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.     

Prostatic kallikreins: biochemistry and physiology

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 kallikreins. The rat prostate expresses two different kallikreins, S3 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 kallikrein. It has no known physiological substrate.     

The epigenetic basis for the aberrant expression of kallikreins in human cancers

The tissue kallikrein gene family consists of 15 genes tandemly arranged on human chromosome 19q13.4. Most kallikrein genes are characterized by aberrant expression patterns in various human cancers, a feature that makes them ideal cancer biomarkers. In the present study, we investigated the effect of the epigenetic drug compound 5-aza-2'-deoxycytidine on the expression of downregulated kallikrein genes in prostate, breast, and ovarian cancer cell lines. Reactivation of multiple kallikrein genes was observed, although some of these genes do not contain CpG islands in their genomic sequence. Epigenetic regulation provides a new mechanism for the pharmacological modulation of kallikreins in human cancers with putative therapeutic implications.     

Molecular cloning of the human kallikrein 15 gene (KLK15). Up-regulation in prostate cancer

Kallikreins are a subgroup of serine proteases with diverse physiological functions. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. By using molecular cloning techniques, we identified a new human kallikrein gene, tentatively named KLK15 (for kallikrein 15 gene). This new gene maps to chromosome 19q13.4 and is located between the KLK1 and KLK3 genes. KLK15 is formed of five coding exons and four introns, and shows structural similarity to other kallikreins and kallikrein-like genes. KLK15 has three alternatively spliced forms and is primarily expressed in the thyroid gland and to a lower extent in the prostate, salivary, and adrenal glands and in the colon testis and kidney. Our preliminary results indicate that the expression of KLK15 is up-regulated by steroid hormones in the LNCaP prostate cancer cell line. The KLK15 gene is also up-regulated, at the mRNA level, in prostate cancer in comparison to normal prostatic tissue. KLK15 up-regulation was found to be associated with more aggressive forms of prostate cancer. This newly discovered gene has the potential of being used as a diagnostic and/or prognostic marker for prostate cancer.     

Not more than three tissue kallikreins identified from organs of the guinea pig

The large and varied multigene families of tissue kallikreins of rat and mouse are considered to selectively release as many bioactive peptides. In order to determine whether a similar family of enzymes is expressed in the organs of the guinea pig purification studies were performed. Tissue kallikreins from the submandibular gland, coagulating gland/prostate complex and the pancreas were separated by affinity chromatography on benzamidine-Sepharose. Amino-terminal sequences, the patterns of hydrolysis rates of a number of peptide p-nitroanilides, inactivation rates by active site-directed irreversible inhibitors, specific kininogenase activities and types of kinin released were used to probe the identity of the isolated enzymes. Guinea pig tissue kallikreins 1 and 2 have been reported previously. In the present study we have identified a third type, designated tissue kallikrein 1a because of its sequence similarity to kallikrein 1, which differs from the latter in the catalytic properties. The inferred occurrence of not more than two or three independent tissue kallikrein genes in the guinea pig contrasts with the varied family of enzymes expressed by the large number of such genes present in rats and mice. Expression in the guinea pig (and also in humans) of only a small number of tissue kallikreins makes specific processing of a multitude of biologically active peptides by such enzymes unlikely.     

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.     

KLK12 is a novel serine protease and a new member of the human kallikrein gene family-differential expression in breast cancer

Kallikreins are a subgroup of serine proteases that are involved in the posttranslational processing of polypeptide precursors. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. In rodents, kallikreins are encoded by a large multigene family, but in humans, only three genes have been identified. By using the positional candidate approach, we were able to identify a new kallikrein-like gene, tentatively named KLK12 (for kallikrein gene 12). This new gene maps to chromosome 19q13.3-q13.4, is formed of five coding exons, and shows structural similarity to serine proteases and other known kallikreins. KLK12 is expressed in a variety of tissues including salivary gland, stomach, uterus, lung, thymus, prostate, colon, brain, breast, thyroid, and trachea. We identified three splicing forms of KLK12 that are expressed in many tissues. Our preliminary results indicate that the expression of KLK12 is down-regulated at the mRNA level in breast cancer tissues and is up-regulated by steroid hormones in breast and prostate cancer cell lines. This gene may be involved in the pathogenesis and/or progression of certain cancer types and may find applicability as a novel cancer biomarker.     

KLK12 Is a Novel Serine Protease and a New Member of the Human Kallikrein Gene Family—Differential Expression in Breast Cancer

Kallikreins are a subgroup of serine proteases that are involved in the posttranslational processing of polypeptide precursors. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. In rodents, kallikreins are encoded by a large multigene family, but in humans, only three genes have been identified. By using the positional candidate approach, we were able to identify a new kallikrein-like gene, tentatively named KLK12 (for kallikrein gene 12). This new gene maps to chromosome 19q13.3–q13.4, is formed of five coding exons, and shows structural similarity to serine proteases and other known kallikreins. KLK12 is expressed in a variety of tissues including salivary gland, stomach, uterus, lung, thymus, prostate, colon, brain, breast, thyroid, and trachea. We identified three splicing forms of KLK12 that are expressed in many tissues. Our preliminary results indicate that the expression of KLK12 is down-regulated at the mRNA level in breast cancer tissues and is up-regulated by steroid hormones in breast and prostate cancer cell lines. This gene may be involved in the pathogenesis and/or progression of certain cancer types and may find applicability as a novel cancer biomarker.     

The T-cell receptor β chain-encoding gene repertoire of a New World primate species,the cotton-top tamarin

 The New World primate, the cotton-top tamarin (Saguinus oedipus), expresses major histocompatibility complex (MHC) class I molecules with limited diversity. The uniqueness of the cotton-top tamarin MHC class I loci may contribute to this species’ unusual susceptibility to viral infections and high incidence of ulcerative colitis. As a prelude to examining the effect of this limited MHC class I diversity on the tamarin CD8⁺ T-cell receptor (TCR) repertoire, we identified expressed tamarin TCR β chain (TCRB) cDNAs by anchored and inverse polymerase chain reaction. Sequence alignments and phylogenetic comparisons with human and rhesus macaque sequences identified homologues of 21 human variable (V) gene families. Only single variable region genes were identified in each of these tamarin VB families, with the exception of the VB 5, 9, and 13 families which were comprised of two or three distinct members. The multiple genes within these three VB families do not appear to have separate human homologues, but rather aligned equally well to a single human gene from their respective VB families. These genes appear to have arisen, therefore, by duplication of certain VB genes in the tamarin ancestors following their divergence from the lineage leading to Old World primates and hominoids. Homologues of 12 of the 13 human joining (J) region genes were also identified in the tamarin. Comparison of the proportion of nonsynonymous (p_N) and synonymous (p_S) substitutions occurring per site within tamarin variable region genes demonstrated a reduction in p_N in the framework regions compared with p_N in the presumed MHC contact regions (CDR1 and CDR2). Taken together, these findings illustrate that the TCR β chain-encoding genes of the cotton-top tamarin are similar in structure and degree of complexity compared with their Old World primate and human counterparts. Received: 19 July 1996 / Revised: 12 August 1996     

The new kallikrein-like gene, KLK-L2. Molecular characterization, mapping, tissue expression, and hormonal regulation

Since in rodents the kallikreins are represented by a large multi-gene family, the restriction of this family in humans to three genes is somewhat surprising. In an effort to identify new human kallikrein genes, we examined a genomic area of about 300 kilobases on chromosome 19q13.3-q13.4, a region that contains most of the currently known kallikreins. By using the positional candidate approach, we were able to identify a new gene named KLK-L2 (for kallikrein- like gene 2). Screening of human EST libraries allowed us to delineate the full genomic and cDNA structure of the new gene. KLK-L2 consists of 5 coding exons and 4 introns and has significant similarities to other members of the kallikrein multi-gene family. Homology studies suggest that the protein is likely secreted. KLK-L2 is expressed mainly in breast, brain, and testis and to a lesser extent in many other tissues. KLK-L2 is up-regulated by estrogens and progestins in the breast cancer cell line BT-474.     

Reflections on the tissue kallikrein and kallikrein-related peptidase family - from mice to men - what have we learnt in the last two decades?

The genes encoding the kininogenase, glandular tissue kallikrein, in rodents and man were first described in the mid-1980s. Remarkably, they appeared to be part of a much larger highly conserved family of genes (GK) in rodents, but only had two paralogs in man. This discrepancy was not rectified until the late 1990s/2000 with the identification of a cluster of 12 more kallikrein-related (KLK) genes in the human 19q13 locus and the subsequent identification of their rodent homologs. Interestingly, there are remarkable similarities in expression patterns, hormonal regulation and functional attributes of the old (GK) and new (KLK) families which underscore the evolutionary conservation across these loci and species. This historical perspective focuses on the lessons learned from earlier studies on the rodent GK gene families and the striking similarities of some attributes, yet uniqueness, of others. These earlier findings have all contributed to the current status of the KLK serine peptidase-encoding gene family as an exciting source of new biomarkers and therapeutic targets.     

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.     

Human tissue kallikrein 9: production of recombinant proteins and specific antibodies

Human tissue kallikreins (genes, KLKs; proteins, hKs) are a subgroup of hormonally regulated serine proteases. Two tissue kallikreins, namely hK2 and hK3 (prostate-specific antigen, PSA), are currently used as serological biomarkers of prostate cancer. Human tissue kallikrein 9 (KLK9) is a newly identified member of the tissue kallikrein gene family. Recent reports have indicated that KLK9 mRNA is differentially expressed in ovarian and breast cancer and has prognostic value. Here, we report the production of recombinant hK9 (classic form) using prokaryotic and mammalian cells and the generation of polyclonal antibodies. Total testis tissue mRNA was reverse-transcribed to cDNA, amplified, cloned into a pET/200 TOPO plasmid vector, and transformed into E. coli cells. hK9 was purified and used as an immunogen to generate polyclonal antibodies. Full-length KLK9 cDNA was also cloned in the vector pcDNA3.1 and was expressed in CHO cells. The identity of hK9 was confirmed by mass spectrometry. hK9 rabbit antiserum displayed no cross-reactivity with other tissue kallikreins and could specifically recognize E. coli- and CHO-derived hK9 on Western blots. hK9 was mainly detected in testis and seminal vesicles by Western blotting. The reagents generated here will help to define the physiological role of this tissue kallikrein and its involvement in human disease.     

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.