Glandular Kallikrein Research Articles

Salivary kallikrein output during the stress response to surgery

Salivary kallikrein output during the stress response to surgery

Epitope mapping of prostate-specific antigen with monoclonal antibodies

Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. We identified and characterized the epitopes of two anti-PSA monoclonal antibodies (mAbs) designated B80 and B87. The epitopes were initially mapped as nonoverlapping by developing a sandwich immunoassay to measure PSA with the two anti-PSA mAbs. The two antibodies do not cross-react with homologous pancreatic kallikrein, but recognize epitopes unique to PSA. B80 and B87 can recognize both free and complexed PSA and hence measure total PSA. Epitope scanning and bacteriophage peptide library affinity selection procedures were used to identify and locate an epitope on PSA. A possible epitope for B80 was identified as being located on or near PSA amino acid residues 50-58 (-GRH-SLFHP-). The epitope for B87 was likely on an exposed nonlinear conformational determinant, unique to PSA, and not masked by the binding of B80 or alpha 1-antichymotrypsin.

Sequential secretion of rat submandibular kallikrein and peroxidase during intermittent sympathetic stimulation

Sequential secretion of rat submandibular kallikrein and peroxidase during intermittent sympathetic stimulation

Sequential secretion of rat submandibular kallikrein and peroxidase during intermittent sympathetic stimulation

Secretion of peroxidase from acinar cells, true tissue kallikrein (rKI) from granular tubules and total protein have been assessed in sequential samples of rat submandibular saliva formed during intermittent periods of sympathetic stimulation in bursts of 50 Hz 1 s every 10 s at 5 V, for 1 or 2 min each with 2 min intervals between periods. This protocol was repeated twice after 1 h rest pauses. The salivary flow remained greater throughout than when using ongoing burst stimulation. Protein secretion was considerable in the first 2 samples (1 min followed by 2 min) reaching the remarkable concentration of 285 ± 14.4 mg/ml, then it gradually decreased with little recovery after 1 h pauses. rKl outputs followed a similar pattern. Peroxidase, however, showed no greater output in initial samples and continued steadily in similar amounts throughout. When intermittent stimulations were used for 1 sequence only (total stimulation time = 9 min) the glands showed 84 ± 2.6% depletion of rK1 compared to the control glands. A correspondingly large depletion of granules occurred from the granular tubules on the stimulated side, which was greater than with ongoing burst stimulation for 1 h (Garrett et al., 1991). Thus, secretion of rK1 from the granules in granular tubules occurs most efficiently with short sharp bursts of high frequency sympathetic stimulation but soon diminishes. In contrast, peroxidase secretion from acinar cells continues steadily and more modestly for long periods of time undiminished.

Temporospatial changes of kallikrein-like enzymes during the estrous cycle and pregnancy in the rat uterus.

We have recently reported the presence of uterine glandular kallikrein in the rat, its mRNA, and its increase in early gestation. This study describes its immunolocalization, by polyclonal antibodies against rat urinary kallikrein, during the estrous cycle and pregnancy, and identifies three members of the kallikrein family. During the estrous cycle, immunoreactivity, represented by an apical rim in the luminal and glandular epithelium, was greater during proestrus. On Day 5 of gestation, the reaction increased, and on Day 6 staining appeared in the apical and basal pole of some cells. On Day 7, staining was markedly increased in the glandular epithelium at the implantation site, frequently occupying the whole cytoplasm, and displaying great intensity in isolated glandular cells; the epithelium of the implantation chamber showed apical immunoreactivity, while the interimplantation zone had a few faintly stained glands. At this stage, the staining represented expression of two of the enzymes, rK1 and rK7. On Days 9 and 10, the staining disappeared, to reappear on Day 12 in the cells underlying the blood vessels of the central subplacental region. On Days 16 and 21, kallikrein staining surrounded the subendothelium of the sinusoids of the whole decidua basalis. This subplacental reaction represented expression of rK1, rK2, and rK7. This study shows important changes in the immunocytochemical expression of three uterine kallikrein-like enzymes during the reproductive cycle, associated with different hormonal milieus and with local stimulation. The localization of these enzymes is associated with areas involved in implantation, trophoblast penetration, and placental blood flow regulation.

Identification of human glandular kallikrein hK2 from LNCaP cells.

Based on studies indicating that human glandular kallikrein (hK2) mRNA is present in the prostate, we prepared a monoclonal antibody to a synthetic peptide corresponding to the 41-56 region of hK2 to try to identify the hK2 protein. Although prostate-specific antigen (PSA) and hK2 share 80% homology, the 41-56 amino acid sequence of hK2 is only 50% homologous with PSA. A monoclonal antibody, HK1A523, was identified that demonstrates high specificity for hK2. In western blot analysis, the antibody has a 1,000-fold greater sensitivity for the detection of hK2 than for PSA. The antibody was used to probe spent media from the prostate carcinoma cell line, LNCaP. An immunoreactive species was N-terminally sequenced and identified as mature hK2. HK1A523 was also utilized to probe prostate tumor cytosols and seminal fluid where putative forms of hK2 were also identified. The hK2 protein therefore is expressed and secreted from prostate carcinoma cells.

Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum

Prostate-specific antigen (PSA) and human prostatic glandular kallikrein (hK2) have 79% identity with the primary structure. When we used recombinant hK2 protein, only 7 of 23 monoclonal anti-PSA IgGs (monoclonal antibodies, MAbs) cross-reacted with hK2, which enabled us to design a novel immunofluorometric MAb-MAb assay for the specific detection of hK2. In the first incubation, an excess of MAb 2H11, which does not cross-react with hK2, is added to prevent both free and complexed PSA from reacting in subsequent immunoreactions. In the second incubation, biotinylated MAb H50, which cross-reacts with hK2 by an epitope overlapping with MAb 2H11, served to bind only hK2 to the microtitration wells coated with streptavidin. In the third step, Eu-labeled MAb H117, which cross-reacts with hK2, detected the immobilized hK2. The hK2 assay was calibrated with recombinant hK2. The detection limit of the assay was 0.1 microgram/L, and the cross-reactivity with recombinant PSA was < or = 0.7%. The concentration of hK2 was measured in serum samples from 334 males with total PSA concentrations ranging from 1 to 3400 microgram/L. Most of the samples (57%) had hK2 concentrations below the detection limit. The proportions of hK2 relative to total PSA were 0-2% in 79%, 2-5% in 14%, 5-10% in 4%, and >10% in 3% of the samples. Gel filtration of 10 serum samples with increased hK2 concentrations showed a single peak of hK2 immunoreactivity with an apparent molecular size of approximately 30 kDa, corresponding to that of recombinant hK2 and free PSA.

The Kallikrein–Kinin and Renin–Angiotensin Systems in the Kidney of an African Lungfish,Protopterus annectens

Renin-like activity (RLA), angiotensin I converting enzyme-like (ACELA), and kallikrein-like activity (KLA), activities of the key enzymes of renin–angiotensin and kallikrein–kinin systems, were sought in the kidney of the African lungfishProtopterus annectensduring the aquatic phase. RLA, examined by RIA (using porcine angiotensinogen as substrate), was 0.38 ± 0.05 ng angiotensin I/mg protein/hr. ACELA and KLA were investigated in assays spectrophotometrically. ACELA, measured at 37 and at 20°, was, respectively, 1.55 ± 0.55 and 0.61 ± 0.23 nmol hippurate/min/mg protein. KLA was 7.34 ± 0.93 mU/mg protein in the crude kidney extract and 31.05 ± 7.50 mU/mg protein after electrophoretic purification. Renal kininogenase activity was inhibited by 100% byD-Phe-Phe-Arg-chloromethyl ketone (10 μM), 98% by phenylmethylsulfonyl fluoride (2 nM), and 91% by aprotinin (1000 kIU). The apparent molecular weight of the renal kininogenase on SDS–PAGE was 27,000 Da. Both the renal enzyme and the purified glandular kallikrein, used as a control, have the same mobility on polyacrylamide gel electrophoresis. Immunoreactivities toward angiotensin II and bradykinin were localized by double immunostaining in the same cells of the proximal tubules. Putative angiotensin II receptors were demonstrated immunohistochemically, in the supranuclear region of proximal tubular cells, using an antibody to the sequence between amino acids 225 and 237 of the mammalian AT1receptor.

Purification and Characterization of Salivary Kallikrein from an Insectivore (Scalopus aquaticus): Substrate Specificities, Immunoreactivity, and Kinetic Analyses

We report the successful one-step separation of tissue kallikrein from the salivary glands of an insectivore, the Eastern Atlantic mole (Scalopus aquaticus) by perfusion chromatography. Purified mole salivary kallikrein was characterized as a 30-kDa serine proteinase with a pIof 5.3 and a pH optimum of 9.0. It was readily recognized by human tissue kallikrein antibody in immunoblot analyses. It preferentially hydrolyzes fluorogenic peptidyl substrates with arginyl residues, rather than lysyl residues at the P1 substrate recognition site, indicating that it is like other mammalian kallikreins. Mole kallikrein efficiently releases kinin from low molecular weight human, dog, and bovine kininogen substrates with specific activities similar to that of human tissue kallikrein. Steady state kinetics performed with the synthetic tripeptidyl substrates, Phe-Phe-Arg-, Pro-Phe-Arg-, and Val-Leu-Arg-7-amino-4-methylcoumarin, gaveKmvalues for mole kallikrein of 3.3, 46.1, and 2.8 μM, respectively, and specificity constants,kcat/Km, of 3818, 165, and 8714 s−1pM−1, respectively. Mole kallikrein, when compared with human and rat tissue kallikreins, more closely resembles human kallikrein based on immunoreactivity and kininogenase activity. Mole kallikrein appears to be a member of a single gene or small multigene family.S. aquaticusis recommended for studying the evolution of mammalian proteins and may offer advantages over rodent models for biomedical research.

Pharmacological characterization of novel tissue kallikrein inhibitors in vivo

In this study we have investigated the effect of novel tissue kallikreins on the plasma protein exudation induced by porcine pancreatic kallikrein (PPK) in the rabbit skin in vivo. The tissue kallikrein inhibitors here described were synthesized based on analogues of peptide substrates for tissue kallikreins. The intradermal injection of PPK and rabbit urinary kallikrein, but not of rabbit plasma kallikrein, significantly increased the microvascular permeability leading to local oedema formation in the rabbit skin. At the dose of 3–200 nmol/site, the intradermal co-administration of the tissue kallikrein inhibitors Bz-F-F-S-R-EDDnp (Ki = 0.1 μM; ESP5), PAC-F-S-R-EDDnp (Ki = 0.7 μM; ESP6), Bz-F-F-A-P-R-NH2 (Ki = 7.8 μM; ESP8), PAC-F-F-R-P-R-NH2 (Ki = 0.3 μM; ESP9) and Bz-F-F-S-R-NH2 (Ki = 0.3 μM; ESP11) dose-dependently inhibited the plasma protein exudation induced by PPK. The most potent compound was ESP6 (IC25 = 7.8 nmol/site) followed by ESP5 (IC25 = 14.2 nmol/site), ESP8 (IC25 = 25 nmol/site), ESP9 (IC25 = 30 nmol/site) and ESP11 (IC25 = 50.4 nmol/site). The compounds Bz-F-F-R-P-R-NH2 (Ki = 0.5 μM; ESPI), Bz-F-F-pNa (Ki = 0.4 μM; ESP3), Bz-F(NH2)-F-R-P-R-NH2 (Ki = 1.1 μM; ESP7) and Bz-F-F-S-P-R-NH2 (Ki = 4.6 μM; ESPI0) had no significant effect on the PPK-induced plasma protein exudation in doses up to 200 nmol/site. ESP6 also inhibited the PPK-induced plasma protein exudation when administered systemically. This compound may constitute a useful tool to further investigate both the physiological and pathological role of tissue kallikreins.

A novel human leukocyte elastase inhibitor from duck egg white

A serine proteinase inhibitor (ovomucoid) has been isolated from duck egg white. The duck ovomucoid effectively inhibited HLE, PPE, chymotrypsin, and HCG in a 1:1 molar ratio, and trypsin in a 1:2 molar ratio. Inhibition of human plasmin and porcine pancreatic kallikrein was not observed. The ovomucoid shows equilibrium dissociation constants of 0.002; 2.4; 2.2; 6.1; and 18.0 nM for HLE, PPE, chymotrypsin, trypsin, and HCG, respectively. The molecule of inhibitor can simultaneously bind two trypsin molecules and one molecule of elastase (or chymotrypsin).

Kallikrein gene expression in human pituitary tissues.

The glandular kallikreins are a family of enzymes involved in the post-translational processing of polypeptides. Three family members have been characterized in the human: glandular kallikrein (KLK1) and two genes expressed in the prostate, KLK2 or hGK1 and KLK3 or prostate specific antigen (PSA). Both kallikrein immunoreactivity and KLK1 mRNA have been detected in the rat anterior pituitary and oestrogen induced prolactinomas. Immunoreactive kallikrein has also been reported in prolactin secreting pituitary tumours in humans. In this study we wished to determine whether KLK1 and/or other KLK genes were expressed in human pituitary tumours. Retrospective analysis of KLK gene expression in pituitary tissue obtained at surgery from 11 patients with a range of pituitary syndromes, 10 of which were tumour induced and included four prolactinomas. Three normal pituitaries, obtained at necropsy, were also analysed. Pituitary total RNA was subjected to both Northern blot analysis, with KLK1 and KLK2 cDNA probes, and KLK-specific reverse transcriptase -- polymerase chain reaction (RT-PCR). No KLK1 gene expression was detected on Northern blot analysis although expression of PRL, GH and pro-opiomelanocortin (POMC) was variously detected. KLK RT-PCR coupled with Southern blot analysis using oligonucleotide probes specific for each of the three KLK genes was positive in many tumours but with varying levels and differential expression. Overall, KLK1 was the most abundant with KLK3 the least. The identity of KLK1 and KLK2 was confirmed by sequencing the PCR products. There was no obvious correlation between KLK1 and hormone gene expression; KLK1 was present in PRL positive and negative tumours and in normal pituitary tissue. We have demonstrated a low level of expression of KLK1 as well as KLK2 and KLK3 in the human pituitary. The expression of KLK2 and KLK3 in the pituitary is a novel finding. The lack of correlation between PRL and KLK1 gene expression is an unexpected finding suggesting that the interaction between these factors may be more complex in the human pituitary than in the rodent model.

Identification of the Major Site of Rat Prolactin Phosphorylation as Serine 177

Phosphorylation of prolactin by endogenous protein kinases within isolated secretory granules was shown to result in the production of both phosphoserine and phosphothreonine residues. The majority of the radiolabel was determined to be present in the C terminus of the molecule after specific cleavage with glandular kallikrein. Glandular kallikrein cleaves in three places at the C terminus, liberating three small peptides, only one of which contains a phosphorylatable residue. Sequencing of this phosphopeptide showed it to be Arg175-Lys185. Thus the major site of prolactin phosphorylation was determined to be serine 177. Using a synthetic peptide equivalent to this region of the molecule (Ser161-Val180), serine 177 was demonstrated to be a substrate for protein kinase A as well as for one of the endogenous granule kinases. Inclusion of the synthetic peptide in an endogenous granule phosphorylation reaction resulted in competition for the kinase and reduced phosphorylation of prolactin. Protein kinase A phosphorylation of purified prolactin resulted in the production of only phosphoserine and primarily the most abundant (monophosphorylated) variant. We conclude that serine 177 is the major in vivo phosphorylation site of rat prolactin and that phosphorylation of this site can be reproduced by protein kinase A in vitro. The minor threonine phosphorylation site was demonstrated by two-dimensional tryptic peptide mapping and mass analysis to be either threonine 58 or 63, both of which are contained within a single peptide.

Apical secretion of rat submandibular tissue kallikrein continues in the absence of external stimulation: evidence for a constitutive secretory pathway.

Sequential samples of saliva evoked by low frequency parasympathetic nerve stimulation were collected from cannulated submandibular ducts of anaesthetized rats following periods of varying length without stimulation. After such rest periods the first samples of saliva were found to contain much higher levels of tissue kallikrein (rK1) activity and protein than the ensuing samples from the same stimulation period and the latter contained levels similar to those found previously in parasympathetically-evoked saliva. rK1 activity in first samples increased with the length of the preceding interval, indicating that a continuous secretion of rK1 occurs from ductal cells into glandular lumina and accumulates there in the absence of stimulation or fluid secretion. The protein secretory pattern following rest pauses was unaffected by alpha-adrenergic receptor blockade and a high percentage of the proteinase activity was resistant to soya bean trypsin inhibitor, showing that the secretion was not caused by exocytosis of storage granules from ductal cells and therefore was likely to be a result of a constitutive secretion of newly synthesized enzyme. The pattern of continuous secretion into lumina detected for total protein in the absence of stimulation, suggests that other secretory proteins may also be secreted similarly but at different rates.

Immunohistochemical localization of kallikrein in salivary glands of the Japanese monkey, Macaca fuscata

The immunofluorescence staining using anti-human urinary kallikrein polyclonal antiserum was intense in the luminal rim of the striated duct cells, but decreased in the excretory duct cells and was weak in the intercalated duct cells. Western blot analysis demonstrated the monospecificity of the antiserum to glandular kallikrein in Macaca submandibular gland homogenate. The staining was observed to a similar degree as that in salivary glands with long and numerous striated ducts in the following order: the submandibular, the parotid and the sublingual gland. These findings suggest that kallikrein is secreted by exocytosis from the duct cells, mainly from the striated duct cells.

Successful separation between benign prostatic hyperplasia and prostate cancer by measurement of free and complexed PSA.

Prostate-specific antigen (PSA) is a serine protease belonging to the human glandular kallikrein gene family [1–3]. The expression of PSA is mainly androgen dependent, and the detection of very high expression levels is restricted to the prostate tissue, but extraprostatic production at much lower levels has been demonstrated in several other tissues such as normal and malignant breast epithelium, endometrium, and bulbourethral glands [4–10]. PSA is synthesized by the columnar epithelium in the glandular ducts and acini of the prostate, but not by any other cells in prostate tissue. It is secreted at high concentrations (0.2-5mg/mL) into seminal fluid [4–6,11]. PSA is synthesized as an inactive precursor [2,3,12]. Like other glandular kallikreins, the PSA-precursor is processed stepwise by release of a leader peptide followed by liberation of an activation peptide that results in conversion of the zymogen into enzymatically active PSA [12]. This process may occur in parallel with the secretory release from the prostate epithelium and most probably occurs prior to the ejaculatory mixing of secretions from the prostate, seminal vesicles, and epididymis, since PSA is active in ejaculates collected from subjects with defective seminal vesicles and deferent ducts [1]. The protease(s) responsible for processing of the PSA precursor have not been identified. The mature 237-amino-acid form of PSA is a single-chain serine protease with extensive structural similarity to the glandular kallikreins [1,12–14]. However, the substrate specificity is uniquely different from that of the trypsin-like glandular kallikreins and resembles that of chymotrypsin, since PSA catalyzes the hydrolysis of peptide bonds’ carboxy-terminal to residues of tyrosine and leucine [15–17]. Synthetic peptide substrates for chymotrypsin can be used to measure PSA activity, but they are hydrolyzed much less efficiently by PSA than by chymotrypsin and are therefore both nonspecific and insensitive in detecting PSA activity [16].

Localization of kallikrein in salivary glands of the Japanese monkey, Macaca fuscata

Although Kallikrein has previously been localized in the duct cells of salivary glands, the manner in which it enters the saliva remains unclear. We used both immunofluorescein and protein A-gold methods at the light microscopic level and the protein A-gold method at the electron microscopic level to determine the distribution of kallikrein antigenicity in monkey salivary glands. The specificity of anti-human urinarykallikrein to glandular kallikrein in the monkey submandibular gland was demonstrated by Western blotting. Light microscopic immunohistochemical staining was intense at the luminal rim of the striated duct cells, but was less intense in the excretory duct cells, and was faint in the intercalated duct cells, Among the major salivary glands, staining intensity paralleled the degree of development of the striated ducts, with the submandibular gland exhibiting the most intense staining, the parotid to a somewhat lesser degree, and the sublingual displaying only minor staining. In the apical cytoplasm of the submandibular striated duct cells, some small granules and vesicles and vacuoles were observed near the luminal plasma membrane. By ultrastructural immunohistochemical staining, kallikrein appeared to be localized in small secretory granules. Because some of the small granules seemed to be attached to the luminal membrane, we suggest that kallikrein is secreted by exocytosis from the duct cells.

Biochemical characteristics and recent biological knowledge on prostate-specific antigen.

Since its identification in seminal fluid in 1971, much new information has been obtained about the biology and expression of prostate-specific antigen (PSA). PSA is a glycoprotein composed of 93% amino acids and 7% carbohydrates, with a molecular weight of about 30,000 Da. Functionally and structurally PSA is a kallikrein-like serine protease, and its physiologic role is degradation of the major proteins of seminal coagulum (semenogelin I and II, fibronectin), which leads to semen liquefaction. The PSA gene is located on the 13q region of chromosome 19, and it has a high degree of homology (more than 80%) with genes of the human glandular kallikrein (hKGK1). PSA production and expression are preferentially but not exclusively associated to the normal, benign hyperplastic and cancerous tissues of the prostate. In fact, it has been demonstrated that PSA is also present in accessory male sex glands and breast cancer. It was recently reported that PSA was also present in milk of lactating women. Many factors may influence PSA synthesis and production, and among them the most important are androgen, retinoic acid and growth factor stimulation. Significant advances have been recently made as regards the molecular isoforms of PSA. In the seminal fluid PSA seems partially bound to a serpine (protein C inhibitor), whereas in serum it is predominantly associated to alpha-1-antichymotrypsin and in a small quantity to alpha-2-macroglobulin. These new findings will have implications for the clinical application of PSA as a tumor marker for prostate cancer.

Isolation and cardiovascular activity of a second bradykinin-related peptide ([Arg0, Trp5, Leu8]bradykinin) from trout.

Previous work has shown that incubation of heat-denatured plasma from the rainbow trout Oncorhynchus mykiss with porcine pancreatic kallikrein generates [Lys0, Trp5, Leu8]bradykinin (trout [Lys0]BK). We have now isolated a second BK-related peptide from kallikrein-treated trout plasma with the primary structure: Arg-Arg-Pro-Gly-Trp-Ser-Pro-Leu-Arg (trout [Arg0]BK). Bolus injections of both trout [Arg0]BK and [Lys0]BK (> 100 pmol/kg) into the dorsal aorta of conscious trout produced multiphasic effects on arterial blood pressure. An initial pressor response of short duration (1-2 min) was followed by a fall in pressure (to below basal values in 11 out of 15 animals) and then by a sustained rise in pressure lasting up to 60 min. The maximum rise in pressure produced by trout [Arg0]BK (10 nmol/kg) was approximately one-fourth of the maximum rise produced by angiotensin II in the same animals. Intracerebroventricular injections of trout [Arg0]BK (500 pmol) into conscious trout had no effect on arterial blood pressure or heart rate. Trout [Arg0]BK did not affect the tension of vascular rings from trout efferent branchial and caeliacomesenteric arteries and anterior cardinal vein. Trout des [Arg9]BK had no effect on cardiovascular parameters, either in vivo or in vitro, indicating that the C-terminal arginine residue of the peptide is important in interaction with the trout kinin receptor(s).

Production of [Asn1, Val5] angiotensin II and [Asp1, Val5] angiotensin II in kallikrein-treated trout plasma (T60K).

Incubation of heat-denatured plasma from the rainbow trout Oncorhynchus mykiss with porcine pancreatic kallikrein generates, in addition to bradykinin-related peptides, previously uncharacterized peptides that contract mammalian and amphibian vascular smooth muscle. Using rings of vascular smooth muscle from the bullfrog systemic arch as bioassay, we have isolated two myotropic peptides whose primary structures were established as: Asn-Arg-Val-Tyr-Val-His-Pro-Phe ([Asn1, Val5]angiotensin II) and Asp-Arg-Val-Tyr-Val-His-Pro-Phe ([Asp1, Val5]angiotensin II). These peptides are the same as those generated in salmon plasma by an extract of kidney. The data raise the possibility that activation of the kallikrein-kinin system in trout generates both bradykinin-related and angiotensin II-related peptides that may act synergistically in the regulation of blood pressure.