Prostatic kallikreins: biochemistry and physiology

Comp. Biochem Physiol. Vol. 107C, No. 1, pp. 13-20, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0742-8413/94 $6.00 + 0.00

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M I N I REVIEW Prostatic kallikreins: biochemistry and physiology J. Y. Dub6 Laboratory of Hormonal Bioregulation, Laval University Hospital Research Centre, Sainte-Foy, Qu6bec, Canada, G1V 4G2 This review describes and compares the properties of seven individual kallikreins present in the prostate of four mammalian species. The first kallikrein discovered in prostate was the one of guinea-pig. That protein has kininogenase activity like classical kailikreins. The rat prostate expresses two different kallikreins, $3 and P1, whose physiological functions remain to be determined precisely. In man, prostate-specific antigen (PSA) is an abundant secretory protein. It is currently used as a prostate cancer marker. The human prostate may also contain renal/pancreatic kallikrein and human glandular kallikrein-1 (hGK-1). Arginine esterase secreted by dog prostate is probably the most abundant kailikrein. It has no known physiological substrate. Key words: Prostatic kallikreins. Comp. Biochem. Physiol. 107C, 13-20, 1994.

Introduction Glandular kallikreins constitute a group of enzymes that were discovered more than 60 years ago through their pharmacological activity on blood vessels and smooth muscles (see the reviews of Schacter, 1980 and Bhoola et al., 1992). This activity is the result of the proteolytic cleavage of the substrate kininogen by kallikreins and the liberation of vasoactive kinins, bradykinin and kallidin. The biological effects of these kinins include smooth muscle contraction, ion transport, cell proliferation, capillary permeability and pain production. It has become evident that not all kallikreins were able to liberate kinins from kininogen. In some cases, kallikreins have rather been shown to activate prohormones or proenzymes by limited proteolytic cleavage. Finally, the actual in vivo substrates of many kallikreins are not known.

As in many other fields, the recent advent of molecular biology technology has considerably expanded and modified our knowledge on the tissue and species distribution of kallikreins. In particular, these studies have shown that there were striking differences between different animal species with respect to the number of kallikrein genes, the tissue pattern of kallikrein expression and kallikrein levels. Classical kallikrein-containing organs are the pancreas, the salivary glands and the kidney. Early studies of Bhoola et al. (1962) have also shown that the prostate and coagulating gland of guinea-pig contained large amounts of a kinin-releasing substance. Such a substance was not present in the prostate gland of rat, rabbit, dog and man. Undoubtedly because of these results, the prostate was not generally considered as a kallikrein-containing organ. However, in the last 15 years, the prostate of several mammalian species was shown to secrete huge amounts of kallikrein-like substances which are now considered as bona fide members of the kallikrein family because of their homology with kallikreins having kininogenase activity and

Correspondence to: J. Y. Dub6, Laboratory of Hormonal Bioregulation, Laval University Hospital Research Centre, Sainte-Foy, Qu6bec, Canada, GIV 4(32. Fax 418-654-2714. Received 19 July 1993; accepted 17 September 1993. caP(c) ,o~/i--s

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because of their clustering with the other kallikrein genes on specific chromosomes. The present review will summarize the biochemistry and physiology of the seven individual kallikreins known or suspected to be present in prostatic tissues of four mammalian species. The main objectives are to discuss the potential roles of these substances in mammalian reproduction and to underline the remaining questions.

Rat prostate kaHikreins The rat genome is estimated to contain between 15 and 20 kallikrein genes. The submandibular gland expresses the highest levels as well as the highest number of different kallikreins. Individual kallikreins are found in many tissues (see the review of Bhoola et al., 1992) and the prostate expresses two of them, namely kallikrein $3 and kallikrein P1. These proteins are not specific to the prostate since they are also found in the submandibular gland.

Guinea-pig prostate kallikrein As mentioned previously, the first prostatic kallikrein was discovered in the guinea-pig (Bhoola et al., 1962). The protein responsible for the kinin-releasing activity was isolated by Moriwaki et al. (1974) and by Dunbar and Bradshaw (1985). It is a single chain enzyme of 37kDa on SDS polyacrylamide gel. After chemical deglycosylation, the protein migrates as a 26 kDa band on SDS gels. The protein has lower esterase activity than y-NGF towards TAME and TLME and no activity towards BAPNA. It binds covalently 3H-DFP as other members of the serine-protease family. In their study, Dunbar and Bradshaw (1985) have focussed their attention on the possible complex formation of the guinea-pig kallikrein with flNGF and EGF which are present at high concentrations in the prostate (Harper et al., 1979). No evidence was found for the formation of such complexes previously demonstrated in mouse salivary glands (Thomas et al., 1981). Since the guinea-pig prostate kallikrein has 60% homology with 7-NGF (Dunbar and Bradshaw, 1987), it has also been suggested that the kallikrein, as in mouse salivary glands, could have a role in the processing of the precursors of NGF and EGF. However, the hypothesis has not been tested experimentally. Therefore, the only known potential physiological activity of the guinea-pig prostate kallikrein is the one related to its ability to form kinins. However, it is not certain that the enzyme ever comes into contact with kininogens in vivo. In the case of rat salivary glands, it has been proposed that at least part of the secreted kallikrein could be involved in local blood flow regulation after its release into the vascular compartment and the interstitial space (BergOrstavik et al., 1982). However, the endocrine secretion of prostatic kallikreins, at least in man and dogs, appears minimal and a very high proportion of the secretory material is destined for the seminal plasma. Thus, the exact endogenous substrate of guinea-pig prostate kallikrein as well as its role remain to be determined.

Kallikrein $ 3

Kallikrein $3 was isolated recently by at least five independent research groups either from the prostate (Winderickx et al., 1989; Wang et al., 1992) or from the submandibular gland (Yamaguchi et al., 1991; Berg et al., 1992; Moreau et al., 1992). The properties of kaUikrein $3 can be summarized as follows. The protein migrates approximately as a 30 kDa band on SDS gel in the absence of mercaptoethanol and as two bands of 18 and 12kDa in the presence of mercaptoethanol. These two bands probably arise by autocatalytic cleavage in the kallikrein autolysis loop. The protein represents from 3 to 6% of the whole cytosolic proteins in the prostate. It shows only little enzymatic activity towards a variety of synthetic substrates. However, the use of protein substrates as well of synthetic substrates clearly indicated that $3 specificity was different from that of classical kallikreins. Indeed, the protein had both trypsin-like and chymotrypsin-like specificity cleaving after basic as well as aromatic residues (Moreau et al., 1992). It preferred a proline residue in position P2 whereas classical kallikreins prefer at that position a bulky hydrophobic residue such as phenylalanine in the kininogen sequence Pro-Phe-Arg-. The proline preference at position P2 has been suggested to be important in the processing of peptide precursors (Schwartz, 1986). Indeed, Winderickx et al. (1989) have indicated in their paper that protein $3 could specifically cleave the C2 component of the major rat prostate secretory protein, prostatic binding protein. It could also be involved in the proteolysis of an abundant protein of seminal vesicle secretion involved in the formation of the copulation plug. However, there have been no experimental details presented for these results which, if confirmed, would suggest that one of the physiological roles of rat prostate $3 would be to liberate spermatozoa entrapped in the copulation plug. Processing of other proteins would not be excluded either since $3 has been shown to have a vasoconstrictor activity in vitro on isolated

Prostatic kallikreins: biochemistryand physiology rabbit or rat thoracic aortic rings (Yamaguchi et al., 1991; Berg et al., 1992). This activity was not blocked by the angiotension II antagonist, indicating that the activity was not due to the formation of angiotension II from angiotensinogen. The substrate responsible for the vasoconstrictor activity was not identified. Furthermore, the importance of this phenomen in prostatic physiology remains unknown. Kallikrein P 1 or r k 8

Kallikrein P1 (also designated rk8) was isolated only recently by Elmoujahed et al. (1990) from the rat submaxillary gland. There has been no report yet of its isolation from rat prostate, but the mRNA is present in that organ (Clements et al., 1988 and Brady et al., 1989). Similarly to kallikrein $3, kallikrein P1 migrates as two bands (19 and 9 kDa) on SDS-mercaptoethanol gel electrophoresis. The enzymatic activity was studied with fluorogenic substrates having an arginine residue at position P1 and various residues at position P2. These experiments indicated that the protein preferentially cleaved Z-Phe-Arg-NHMec rather than the kininogen-like substrate Pro-Phe-Arg-NHMec. However, the actual kininogenase activity was not tested. But this type of activity certainly remains possible since all the necessary amino acids thought to be required for the kininogenase activity are present in kallikrein P1 (Brady et al., 1989). Further studies are necessary, first to demonstrate that kallikrein P1 is really present in the prostate, and secondly to determine its physiological substrate in that organ.

Human prostate kallikreins Three kallikrein genes have been characterized in the human genome. These are the prostate specific antigen (PSA) gene, the human glandular kallikrein-1 (hGK-1) gene and the true tissue kallikrein gene (hRPK) expressed in the kidney, the pancreas and the salivary glands. PSA is one of the major secretory proteins in the prostate. There are also some evidences that the proteins encoded by hGK-1 and hRPK genes could be present in the prostate and prostatic secretions. PSA

PSA was first isolated by three independent groups in the 1970s on the basis of its antigenicity (Li and Beling, 1973) or of its utility in forensic science (Hara et aL, 1971; Sensabaugh, 1978). The term "prostate specific antigen" was first used by Wang et al. (1979) who suggested the usefulness of this protein in the monitoring of prostate cancer patients. Thereafter, PSA has

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rapidly become the most useful marker of cancer and has replaced the long used acid phosphatase (Gutman and Gutman, 1938). For the clinical aspects of PSA, the reader is referred to the recent review of Oesterling (1991). Only the biochemical and physiological aspects will be dealt with in this paper. PSA is a single chain enzyme of 34 kDa on SDS-mercaptoethanol gels. Its amino acid sequence was first published by Watt et al. (1986) and later on by Lundwall and Lilja (1987) and Riegman et al. (1988). It showed that the protein belonged to the kallikrein family of serine proteases. The enzymatic activity of PSA was studied with some details by Ban et al. (1984), Lilja (1985), Watt et al. (1986), Akiyama et al. (1987) and Christensson et al. (1990). These studies clearly showed that when contaminating proteases were removed by affinity chromatography, PSA demonstrated a very strict chymotrypsin like specificity towards synthetic substrates and proteins during in vitro incubations. The hydrolysis of proteins occurred on the carboxylic side of tyrosine, leucine and phenylalanine. This type of specificity is totally different from more habitual trypsin-like specificity of kallikreins, and indicates that PSA has no kininogenase activity. The in vivo substrate of PSA is the predominant seminal vesicle protein called semenogelin (Lilja, 1985; McGee and Herr, 1988; Lee et al., 1989). The fragmentation sites of semenogelin by PSA were identified as Tyr-44, Leu-84 and Tyr-136 of semenogelin (Lilja et al., 1989). On the basis of its action on semenogelin, the main component of the seminal clot formed after ejaculation, PSA's physiological role could be the release of spermatozoa entrapped in this clot. Since, it was also observed that the spermatozoa become progressively motile as the gel liquifies, it was hypothesized that the proteolytic action of PSA on semenogelin or other proteins in the seminal plasma could liberate a mobilityactivating peptide acting on the spermatozoa. The demonstration of this type of activity remains to be made. The formation and liquefaction of the seminal clot probably involves several other components. Indeed, fibronectin has been shown to be part of the clot and to be fragmented by PSA during liquefaction (Lilja et al., 1987). Another suspected component of the coagulation-liquefaction phenomenon is zinc. In a previous paper, we have shown that zinc interacted strongly with semenogelin in freshly ejaculated sperm (Frenette et al., 1989). It is also known from several studies that zinc, at concentrations that are present in the prostatic fluid, is a very effective inhibitor of PSA activity. Based on these informations, the sequence of events

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leading to sperm liquefaction could be viewed as follows. In prostatic secretory granules, in prostatic acini and in prostatic fluid, the enzymatic activity of PSA is inhibited by high concentrations of zinc, and is therefore not noxious for companion proteins. After ejaculation, zinc binds to semenogelin and relieves the inhibition of PSA. At the same time, semenogelin interacts with spermatozoa and fibronectin to form the seminal clot. Then, PSA progressively hydrolyses the seminal vesicle proteins and liberates entrapped spermatozoa. During that process, some zinc could be liberated and inhibit PSA. Another mechanism of PSA inhibition after sperm liquefaction appears to be through proteolysis. Indeed, Watt et al. (1986) have shown that at least 10-20% of PSA molecules contained internal splits after Arg-85, Lys-148 and Lys-185. Since PSA has a strict chymotrypsinlike specificity, these cleavages must be done by trypsin-like proteases. The renal-pancreatic kallikrein and hGK-1 are good candidates for this activity (see later). Another type of proteolytic cleavage performed by PSA in vitro may be of physiological significance. It is the hydrolysis of insulin-like growth factor binding proteins (IGFBP). Indeed, Cohen et al. (1991) have shown that even low concentrations of PSA could cleave IGFBP3 but not IGFBP-2 and IGFBP-4. IGFBP-1 was cleaved only to a minor extent. The IGFBP-3 is the major circulating IGFBP, but it is present in only very low amounts in seminal plasma. Based on this information, it is hypothesized that the proteolysis of IGFBP-3 at the level of prostatic metastases could increase the availability of insulin-like growth factors for their receptors and stimulate the growth of cancer cells in PSA secreting metastases. The confirmation of the in vivo occurrence of this phenomenon could stimulate new treatment approaches for prostate cancer patients having metastases. R e n a l pancreatic kallikrein

Renal pancreatic kallikrein isolated from urine as human urinary kallikrein is one of the best characterized glandular kallikrein (Geiger and Fritz, 1981). The protein has a rather high molecular weight (30,000 to 50,000) as determined by various procedures such as gel filtration, SDS gel electrophoresis and ultracentrifugation. Since the molecular weight deduced from the amino acid sequence is only 26,500, this behaviour must be ascribed to its high carbohydrate content. As with many other kallikreins, internal cleavages may occur and yield a 2- or 3-chain enzyme. Because of its kininogenase activity, the enzyme can produce measurable biological actions at very low concentrations (50 ng in the blood pressure test and

less than 1 ng in the rat uterus contraction assay). Evidence for the presence of true tissue kallikrein in human seminal plasma and prostatic tissue was obtained by Fink et al. (1985). The immunoreactive tissue kallikrein in seminal plasma had an apparent molecular mass of 48 kDa by gel filtration. Its concentration was 609 ng/ml in prostatic fluid and 57 ng/g in prostatic tissue. The partly purified material had specific kininogenase activity equivalent to one sixth of the value of purified human urinary kallikrein. As for other kallikreins with kininogenase activity, it was proposed that the tissue kallikrein-kinin system might regulate local blood flow as mentioned previously. Furthermore, the kinins released from seminal plasma kininogens by the action of kallikrein could be responsible for the increased motility of human ejaculated spermatozoa observed after the addition of pig pancreatic kallikrein (Schill and Haberland, 1974; Sato and Schill, 1987). This action would be mediated through bradykinin receptors on the spermatozoa membrane (Miska et al., 1990). Several other experimental evidences point to the in vivo significance of the kallikrein-kinin for male reproductive function (see the recent review of Schill and Miska, 1992). It is also believed that kinins may directly stimulate tubular function and improve sperm counts. This conclusion was derived from clinical trials showing that oral administration of kallikrein was useful for male subjects with idiopathic oligospermia (Schill, 1979). Although prostatic immunoreactive kallikrein was found to be localized in the glandular epithelial cells (Saitoh et al., 1985), another potential source of kallikreins in the prostate is the polymorphonuclear leucocytes. Indeed, circulating polymorphonuclear leucocytes have been shown to contain measurable amounts of immunoreactive kallikrein (Figueroa et al., 1989). It has been proposed that tissue kallikreins could be important mediators in inflammatoryjoint disease (Worthy et al., 1990). I have recently made the hypothesis (Dub6, 1992) that similar mechanisms could be operative in prostatic inflammation, a condition in which polymorphonuclear leucocytes are particularly abundant. The kinins formed by the action of kallikreins could contribute to the maintenance of inflammation through increased vascular permeability. Furthermore, kinins are known to be an important factor in the production of pain (Worthy et al., 1990) and could thus explain some of the symptoms of prostatitis. In all the studies cited in this section, the renal pancreatic kallikrein was identified by its

Prostatic kaUikreins: biochemistryand physiology immunoreactivity with an antiserum to kallikrein or by its kinin-releasing activity. However, there was not a single instance in which the protein was positively identified by its amino acid sequence. Similarly, the presence of renal pancreatic mRNA in human prostate has not yet been reported. This remark is not trivial because there is an important amino acid homology (66%) between renal pancreatic kallikrein and hGK-1 (Schedlich et al., 1987). Furthermore, the specificity of the various antisera prepared against human urinary kallikrein is not known because the protein encoded by hGK-1 has never been isolated. Finally a recent paper by Wu et al. (1993) shows that human polymorphonuclear leucocytes contain the mRNA for hGK-1 but not for renal pancreatic kaUikrein. The question can therefore be raised as to the real identity of the kallikrein mediating inflammation and also of the kallikrein with kininogenase activity in human prostate. hGK-1

The hGK-1 gene is the first human glandular kaUikrein gene to have been cloned and sequenced (Schedlich et al., 1987). The presence of hGK-1 mRNA was first demonstrated by our group (Chapdelaine et al., 1988b). Several other groups confirmed these findings which indicated that hGK-1 mRNA was 0.1-0.6 times as abundant as PSA mRNA in BPH prostate (Morris, 1989; Henttu et al., 1990; Riegman et al., 1991; Young et al., 1992). However, all attempts to isolate the protein from prostatic tissue or seminal plasma have so far been unsuccessful (Paradis et al., 1989; Christensson et al., 1990). Candidate proteins have been found by 2-dimensional gel electrophoresis (Paradis et al., 1989). Their characterization is presently underway. The only things that can be said of the putative hGK-1 protein are those that can be deduced from the predicted amino acid sequence (Schedlich et al., 1987). The presence of an aspartic residue at position 189 indicates that the protein should have a trypsin-like specificity in contrast to the chymotrypsin-like specificity of PSA which has a serine residue at position 189. However, the kininogenase activity of the hGK-1 protein is uncertain since at least one of the amino acids thought to be necessary for this type of activity is absent. Indeed, tyrosine-93 of bona fide kininogenases has been replaced by serine at the same position in hGK-1. As mentioned previously, one of the other potential substrates of hGK-1 protein could be PSA since it has been found to contain internal cleavages at amino acids compatible with trypsin-like cleavage.

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Another reason for the interest raised by the discovery of hGK- 1 is its potential expression in prostate cancer cells and interaction with antisera to PSA commonly used for radioimmunoassays. If PSA and hGK-1 are expressed in a differential manner during prostatic cancer evolution, the measurement of hGK-1 could be useful in clinical situations.

Dog prostate kallikrein Arginine esterase is one of the most quantitatively important kallikreins secreted by an organ (Isaacs and Shaper, 1983; Chapdelaine et al., 1984; Isaacs and Coffey, 1984). Its concentration in seminal plasma is approximately 10mg/ml. This single protein represents more than 90% of whole seminal plasma proteins and 30% of whole dog prostate homogenate proteins (Frenette et al., 1985a). Partial amino acid sequencing (Lazure et al., 1984) and later complete sequencing of the mRNA (Chapdelaine et al., 1988a) showed that the protein was a member of the kallikrein family. Its homology with the other prostatic kallikreins described in this review, guinea-pig prostate kallikrein, rat $3, rat P1, human PSA, human hGK-1 and hRPK, is respectively 57, 51, 53, 58, 61 and 58%. It has a strict trypsin-like specificity and a marked preference for synthetic substrates containing arginine. It can also hydrolyze extensively in vitro several proteins such as actin, fibronectin, fibrinogen, collagen and casein but not albumin, ovalbumin, v-globulin and human prostatic acid phosphatase (Frenette et al., 1986; Isaacs and Coffey, 1984). The hydrolysis of casein by arginine esterase was estimated to be approximately 1000-fold less than that observed for equal quantities of bovine trypsin. The in vivo significance of this proteolytic activity is presently unknown. In contrast with many other mammalian species, the dog has no seminal vesicles and the ejaculate does not form a coagulum that needs to be hydrolyzed. Furthermore, arginine esterase has probably no kininogenase activity since it is inactive in the blood pressure test in the dog and in the rat uterus contraction test (Frenette et al., 1985b). Therefore, other functions must be sought for the huge amounts of arginine esterase in the prostatic secretion. The in vivo substrates of arginine esterase could be located anywhere from within the prostatic secretory granules to the female genital tract. Indeed, we have found that arginine esterase is already in a fully enzymatically active form in the secretory granules (Frenette et al., 1985a). Although it is possible that like other kaUikreins, arginine esterase could be involved in the processing of prohormones or proenzymes within the secretory

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granules (Lazure et al., 1983), the most probable sites of origin of potential substrates should be the seminal plasma (various secretions, spermatozoa and sperm binding proteins) and proteins of the female genital tract. It may be significant that arginine esterase has the ability to bind canine spermatozoa (Isaacs and Coffey, 1984). Because of these results, it has been hypothesized that the enzyme could catalyze the cleavage of proteins on the sperm surface itself or alternatively that it could be transported as a bound protein and act on distant sites (Isaacs and Coffey, 1984). However, these experiments have not been pursued and the question of the physiological substrate of canine prostate arginine esterase still remains opened.

Conclusions Experimental data gathered on prostatic kallikreins from four different mammalian species show that it is not possible to make generalizations on the physiological roles of prostatic kallikreins in different animal species. The only generalization that can be made is that kallikreins are abundant secretory products in the prostatic fluid and that these proteins generally demonstrate very low enzymatic activity towards synthetic substrates. These experiments suggest that prostatic kallikreins are not large spectrum proteolytic enzymes and that specific amino acid sequences are required in the vicinity of the cleavage site in order that proteolysis occurs. The only well demonstrated in vivo substrates of prostatic kallikreins are the proteins originating from the seminal vesicles and forming the seminal coagulum in humans. Proteins of the copulatory plug in rodents are also potential substrates. Finally, although this aspect has not been really investigated, logical substrates of kallikreins could be the sperm binding proteins originating from various accessory sex glands, particularly the epididymis. Indeed, the hydrolysis of sperm binding proteins is known to be associated with sperm capacitation (Hinrichsen-Kohane et al., 1984). It is evident from this review that the basic biochemistry of prostatic kallikreins is now beginning to be well understood. However, in most cases, the physiological substrates and the biological roles of prostatic kallikreins are still totally unknown. This has not precluded the use of prostatic kallikrein genes as molecular models for the study of the mechanisms of action of androgens (Wolf et al., 1992; Gauthier et al., 1993) and of tissue specificity (Southard Smith et al., 1992). These studies are just beginning and it is clear that prostatic kallikreins still have many secrets to reveal.

Acknowledgements--The author is grateful to Mrs Lucie Turcotte for excellent secretarial assistance in the preparation of this manuscript and to the Medical Research Council of Canada for continued support of the research projects originating from our laboratory and described here.

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