Immunolocalization of renal kallikrein-like substance in rat urinary bladder

Summary The renal origin of kallikrein is now clearly established. However, the presence of kallikrein in urine raises questions about a possible physiological role of this enzyme at the urinary level. We have already demonstrated the presence of kallikrein-like substance in rat ureter. For establishing the continuity of the presence of kallikrein-like substance along the urinary tract we have studied the localization of immunoreactive kallikrein-like substance in urinary bladder of the normal rat by immunohistochemical methods for light- and electron-microscopy, using an antibody against rat urinary kallikrein. By light microscopy, kallikrein-like substance was found to be associated with the lamina propria, which is the connective tissue component which constitutes one layer of the bladder wall. Weak staining was present in the smooth-muscle layer. By immuno-electron microscopy, kallikrein-like substance was localized in fibroblasts which were present in the connective tissue and which penetrated into the layer of smooth muscle; immunoreactivity was observed in endoplasmic reticulum, Golgi apparatus and free polyribosomes. Immunolabelling was demonstrated in no other part of the wall bladder and in no other cellular component. The continuity of the presence of kallikrein-like substance from the kidney to the urinary bladder gives new indications concerning the significance of this system in renal physiology.     

Androgen dependence of specific kallikrein gene family members expressed in rat prostate

We have used oligonucleotide probes specific for members of the rat kallikrein/tonin gene family (PS, S1, S2, S3, K1, and P1) to establish which arginyl esteropeptidase (kallikrein-like) genes are expressed in the prostate. We have also compared the expression and androgen dependence of these genes in prostate, submaxillary gland (SMG) and kidney. Only S3 (tonin-like) and P1 (kallikrein-like) are expressed in the prostate, with S3 very much more abundant. Prostatic S3 mRNA disappears after 8 days castration and is restored to intact levels by dihydrotestosterone (DHT) but not estradiol benzoate (EB) for 8 days. Prostate P1 mRNA levels were similarly but not identically affected. All six genes are expressed in the SMG, with PS (true kallikrein) the most abundant. Levels of PS mRNA in SMG are unaffected by castration, DHT, or EB treatment, although mRNA levels of other kallikrein-like (S1, K1, and P1), tonin (S2), and tonin-like (S3) genes fall 40-60% after castration, and are unaffected or partially restored by DHT and/or EB administration. Only PS and K1 are expressed in the kidney, at much lower levels than in the SMG and unaffected by castration or steroids. These studies thus confirm and extend the concept of tissue specificity of arginyl esteropeptidase gene expression, and further demonstrate that the same gene(s) is differentially regulated by androgens in the rat prostate, SMG, and kidney.     

The expression of two kallikrein gene family members in the rat kidney

The mRNAs for two kallikrein gene family members expressed in the rat kidney have been characterized. One mRNA (PS) has previously been found in the pancreas and submaxillary gland and encodes true kallikrein. The second mRNA (K1) encodes a novel kallikrein-like enzyme expressed in the kidney and submaxillary gland that retains many of the key amino acid residues for the characteristic enzymatic cleavage specificity of kallikrein. Two oligonucleotide hybridization probes specific for the K1 mRNA demonstrate that the K1 mRNA is expressed in the kidney and submaxillary gland, but in none of the other eight tissues known to express one or more members of the rat kallikrein gene family. The K1 mRNA is the dominant kallikrein-related mRNA of the kidney, expressed at roughly 10 times the level of the true kallikrein (PS) mRNA. In the submaxillary gland the K1 mRNA is expressed at roughly one-fourth the level of true kallikrein mRNA.     

Immunological identification of rat tissue kallikrein cDNA and characterization of the kallikrein gene family

A tissue kallikrein cDNA was identified by direct immunological screening with affinity-purified anti-rat tissue kallikrein antibody from a rat submandibular cDNA library constructed with the expression vector pUC8. Sequence analysis of the kallikrein cDNA revealed an encoded protein 97% homologous to the partial amino acid sequence of rat submandibular kallikrein. This cDNA was used to hybrid-select kallikrein-specific RNA from submandibular gland. Translation of the hybrid-selected RNA in a cell-free assay system resulted in the production of a 37 kDa peptide representing the preproenzyme. In addition, hybrid-selection of RNA under less stringent conditions showed cross-hybridization with other submandibular gland mRNA species. In correlation with these results, analysis of rat genomic DNA showed extensive hybridization, suggesting a family of closely related kallikrein-like genes. Consequently, a Charon 4A rat genomic library was screened for kallikrein genes by hybridization with rat tissue kallikrein cDNA. Thirty-four clones were isolated and found to be highly homologous by hybridization and restriction enzymes analyses. Fourteen unique clones were identified by restriction enzyme site polymorphisms within DNA segments which hybridized to the kallikrein cDNA probe and it was estimated that at least 17 different kallikrein-like genes are present in the rat. Sequence and structural analysis of one of the genomic clones revealed a gene structure similar to that of other serine proteinases. Comparison of the partially sequenced exon regions of the gene with the sequence of rat tissue kallikrein cDNA reveals 89% identity when aligned for the greatest homology. However, the genomic sequence predicts termination codons in all three translational reading frames, implying that this gene is nonfunctional, i.e., a pseudogene. Comparison of the rat genomic sequence to a kallikrein-like gene from the mouse reveals extensive preservation of exons, less identity within introns and no significant homology between extragenic regions.     

Localization of human glandular kallikrein-1 gene to chromosome 19q13.3-13.4 by in situ hybridization.

Humans possess 3 fully characterized kallikrein-like genes. The gene expressed in kidney, pancreas and salivary gland (KLK), and the gene encoding prostate-specific antigen (APS) have been localized to chromosome 19q13.2-qter. The present study describes the localization of the remaining gene, hGK-1, which has highest homology to and a similar tissue specificity of expression as the APS gene. Using a [3H]-labeled probe derived from a hGK-1 genomic clone, we demonstrated hybridization confined to the q13.3 and q13.4 bands of chromosome 19 and suggest that kallikrein genes may possibly be located near the border of these two bands.     

Characterization of genes encoding rat tonin and a kallikrein-like serine protease

Tissue kallikreins are a group of serine proteases which may function as peptide hormone processing enzymes. Two rat kallikrein genomic clones (RSKG-5 and RSKG-50) were sequenced and characterized. The rat tonin gene and a kallikrein-like gene were found in clones RSKG-5 and RSKG-50, respectively. The tonin gene is 4146 base pairs in length, with both the variant CCAAA and TTTAAA boxes in the 5'-end region and an AATAAA polyadenylation signal at the 3' end of the gene. It has five exons which are separated by four introns. Sequence analysis of 3.7-kb 5' upstream and 7.5-kb 3' downstream of the tonin gene failed to reveal a second kallikrein gene. Sequence comparisons of the RSKG-5 exons with tonin cDNA revealed that only one base in the 3'-noncoding region was different from that in the previously reported rat tonin cDNA. Characteristic TC- and TG-repeated sequences were also found in the first and second introns of the tonin gene. The tonin gene encodes a preprotonin of 259 amino acids (aa). The active enzyme consists of 235 aa and is preceded by a deduced signal peptide of 17 aa and a profragment of 7 aa. Northern blot analysis indicates that RSKG-5 is expressed in a sex-dependent manner in rat submandibular gland, with a higher level expressed in males. The RSKG-50 gene was truncated at an EcoRI site in the second intron, excluding its 5' end. Compared to the coding sequence of pancreatic kallikrein, 12 nucleotides have been deleted in exon 3 of the RSKG-50 gene. The nucleotide sequences of the third, fourth, and fifth exons of the RSKG-50 gene encode a polypeptide of 188 aa residues. The translated peptide is 80% homologous to rat pancreatic kallikrein and 75% homologous to rat tonin in the corresponding regions. Key residues in the RSKG-50 gene product indicate a serine protease with kallikrein-like cleavage specificity at basic amino acids.     

Oestrogen administration and the expression of the kallikrein gene family in the rat submandibular gland

Using a series of oligonucleotide probes (18-21 mers) specific for members of the rat kallikrein/tonin (arginyl-esteropeptidase) gene family (PS, S1, S2, S3, K1, P1), we have shown by Northern blot analysis that all six genes are expressed in the submandibular gland (SMG), with PS (true kallikrein) the most abundant in both male and female rats. Though female levels of PS mRNA are similar to that in the male, levels of mRNA from both the kallikrein-like (S1, K1, P1) and tonin (S2)/tonin-like (S3) genes are all substantially lower in the female than in the male rat. In contrast with the oestrogen dependence of anterior pituitary kallikrein (PS) gene expression, oestrogen administration (6 micrograms/day for 8 days) to castrate male or female rats is without effect on PS or S1, S2, S3, K1, P1 mRNA levels in the SMG. These findings suggest a tissue-specificity in the oestrogen regulation of true kallikrein gene expression in the two tissues. In intact male rats, oestrogen administration lowers SMG levels of S1, S2, S3, K1, and P1 but not PS mRNA to castrate levels, presumably by suppression of the pituitary/gonadal axis, consistent with the previously reported androgen dependence of SMG expression of these genes with the exception of PS.     

The expanded human kallikrein gene family: locus characterization and molecular cloning of a new member, KLK-L3 (KLK9)

In rodents, kallikreins are encoded by a large multigene family but in humans, only three kallikrein genes were thought to exist. Based on the homology between the human and the rodent kallikrein loci, we defined a 300-kb human kallikrein gene region on chromosome 19q13. 3-q13.4. By using linear sequence information, restriction analysis, PCR, and blotting techniques, we were able to construct the first detailed map of the human kallikrein gene locus. Comparative analysis of genes located in this area enabled us to expand the human kallikrein multigene family with some recently identified serine proteases and establish common structural features. We further identified a new kallikrein-like gene, named kallikrein-like gene 3 (KLK-L3; HGMW-approved symbol KLK9). We describe the structural characterization of the KLK-L3 gene, together with its precise chromosomal localization in relation to other kallikreins and its tissue expression pattern and hormonal regulation.     

Human prostate-specific antigen (APS) is a member of the glandular kallikrein gene family at 19q13

The amino acid sequence of human prostate-specific antigen (APS) suggests that it is a member of the glandular kallikrein subfamily of serine proteases. In the mouse, the kallikrein-like family is localized in a single locus on chromosome 7, while other serine proteases are distributed over a variety of different chromosomes. To investigate the physical relationship between the human kallikrein genes, we have used in situ hybridization and Southern analysis of a human x mouse somatic cell hybrid panel to map the APS gene to 19q13, concordant with the renal kallikrein KLK1 gene. This finding indicates that APS is a member of a human kallikrein-like gene family with analogous organization to that of the mouse.     

Immunocytochemical and enzyme histochemical localization of kallikrein-like enzymes in colon, intestine, and stomach of rat and cat

Kallikrein was localized in goblet (or mucous) cells of rat colon and in rat and cat small intestine and stomach by two immunocytochemical techniques. A kallikrein-like enzyme was also localized by enzyme histochemistry in mast cells of colon, intestine, and stomach of the cat, where they appeared to be associated with blood vessels in the lamina propria. The mast cell enzyme, however, was not detected by immunocytochemistry using antibodies to kallikrein. Modification in the enzyme histochemical procedure (pH, fixation) yielded positive results for a kallikrein-like protease in goblet cells of the intestine and colon. The possible physiological and pathological significance of kallikrein-like enzyme in the gastrointestinal tract and elsewhere is discussed.     

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.     

Enzyme- and immuno-histochemical localization of kallikrein

Summary 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.     

Nucleotide sequence of cloned cDNA for human pancreatic kallikrein

Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.     

Enzyme-and immuno-histochemical localization of kallikrein

Summary 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 reveated 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.     

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.     

Histochemical localization of kallikrein-like pro-phe-arg-naphthylester esterase activity in the rat kidney

Summary In the rat kidney the presence of the kallikrein-like pro-phe-arg-naphthylester esterase activity was demonstrated by a simultaneous coupling azo dye method. The enzyme was identified as a serine-protease because it was inhibited by preincubation with diisopropyl-fluorophosphate and unaffected by sodium iodoacetate. Since kallikrein is a serine-protease and pro-phe-arg-naphthylester is a synthetic and sensitive substrate for kallikrein, the enzyme activity revealed by this method was considered to represent kallikrein, although non-kallikrein esterase activity is not totally excluded. The enzyme activity was localized mainly in the outer stripe of the outer medulla, with focal extensions primarily only in the lower half of the cortex corresponding to the medullary rays.     

Genomic organization, mapping, tissue expression, and hormonal regulation of trypsin-like serine protease (TLSP PRSS20), a new member of the human kallikrein gene family

The cDNA for the trypsin-like serine protease gene (TLSP, HGMW-approved symbol PRSS20) has been recently identified. TLSP is expressed in brain and skin tissues but little else is known about this new serine protease gene. In this paper, we describe the complete genomic organization and precise mapping of the TLSP gene. This gene spans 5.3 kb of genomic sequence on chromosome 19q13.3-q13. 4. The gene consists of six exons, the first of which is untranslated. All splice junctions follow the GT/AG rule, and the intron phases are identical to those of other kallikrein-like genes, including zyme (PRSS9), NES1 (PRSSL1), and neuropsin (PRSS19). Fine-mapping of the area indicates that TLSP lies downstream from the PSA, zyme, neuropsin, and NES1 genes. Significant sequence homologies were found between TLSP and other human kallikreins. Furthermore, there is conservation of the catalytic triad (histidine, aspartic acid, serine) and of the number of coding exons (five; the same in all members of the kallikrein gene family). We thus suggest that TLSP is a new member of the human kallikrein gene family. TLSP is expressed in many tissues including cerebellum, prostate, salivary glands, stomach, lung, thymus, small intestine, spleen, liver, and uterus. TLSP expression appears to be regulated by steroid hormones in the breast carcinoma cell line BT-474.