Human tissue kallikrein. II. Isolation and characterization of human salivary kallikrein

Human salivary kallikrein was isolated from saliva using affinity chromatography on aprotinin-Sepharose and anti-human urinary kallikrein IgG-Sepharose followed by ion exchange chromatography on DEAE-Sepharose. The enzyme preparation had a specific activity of 950 U/mg protein towards the synthetic substrate Ac-Phe-Arg-OEt, a specific biological activity of 2000 KE/mg protein (measured in the dog blood pressure assay) and 0.64 HMW-kininogen-U/mg, corresponding to the liberation of 679 micrograms bradykinin equivalents per mg enzyme per min from HMW-kininogen (using the rat uterus test). In sodium dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 32 kDa was obtained. The amino-acid composition was determined and isoleucin was found as the only N-terminal residue. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 8 l x mol-1 x min-1. The dissociation constant Ki of the human salivary kallikrein-aprotinin complex was calculated to be 0.7 x 10(-10)M. The Km and Vmax values for the hydrolysis of the synthetic substrates Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. In the enzyme immunoassay for human urinary kallikrein parallel binding curves were obtained.     

Immunohistochemical localization of kallikrein in human pancreas and salivary glands

The localization of kallikrein in human exocrine organs was studied with a direct immunofluorescence method. In the submandibular and parotid salivary glands, kallikrein was found apically in the striated duct cells whereas it was absent from the main excretory ducts or present only as a weak luminal rim. Kallikrein was not found in the acinar cells or in cells of the intercalated ducts. In the pancreas, kallikrein-specific fluorescence was seen in the granular portion of the acinar cells, whereas the islets of Langerhans and ductal cells were unstained.     

Human kallikrein 6 expression in salivary gland tumors.

Human kallikrein 6 (hK6), also known as zyme/protease M/neurosin), is expressed in many normal glandular tissues. The aim of this study was to determine whether hK6 is expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), using an immunohistochemical method. Pleomorphic adenomas (PA), adenoid cystic carcinomas, polymorphous low-grade adenocarcinomas, acinic cell carcinomas, mucoepidermoid carcinomas, and adenocarcinomas not otherwise specified of both minor and major salivary glands were examined. Cells lining duct-like structures and non-duct-like cells were scored. Only in PA of minor salivary gland origin was overall staining higher in duct-like than in non-duct-like cells. In all other tumors exhibiting both types of cells, hK6 staining was similar in both duct-like and non-duct-like cells. Tumors that exhibited non-duct-like cells only also exhibited cytoplasmic staining. Results of this study show that salivary gland tumors express hK6, apparently downregulated in comparison with normal salivary gland tissue, and that this expression is not specific for any of the tumors studied.     

Human kallikrein 3 (prostate specific antigen) and human kallikrein 5 expression in salivary gland tumors

The human kallikrein 5 protein (hK5) is expressed in many normal tissues, most notably in skin, breast, salivary gland and esophagus. It has also been shown to be a potential biomarker for breast, ovarian and testicular cancer. Human kallikrein 3 (hK3; prostate-specific antigen) is the most useful marker for adenocarcinoma of the prostate gland. The aim of this study was to determine whether hK3 and hK5 are expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), in order to compare normal with tumor tissues. Pleomorphic adenomas, adenoid cystic carcinomas, polymorphous low-grade adenocarcinomas, acinic cell carcinomas, mucoepidermoid carcinomas and adenocarcinomas not otherwise specified of both minor and major salivary glands were examined. The results of this study indicate that most salivary gland tumors do not show high levels of expression of hK5. Staining was most prominent in keratinizing epithelia in pleomorphic adenomas. hK3 is not expressed in salivary gland tumors.     

Human kallikrein 13 expression in salivary gland tumors

The human kallikrein 13 protein (hK13) is expressed in many normal tissues. Petraki et al have previously described presence of hK13 in salivary gland tissue, localized to duct epithelia and some acinar cells. The aim of this study was to determine whether hK13 is expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), in order to compare normal with tumor tissues. Pleomorphic adenomas (PA), adenoid cystic carcinomas (ACC), polymorphous low grade adenocarcinomas (PLGA), acinic cell carcinomas (ACI), mucoepidermoid carcinomas (MEC) and adenocarcinomas not otherwise specified (ANOS) of both minor and major salivary glands were examined. The results of this study indicate that most salivary gland tumors show high levels of expression of hK13. Overall, staining in PA was significantly less than that seen in normal salivary gland tissue. PLGA, ACC and ANOS each stained significantly more than normal salivary gland tissue while MEC and ACI did not. Ductal cells and cells lining duct-like structures showed a higher intensity of staining than non-ductal cells in most tumors. Tumors which exhibited only non-ductal cells also exhibited cytoplasmic staining. In conclusion, we demonstrate the high expression of hK13 in several common salivary gland tumors.     

Similarity between a kininogenase (kallikrein) from human large intestine and human urinary kallikrein.

A human colon kininogenase (kallikrein) was isolated by gel filtration on Sephacryl S-200 and affinity chromatography on Trasylolbound Sepharose, yielding a material with a specific activity of 1.3 U/mg (substrate: AcPheArgOEt). The molecular weight of the enzyme as estimated by gel filtration is approximately 70 000. After reduction with mercaptoethanol two bands were obtained in dodecyl sulfate eletrophoresis with molecular weights of 27 000 and 70 000. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 4 l x mol-1 x min-1. The preparation was characterized by immunological and enzymatic methods. Using the radioimmumoassay for human urinary kallikrein cross-reactivity and parallel binding curves were obtained. Kinin liberation from human high Mr-kininogen was totally inhibited by antibodies directed against human urinary kallikrein. Trasylol and diisopropyl fluorophosphate, but not by antibodies directed against human trypsin and plasma kallikrein. The effect on dog blood pressure was comparable to that obtained with human urinary kallikrein. The amino acid composition of human large intestine kallikrein is very similar to that of human urinary kallikrein.     

Characterization of the human kallikrein locus.

The human kallikrein gene family is composed of three members: tissue kallikrein (KLK1), prostate-specific antigen (PA or APS), and human glandular kallikrein-1 (hGK-1 or KLK2). The three genes have previously been isolated and mapped to chromosome 19q13.2-q13.4. Further analysis of an area of 110 kb surrounding the kallikrein genes by CHEF electrophoresis and chromosome walking showed clustering of the three genes. The KLK1 gene is positioned in the opposite orientation of the APS and KLK2 genes in the order KLK1-APS-KLK2. The APS and KLK2 gene are separated by 12 kb; the distance between KLK1 and APS is 31 kb. A CpG island was detected in the region between KLK1 and APS. Preliminary data indicate that this CpG island is located directly adjacent to a gene that is unrelated to the kallikreins and seems to be ubiquitously expressed.     

Human tissue kallikrein. I. Isolation and characterization of human pancreatic kallikrein from duodenal juice

Human pancreatic kallikrein was purified from duodenal juice by ion exchange chromatography on DEAE-Sepharose and immunoaffinity chromatography. Thus, an enzyme preparation with a specific activity (using Ac-Phe-Arg-OEt as substrate) of 1 000 U/mg protein was obtained. A specific biological activity of 1310 KE/mg protein was measured in the dog blood pressure assay and of 0.361 HMW kininogen-U/mg, corresponding to the liberation of 383 micrograms bradykinin-equivalents per mg enzyme per min from HMW kininogen in the rat uterus assay. In dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 27 kDa was obtained. Using gel filtration on Ultrogel AcA-44 a molecular mass of 40 kDa was measured. The amino-acid composition was determined and isoleucine and alanine were identified as the only N-terminal amino-acid residues. On isoelectric focusing four protein bands with isoelectric points of 5.60, 5.65, 5.70 and 5.85 were separated. The bimolecular velocity constant for the inhibition by diisopropyl fluoro phosphate was determined as 10.5 l x mol-1 x min-1. The dissociation constant Ki of the human pancreatic kallikrein-aprotinin complex was calculated to be 1.5 x 10(-10)M. The kinetic constants for the kallikrein-catalysed hydrolysis of Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. Immunological studies showed a close relationship between the human pancreatic kallikrein and other human tissue kallikreins, especially with human urinary kallikrein. Detergents such as Triton X-100, Tween 20 and lysolecithin, as well as human serum albumin, activated the human pancreatic kallikrein preparation.     

Inhibition of kallikrein from human plasma by salivary gland secretion and extracts of the medicinal leech Hirudo medicinalis

It has been shown for the first time that the salivary gland secretion of the medicinal leech (Hirudo medicinalis) contains a human blood plasma kallikrein inhibitor which is capable of blocking the amidolytic activity of the enzyme in an irreversible manner (with D-Pro-Phe-Arg-pNA as substrate) and which also suppresses the kininogenase activity of kallikrein. The inhibition of the amidolytic activity of highly purified kallikrein preparations from human blood plasma obeys the pseudo-first order kinetics. The experimental results suggest that in the salivary-gland secretion of H. medicinalis the inhibitor concentration exceeds by one order of magnitude that in whole leech homogenate extracts, which indicates that the inhibitor biosynthesis may be localized in leech salivary glands.     

Tissue-specific and hormonal regulation of human prostate-specific glandular kallikrein.

Kallikreins are involved in the posttranslational processing of a number of specific polypeptide precursors. Previously, human glandular kallikrein (hGK-1) mRNA has been identified in the prostate; however, the hGK-1 protein has not been identified and characterized. Therefore, its physiologic function in the prostate is not known. In this study, we have isolated a full-length hGK-1 cDNA from a human adenocarcinoma cell line, LNCaP. In vitro translation experiments demonstrated that the molecular size of the hGK-1 protein generated from this cDNA is similar to that of prostate-specific antigen (PSA), a protein which is produced exclusively in the prostate. In situ hybridization with a hGK-1-specific oligonucleotide probe (77 bases), which can differentiate hGK-1 mRNA from PSA mRNA, demonstrated the hGK-1 mRNA to be located in the prostate epithelium. The hGK-1 mRNA was colocalized with PSA mRNA in prostatic epithelia. Moreover, in situ hybridization studies revealed that the level of hGK-1 mRNA in human benign prostatic hyperplasia tissues is approximately half that of PSA mRNA. Furthermore, we have demonstrated that hGK-1 mRNA is under androgenic regulation in LNCaP cells. Time course analysis revealed that hGK-1 mRNA levels increased significantly at 5 h of mibolerone treatment and reached maximal levels by 9 h. In addition, hGK-1 mRNA levels were increased by dihydrotestosterone, but not by dexamethasone or diethylstilbestrol treatments. Flutamide, a nonmetabolized anti-androgen, repressed the androgenic effects. These studies suggest that expression of hGK-1 mRNA is regulated by androgen via the androgen receptor.     

Cigarette smoking reduces human salivary eicosanoids.

The effect of cigarette smoking on salivary eicosanoid levels was investigated in 10 smoker and 10 non-smoker volunteers. The smokers consumed an average of 20 cigarettes/day for the past 5 years or longer. The smoking status was validated by salivary cotinine level. Eicosanoids were extracted from saliva with ethanol, and the radioimmunoassay was performed to determine the concentrations of four major eicosanoids, i.e. prostaglandin E2 (PGE2), PGF2 alpha, 6-sulphidopeptide-containing leukotrienes (LTs) and 12-hydroxyeicosatetraenoic acid (12-HETE). The levels of PGE2, PGF2 alpha, and LTs were significantly lower in the saliva of smokers as compared to that of the non-smokers (1.74 +/- 0.32 vs 2.41 +/- 0.64, p = 0.006; 0.36 +/- 0.12 vs 0.54 +/- 0.18, p = 0.04; 2.24 +/- 0.96 vs 4.92 +/- 1.29, p = 0.006; mean +/- SD, ng/ml saliva). No significant differences were found in the levels of 12-HETE between the two groups. The results suggest that cigarette smoking reduces the concentrations of both the cyclooxygenase and 5-lipoxygenase products in saliva.     

Enzyme- and immuno-histochemical localization of kallikrein. II. The human kidney

Localization of kallikrein in the human kidney was investigated by two markers: kallikrein-like activity and kallikrein antigenicity. Kallikrein-like activity was demonstrated enzyme-histochemically by using a synthetic substrate for kallikrein, pro-phe-arg-naphthyl-ester. Kallikrein antigenicity was demonstrated by the unlabelled antibody peroxidase-antiperoxidase method using an antiserum against human urinary kallikrein. The kallikrein-like activity was localized in the proximal tubular cells without any corresponding kallikrein antigenicity. Neither kallikrein-like activity nor kallikrein antigenicity was noticed in any other tubular cell. These results are contrary to those in the ductal cells of the human parotid gland where the kallikrein-like activity and the kallikrein antigenicity were identical in their locations. The peroxidase-antiperoxidase method revealed, for the first time, kallikrein antigenicity both in the interstitium and in the basement membrane region of Bowman's capsule and of all the tubules, possibly representing circulating glandular kallikreins deposited in the renal tissue. Thus, the present findings are consistent with the hypothesis that the urinary (renal) kallikreins are derived from circulating glandular kallikreins.     

Cloning and expression of human salivary-gland kallikrein in Escherichia coli.

CDNA clones for human kallikrein have been identified in a cDNA library constructed from mRNA of human salivary gland. The entire coding sequence for preprokallikrein and for the 5'- and 3'-untranslated regions were isolated by using a mixture of oligonucleotides corresponding to amino acids 51-56 of human urinary kallikrein and one oligonucleotide corresponding to amino acids 233-238 of human pancreatic kallikrein. The DNA sequence proved that, with the exception of two amino acid exchanges, kallikrein of the human salivary gland is identical with pancreatic kallikrein. Salivary gland and renal kallikrein was expressed in Escherichia coli from plasmid pKK223-3 under the control of the tac promoter. The protein was identified by Western-blot analysis and by demonstration of its specific proteolytic activity.     

Enzyme- and immuno-histochemical localization of kallikrein. I. The human parotid gland

Localization of kallikrein in the human parotid gland was investigated simultaneously by two markers: kallikrein-like (enzyme) activity and kallikrein antigenicity. Kallikrein-like activity was histochemically demonstrated by using a synthetic substrate, pro-phe-arg- naphthylester . Kallikrein antigenicity was demonstrated by an unlabelled antibody peroxidase-antiperoxidase method, where monospecific antiserum against highly purified urinary kallikrein was used as the primary antiserum. The results showed that kallikrein-like activity and kallikrein antigenicity were identical in their locations in the ductal cells, being localized in the luminal part of the striated ducts and to a lesser degree in the excretory ducts. This indicates the presence of active kallikrein in these regions. No enzyme activity nor antigenicity was observed either in acini or in intercalated ducts. Moreover, the peroxidase-antiperoxidase method revealed kallikrein antigenicity for the first time extracellularly in the basement-membrane region of acini and of ducts as well as in the interstitium surrounding ducts and major vessels.     

Immunohistochemical localization of tonin and its relation to kallikrein in rat salivary glands

Tonin and kallikrein are serine proteases present in high concentrations in the submandibular gland of the rat. These enzymes release the vasoactive peptides angiotensin II and lysyl-bradykinin from the precursors angiotensinogen and kininogen, respectively. Tonin and kallikrein were purified from homogenates of rat submandibular gland, and antisera against each protein were raised in rabbits. The anti-kallikrein antibody also reacted with tonin, showing partial cross-reactivity between kallikrein and tonin when tested by double immunodiffusion and by immunoelectrophoresis. The anti-tonin antibody did not appear to react with kallikrein in immunodiffusion systems. The cellular localization of tonin was investigated by the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. In the granular tubular cells tonin-specific staining was abundantly present with a granular distribution; in the striated duct cells tonin-specific staining was observed as a thin luminal rim. Tonin was not detected in any other structures of the gland. When the localization of tonin was compared with that of kallikrein, both enzymes were found within the same granular tubular cells. However, more kallikrein than tonin was detected in the striated duct cells. Furthermore, kallikrein but not tonin was found in the ductal cells of the parotid and sublingual glands.     

Relationships among urinary kallikrein, mineralocorticoids and human hypertensive disease.

Urinary kallikrein excretion is reduced in patients with hypertension of unknown etiology. In addition, the excretion of this renal, kinin-forming enzyme was found to be elevated in hypertensive patients with primary aldosteronism. Aldosterone regulates kallikrein excretion, as normal subjects show increased kallikrein excretion in response to a low sodium intake, high potassium intake, or the synthetic mineralocorticoid, fludrocortisone, whereas kallikrein excretion falls during treatment with spironolactone. The relationship between kallikrein excretion and aldosterone activity may directly reflect the intrarenal activity of the kallikrein-kinin system, as determined by studies of kallikrein levels from isolated renal cells or of plasma kinin levels in man in response to postural changes or saline loads. Some patients with essential hypertension do not show a normal increase in kallikrein excretion in response to low dietary sodium intake despite an apparently normal aldosterone response, suggesting that there may be a defect in the renal kallikrein-kinin system in these patients. Whether these findings are of pathogenetic significance in human hypertensive disease remains to be determined.     

Studies on the temperature-dependent autoinhibition of human plasma kallikrein I.

At 37 degrees C, human plasma kallikrein I follows Michaelis-Menten behaviour and exhibits a normal linear relationship between the initial velocity of hydrolysis of Ac-Pro-Phe-Arg-OMe,HCl and enzyme concentration in the range 0--150 pM. At temperatures of 30 degrees C and below substantial deviations from linearity are observed over the same enzyme concentration range. The temperature-dependent autoinhibition of kallikrein I activity is reversible and is not due to low-molecular-weight endogenous inhibitors or cofactors. The kinetic effect is apparently due to aggregation and can be abolished by the addition of sodium deoxycholate.