pri-

mary cultures of fetal mouse brain [104] also exhibit this activity. To our knowledge, a PP II activity has yet to be described in human brain. In marked contrast to PP I, PP II has been found to exhibit a large relative molecular mass (V230 kDa) and is readily inhibited by chelating agents such as 1,10-phenanthroline, 8-hydroxyquinoline, and EDTA [96,100,101]. No sensitivity to sulfhydryl-blocking reagents [96] or to the speci¢c PP I inhibitor, pGlu diazomethyl ketone [98], could be observed. Bauer [101] has also recently demonstrated that PP II puri¢ed to homogeneity from rat and porcine brain is comprised of two identical subunits of 116 kDa each, this dimeric architecture being a general feature of membrane anchored ectoenzymes. Mammalian PP II is located primarily in the central nervous system with signi¢cantly smaller levels observed in other tissues [98,105]. Unlike PP I, PP II appears to have a relatively heterogeneous distribution within the CNS (principle locations include anterior pituitary, hypothalamus, amygdala, frontal cortex, and hippocampus) [106,107] where it appears to be associated almost exclusively with neurons, presumably on postsynaptic membranes [104,108]. The observation that PP II has a substrate specificity restricted to TRH, TRH analogs, and TRH-like peptides (tripeptides, tripeptide amides, and tetrapeptides commencing with the sequence pGlu-His) is highly signi¢cant [100,102,103,109^111] (one notable exception here is a recent observation by Bauer [101] who reports on the unusual ability of rat/porcine brain PP II to hydrolyze pGlu-LNA, a substrate typically used to assay for PP I, albeit at a considerably reduced rate compared to TRH). This unprecedented degree of speci¢city for a particular peptide con¢guration has allowed researchers to postulate that membrane anchored PP II is responsible for selectively inactivating neuronally released TRH within the extracellular vicinity of target cells, and should therefore be considered the ¢rst `neuropeptide-specific peptidase' [112]. TRH (Fig. 1) was initially isolated and characterized from porcine [13] and ovine [14] hypothalamic tissue fragments and subsequently designated thyrotropin-releasing hormone or thyroliberin by virtue of its capacity to stimulate the release of thyroid-stimulating hormone (TSH, thyrotropin) from the mammalian anterior pituitary. Its importance in the regulation of the hypothalamic/pitui-

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tary/thyroid axis (Fig. 2) in mammals has since been well established [113]. Many researchers also feel that TRH quali¢es for serious consideration as a neurotransmitter/neuromodulator. Its extrahypothalamic

7

distribution in the brain combined with its localization at the synaptic level, release at synaptic terminals, attachment to high a¤nity receptors which show a remarkable degree of anatomical localization,

Fig. 2. The mammalian hypothalamic/pituitary/thyroid axis. TRH is released from the paraventricular nucleus of the hypothalamus in response to physiological stimuli through median eminence nerve terminals. The hormone is carried via the hypophysial portal blood supply to receptors on target thyrotroph and lactotroph cells of the anterior pituitary (adenohypophysis) where it induces TSH and prolactin secretion. TSH, thyroid-stimulating hormone; TRH, thyrotropin-releasing hormone; T3 , triiodothyronine; T4 , thyroxine ; +/3, positive/negative in£uence.

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speci¢c e¡ects on neuronal activity, and its stimulation of a wide range of centrally mediated behavioral e¡ects, provide a formidable list of criteria consistent with such a neuroregulatory role [114,115]. In support of a role for PP II in the metabolism of TRH in vivo are the following observations: (1) the active site of PP II is located extracellularly [116]; (2) PP II is primarily located within neuronal elements of the CNS where it exhibits a heterogeneous distribution; (3) the inhibition of PP II speci¢cally increases recovery of TRH released from rat brain tissue [117]; (4) in vitro hybridization indicates colocalization of PP II mRNA with TRH receptor mRNA in speci¢c rat brain regions [118]. Numerous exceptions do exist, however [118,119], suggesting that additional mechanisms for TRH inactivation may operate; and (5) Bauer [120,121] has previously reported that rat adenohypophysial PP II activity is stringently controlled by thyroid hormones, whereas brain PP II activity is not. Such tissue-speci¢c regulation of PP II by thyroid hormones suggests that the enzyme could serve as a regulatory control element to in£uence the extent and duration of the endocrine activities of TRH at adenohypophyseal target sites. Elevation of PP II activity speci¢cally in the rat adenohypophysis has also been reported by other workers [122,123] following treatment with thyroid hormones (interestingly, Suen and Wilk [123] also report PP II elevation in the frontal cortex following triiodothyronine (T3 ) treatment). More recently, the downregulation of PP II activity in cultured adenohypophyseal cells by TRH has been observed [107] whilst Lin and Wilk [124] have demonstrated increased levels of PP II mRNA in rat pituitary following T3 treatment. Schomburg and Bauer [125] have recently shown the ability of thyroid hormones to regulate the mRNA transcript levels of both the TRH receptor and PP II in a reciprocal fashion in rat pituitary, again suggesting that PP II represents an important regulatory element controlling the stimulation of TRH target cells. The tissue-speci¢c regulation of PP II activity by estrogen has also been observed. Bauer [121] has previously demonstrated that estrogen treatment decreases adenohypophyseal, but not brain, PP II activity in ovariectomized rats, likely explaining the signi¢cant sex di¡erence in adenohypophyseal membrane PP II activity observed (male rats display four-

fold more anterior pituitary PP II activity than females). More recently, workers have reported that estrogen treatment decreases, in a tissue speci¢c manner, PP II activity and transcript levels in rat pituitary [126]. In vivo, and with GH3 cells in vitro, estrogen also e¡ectively counteracted the increase in enzymatic activity induced by T3 , suggesting their coordinate action on PP II regulation. Within the past year, Charli and coworkers have also demonstrated that, in adenohypophyseal cell cultures, PP II expression and activity is subject to multifactorial regulation by TRH and various other hypophysiotropic factors (somatostatin, PACAP, dopamine etc.) likely acting through cAMP and protein kinase C signaling pathways [127,128]. Finally, the hydrolysis of TRH by PP II yields His-Pro-NH2 , which spontaneously cyclizes at physiological pH to yield cyclo(His-Pro) (Fig. 1) [129], a molecule reputed to play an important role within the mammalian CNS [130,131]. As such, PP II may represent a possible determinant of endogenous cyclo(His-Pro) concentration. 3.3. Serum thyroliberinase (EC 3.4.19.-) In the late 1970s, an enzyme that cleaved the pGlu-His bond of TRH was partially puri¢ed from porcine [132] and rat [133] serum. Unlike the previously characterized cytosolic PP I activity, the serum enzyme was not inhibited by sulfhydryl-blocking reagents, but could be inhibited by metal chelators. This enzyme, optimally active at neutral pH, was reported to have a relative molecular mass of 260 kDa, one order of magnitude greater than that of PP I. In a subsequent study probing the substrate speci¢city of the serum enzyme, Bauer and coworkers [134] have demonstrated that the selectivity of this enzyme is directed towards the pyroglutamyl bond of TRH and TRH-like peptides. Other pGlu substrates such as pGlu-LNA, LHRH, and neurotensin were not cleaved. As a consequence of this narrow substrate speci¢city, the name `thyroliberinase' was subsequently proposed by these researchers to identify this enzyme. Given the close similarities observed between this enzyme and membrane bound PP II, one might speculate that they are products of the same gene, although this has yet to be shown.

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Based on its anatomical localization and high degree of substrate speci¢city, workers have postulated that the serum enzyme may be involved in regulatory mechanisms. The enzymatic degradation of TRH by serum thyroliberinase, during its transport via the hypophyseal portal vasculature to the anterior pituitary for example, might therefore represent a functional control element regulating TRH availability to receptors on target trophic cells. In support of this hypothesis, studies indicate that the activity of thyroliberinase, like PP II, is under the in£uence of thyroid hormones [135^138]. Treatment of rats with a mild goitrogenic agent, for example, has been shown to decrease serum thyroliberinase activity, whilst activity was increased in a dose-related manner after injection of thyroid hormones. Moreover, a signi¢cant sex di¡erence was also observed. Euthyroid female rats showed only 85% of the enzymatic activity of euthyroid males [135]. Thyroliberinase activity also drastically alters with developmental changes [139^142], being absent from rat and human plasma during the neonatal period and appearing during development, which is marked by dramatic changes in thyroid physiology [143,144]. Friedman and coworkers have also recently reported that serum thyroliberinase may be regulated by, or regulates, body weight [145]. Presently, the source(s) of the serum enzyme is unknown. Earlier work by Scharfman and coworkers, however, points to the liver as the potential source of thyroliberinase, as the liver is a potential source of many plasma enzymes [146,147]. The recent ¢ndings of Lin and Wilk, who clearly demonstrate that T3 treatment of rats markedly increases the levels of PP II mRNA in pituitary and liver, add further support to this hypothesis [124]. 4. Pyroglutamyl peptidase: molecular properties Characterization of the genetic determinants of pyroglutamyl peptidases is a necessary step in order to improve our understanding of this particular class of enzymes. Nucleotide sequence analysis and comparisons would improve prediction of conserved protein patterns involved in substrate binding and catalysis, and would enable researchers to glean insight into the factors which regulate PP gene expression.

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4.1. Bacterial pyroglutamyl peptidase: molecular characterization Researchers have recently focused on bacterial sources to isolate and characterize the genes which encode for PP activity. To date, bacterial PP genes have been cloned from S. pyogenes [82,83], B. subtilis [77], B. amyloliquifaciens [81], P. £uorescens [75], and most recently, S. aureus [85]. These genes have been designated by various authors as `pcp' genes (for pyrrolidone carboxylyl peptidase). The overexpressed B. amyloliquifaciens enzyme has also been crystallized [81], and therefore a knowledge of PP structure is imminent. The strategy used for isolating these genes was to screen bacterial gene libraries for PP activity in E. coli, a host which does not exhibit PP activity. Clones were selected using a procedure developed by Mulczyk and Szewczuk [148] which relies on the enzymatic liberation of LNA from pGlu-LNA by `PP-positive' colonies, with the subsequent conversion of LNA into a colored compound. Analysis of the aforementioned PP genes reveals that they have a common structure. The size of their open reading frame (ORF) is similar and relatively small. The PP genes from S. pyogenes, B. subtilis, and B. amyloliquifaciens are all 645 nucleotides long and encode for a 215-amino acid protein, whilst those from P. £uorescens and S. aureus are 639 and 636 nucleotides long, respectively, encoding for proteins of 213 and 212 amino acids, respectively. PP also appears to be present as a single copy gene in the bacterial genome (this has been con¢rmed for S. pyogenes, B. subtilis, and P. £uorescens) [75,77,83]. Gene mapping studies have revealed the existence of inverted repeats of the PP gene both upstream and downstream of the ORF in B. amyloliquifaciens, B. subtilis, and S. pyogenes, suggesting the presence of putative regulatory binding sites that would be of great interest to investigate further. The occurrence of extremely low PP primary transcript levels in B. subtilis and S. pyogenes may indicate either poor mRNA stability, or that the PP gene is weakly expressed in these bacteria, subsequently concurring with the possible existence of a transcriptional control mechanism regulating gene expression. Recent studies with P. £uorescens also indicates the existence of multiple inverted repeat sequences as well as a putative ferric uptake regulator (FUR) binding site

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within the PP promoter, suggesting transcriptional regulation by iron [149]. Interestingly, this latter study also demonstrated induction of PP gene expression with the product of the enzyme activity, pyroglutamic acid. Bacterial PP genes show neither any signi¢cant similarity to other known nucleotide sequences, nor do the deduced amino acid sequences compare favorably with those from other prokaryotic or eukaryotic sources [89]. This lack of homology with other proteins, including other proteases, suggests that PPs may represent a new and unique class of peptidases. Alignment of the deduced amino acid sequences of the ¢ve aforementioned bacterial PPs cloned thus far reveals a highly conserved primary structure [75,83,85,89]. The two most highly conserved segments lie between amino acids 81^100 and 131^149 (numbering is for P. £uorescens) (Fig. 3). On the basis of an 80% consensus de¢nition, the identities between these ¢ve PP sequences are V76% and V67%, for domains 1 and 2, respectively. Based on this degree of conservation, these two domains are likely to be involved in the biological activity of the bacterial PPs. A uniquely conserved cysteine residue (Cys144 ) is also present within the second domain of the alignment, consistent with the thiol-dependent nature of this class of enzymes, and therefore likely involved in catalysis. This hypothesis has subsequently been con¢rmed by site-directed mutagenesis of the PP from B. amyloliquifaciens. Mutating

Cys144 to Ser144 resulted in total ablation of enzyme activity [81]. Like other cysteine peptidases, bacterial PPs may exhibit a putative catalytic triad consisting of a Cys and His residue with either Asp, Glu, Asn, or Gln [150]. In this regard, one notes from the sequence alignment that Cys144 and His166 (numbering is for P. £uorescens) are also fully conserved among the ¢ve sequences examined [85,89], as are Glu81 / Asp94 . The involvement of either of these latter residues in catalysis, however, has yet to be determined. Using the method of Kyte and Doolittle [151], assessment of the hydrophobic character of the aforementioned PP sequences reveals that the enzyme charge is uniformly distributed along the polypeptide chain, with a distinct absence of an extended stretch of hydrophobic amino acids. This is consistent with the observation that these enzymes are soluble [82,83]. The absence of a recognizable secretory signal sequence from any of the bacterial PP cDNAs examined thus far is also consistent with an intracellular location for this enzyme. 4.2. Mammalian pyroglutamyl peptidases: molecular characterization The reader is reminded at this point that the bacterial PPs very closely resemble mammalian PP I. To our knowledge, however, no genetic/structural data are currently available for the mammalian type I enzyme, although Schauder et al. [152] have recently

Fig. 3. Alignment of homologous domains likely to be involved in the biological activity of bacterial PPs. Numbering is for P. £uorescens. Residues in bold are common to all ¢ve sequences. The common sequence indicates residues which occur at a frequency of at least 80% within the alignment. The conserved residue in Domain 2, Cys144 , is also underlined.

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cloned, for the ¢rst time, a cDNA encoding for a mammalian PP II activity. The strategy employed by these authors involved screening of cDNA libraries from rat brain and pituitary using synthetic probes predicted from the sequence of the puri¢ed enzyme, subsequently leading to the identi¢cation and isolation of a cDNA that encodes a protein of 1025 amino acids (subunit molecular mass of 117.3 kDa). Transient transfection of COS-7 cells with this cDNA led to the expression of an active ectopeptidase that displayed the characteristics of the TRHdegrading mammalian ectoenzyme. Southern hybridization also revealed that this enzyme is present in the rat genome as a single copy gene. Analysis of the deduced amino acid sequence was consistent with the identi¢cation of the enzyme as a glycosylated, membrane-anchored, zinc metallopeptidase. Hydropathy analysis predicted a transmembrane spanning domain of 22 hydrophobic residues near the amino terminus which is preceded by a basic stop transfer sequence, indicating that mammalian PP II is an integral membrane protein anchored by an uncleaved signal sequence [153]. The authors also report the presence of 12 putative glycosylation sites in addition to an intracellular threonine residue towards the amino terminus which may be a potential regulatory site for phosphorylation by protein kinase C. PP II also displays a HEXXH motif (aa position 441^445), in addition to a glutamate residue at position 464, 19 residues carboxyl to this motif. This consensus sequence serves to coordinate the catalytically essential zinc atom within the active site [154], and is a characteristic of the zinc-dependent metallopeptidase family. Interestingly, one of the residues in this motif is reported to be cysteine, a unique occurrence in this family of peptidases, although the functional signi¢cance of Cys in this position has yet to be determined. 5. Pyroglutamyl peptidase: inhibitors The rational design of potent and speci¢c peptidase inhibitors generally proceeds from a knowledge of the enzymes mechanism of action and from detailed mapping of its substrate speci¢city. This enables structural features to be incorporated into the inhibitor, thus facilitating its interaction with cata-

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lytic subsites on the enzyme. Such inhibitors may be of potential value in a number of studies. For example: (1) they may increase the half life of endogenous neuropeptides such as TRH and therefore be of value in exploring the physiological e¡ects of these neuropeptides; (2) they may potentiate the e¡ects of exogenous neuropeptides; (3) they can be used to prevent the degradation of neuropeptides in radioligand binding assays and radioimmunoassays; and (4) they are also excellent tools for probing the physiological signi¢cance of the targeted enzyme. 5.1. Inhibitors of pyroglutamyl peptidase I Active site-directed PP inhibitors were ¢rst synthesized by Fujiwara and coworkers [155^157] for the B. amyloliquifaciens PP. These were pGlu chloromethyl ketone (pGCK), Z-pGlu chloromethyl ketone (ZpGCK), and Z-pGlu diazomethyl ketone (ZpDMK) (Fig. 4). The chloromethyl ketone derivatives were found to be highly speci¢c, potent, and irreversible inhibitors of this enzyme. In addition, the rate of PP inactivation by pGCK was found to be over 10-fold greater than that of Z-pGCK (this lower potency can be accounted for by the presence of the Z group on the pGlu ring). The aforementioned research group [155] have also achieved complete and rapid inactivation of PP I from rat liver and kidney in vitro with pGCK. Svoboda and Currie [158] have also reported the irreversible inhibition of calf liver PP I by chloromethyl ketone analogs of TRH. Unfortunately, thiol-reducing agents such as DTT and L-mercaptoethanol, had to be excluded from the assay mixture to avoid their reactivity with the above inhibitors, subsequently making accurate kinetic analysis of the inactivation reaction both di¤cult and error prone. The diazomethyl ketone derivative, although far less potent than pGCK or Z-pGCK [157], was also found to be a speci¢c and irreversible PP inhibitor. However, Z-pDMK was found to be relatively inert toward thiol reagents. The in vivo e¡ects of these inhibitors have yet to be ascertained. Wilk et al. [159] have subsequently synthesized pGlu diazomethyl ketone (pDMK) (Fig. 4), an inhibitor found to be signi¢cantly more potent than its ZpGlu derivative described above. Preincubation of partially puri¢ed bovine brain or calf liver PP I

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Fig. 4. Active site-directed inhibitors of PP I: chemical structures of 2-pyrrolidone and the chloromethylketone and diazomethylketone inhibitors.

with nanomolar quantities of this compound led to rapid and irreversible inactivation of the enzyme in a time- and concentration-dependent manner. In addition, pDMK concentrations ¢ve orders of magnitude higher did not inactivate other endo- or exopeptidases tested, including PP II [160], indicating that this inhibitor is highly speci¢c for PP I. This inhibitor was also found to be extremely e¡ective and long lasting in vivo. When administered intraperitoneally to mice, it totally inactivated PP I in all tissues studied (brain, heart, muscle, lung, spleen, liver, and kidney) at doses as low as 0.1 mg/kg, with as much as 50% inhibition still observed in most tissues 24 h after administration. Signi¢cant inhibition was also observed when the inhibitor dose was decreased to 10 Wg/kg. Peptide aldehyde analogs of the acyl portion of protease substrates have been reported to be potent competitive inhibitors of thiol and serine proteases [161,162]. Consequently, Friedman et al. [163] have synthesized 5-oxoprolinal, the aldehyde analog of

pGlu, as an active site-directed, transition state inhibitor of PP I. This inhibitor was shown to be a potent and speci¢c competitive inhibitor of calf liver PP I in vitro (Ki = 26 nM), although far less e¡ective in vivo. Intraperitoneal injection into mice at a dose of 50 mg/kg resulted in greater than 60% inhibition of enzymatic activity in all organs tested 10 min after injection. After 30 min, however, the degree of inhibition had signi¢cantly decreased. This relatively weak and transient action in vivo contrasts with the aforementioned pDMK, and can probably be accounted for by the reversible binding of 5-oxoprolinal and/or its metabolic inactivation. Other selective PP inhibitors worthy of mention include benarthin, pyrizinostatin, and 2-pyrrolidone. The former two inhibitors, benarthin and pyrizinostatin, were isolated from culture ¢ltrates of the genus Streptomyces and represent a new structural class of PP inhibitors [164^167]. Pyrizinostatin has also been synthesized from the antibiotic, 2-methylfervenulone [168]. The latter compound, 2-pyrroli-

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done (Fig. 4), is a pyroglutamyl substrate analog which acts as a potent, reversible, non-competitive inhibitor of mammalian PP I and microbial PPs, although 100% inhibition of enzyme activity has never been obtained with this inhibitor, even at concentrations as high as 100 mM. The ability of 2-pyrrolidone to stabilize PP activity in solution during puri¢cation and storage has also been reported [36,38,69], with complete recovery of activity obtainable following dialysis to remove the inhibitor. 5.2. Inhibitors of pyroglutamyl peptidase II PP II has been identi¢ed as a TRH-speci¢c metallopeptidase, suggesting that an inhibitor should incorporate the structural features of TRH together with a group capable of chelating the active site metal ion. Reductive amination of peptides with an Kketo acid has proved to be a fruitful approach to the synthesis of active site-directed inhibitors of metalloenzymes (enalpril, an inhibitor of angiotensin-converting enzyme [169], for example, is one such compound). Charli et al. [117] have subsequently synthesized a PP II inhibitor via the reductive amination of His(Nim -benzyl)-NA with phenylpyruvate to yield CPHNA (N-[1(R,S)-carboxy-2-phenylethyl]N-imidazole benzyl-histidyl L-naphthylamide). A pGlu containing keto acid would obviously have presented more optimal binding features. Unfortunately, the latter research group were unable to synthesize this intermediate. Irrespective of this, CPHNA has proved to be a potent, reversible inhibitor of puri¢ed PP II with a Ki of 8 WM (Fig. 5).

13

deed, despite the availability of enzymatic and chemical methods to open pGlu rings [172,173], and physical methods, such as mass spectrometry, which are available to overcome sequencing di¤culties due the amino terminal pGlu block [174], PP still remains the deblocking method of choice for many sequencers [175,176]. The functional usefulness of PP has also been elevated by its applicability to the ¢eld of bacterial diagnostics. Although present in some bacterial strains, this enzyme activity is absent in others [4]. Several bacterial diagnostic techniques have subsequently been developed which are based on the use of speci¢c chromogenic and £uorogenic substrates, such as pGlu-LNA, pGlu-Mca, and pGlu-pNA. For example, initial applications of the `PLP' test developed by Mulczyk and Szewczuk [148,177] included di¡erentiation of the enterobacteriacea and staphylococci species whilst, quite recently, PP activity has been exploited in diagnostic tests for the identi¢cation of enterococci, leuconostocs, and pediococci [178]. The recent characterization of bacterial PP genes may also lead to promising applications in this area. Group A streptococci for example have already been identi¢ed in clinical human and animal samples using DNA probes to the PP gene [179]. Using this approach, it may be possible to replace the PP activity tests with molecular probe tests for a more rapid and de¢nitive identi¢cation of bacterial species. The reader is directed to Awade et al. [89] for a more

6. Discussion The impetus behind the initial discovery and isolation of pyroglutamyl peptidase [4] was to isolate an enzyme activity that would render terminal amino groups accessible in pyroglutamyl peptides and proteins. The absence of an K-NH2 group in these substances was for many years a major obstacle in their characterization, since amino terminal analyses, including stepwise degradation methods [170], could not be carried out. Consequently, PP has proven extremely useful in this role (typically commercial calf liver PP I or a bacterial PP preparation) [171]. In-

Fig. 5. Active site-directed inhibitors of PP II: chemical structure of CPHNA.

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complete overview of the usefulness of pyroglutamyl peptidase with regard to bacterial diagnostics. In conclusion, this article has attempted to present a well-rounded overview of the pyroglutamyl peptidase class of enzymes (EC 3.4.19.-) which have been implicated in the metabolism of proteins and peptides at both the cytosolic and plasma membrane level. The primary focus has been the examination of biochemical, enzymatic, and molecular characteristics of pyroglutamyl peptidases, primarily from mammalian species, although prokaryotic sources have also been included. The authors sincerely hope that research will continue with this unique class of enzymes, particularly with respect to the continued delineation of their genetic determinants and physiological roles in neuroendocrine processes.

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