Localization of a new prostate-specific antigen-related serine protease gene, KLK4, is evidence for an expanded human kallikrein gene family cluster on chromosome 19q13.3-13.4.

The human tissue kallikrein (KLK) family of serine proteases, which is important in post-translational processing events, currently consists of just three genes-tissue kallikrein (KLK1), KLK2, and prostate-specific antigen (PSA) (KLK3)-clustered at chromosome 19q13. 3-13.4. We identified an expressed sequence tag from an endometrial carcinoma cDNA library with 50% identity to the three known KLK genes. Primers designed to putative exon 2 and exon 3 regions from this novel kallikrein-related sequence were used to polymerase chain reaction-screen five cosmids spanning 130 kb around the KLK locus on chromosome 19. This new gene, which we have named KLK4, is 25 kb downstream of the KLK2 gene and follows a region that includes two other putative KLK-like gene fragments. KLK4 spans 5.2 kb, has an identical genomic structure-five exons and four introns-to the other KLK genes and is transcribed on the reverse strand, in the same direction as KLK1 but opposite to that of KLK2 and KLK3. It encodes a 254-amino acid prepro-serine protease that is most similar (78% identical) to pig enamel matrix serine protease but is also 37% identical to PSA. These data suggest that the human kallikrein gene family locus on chromosome 19 is larger than previously thought and also indicate a greater sequence divergence within this family compared with the highly conserved rodent kallikrein genes.     

Sequencing and expression analysis of the serine protease gene cluster located in chromosome 19q13 region

The human kallikrein gene cluster, located in the chromosome band 19q13, contains several tissue-specific serine protease genes including the prostate-specific KLK2, KLK3 and prostase genes. To further characterize the gene cluster, we have mapped, sequenced, and analyzed the genomic sequence from the region. The results of EST database searches and GENSCAN gene prediction analysis reveal 13 serine protease genes and several pseudogenes in the region. Expression analysis by RT-PCR indicates that most of these protease genes are expressed only in a subset of the 35 different normal tissues that have been examined. Several protease genes expressed in skin show higher expression levels in psoriatic lesion samples than in non-lesional skin samples from the same patient. This suggests that the imbalance of a complex protease cascade in skin may contribute to the pathology of disease. The proteases, excluding the kallikrein genes, share approximately 40% of their sequences suggesting that the serine protease gene cluster on chromosome 19q13 arose from ancient gene duplications.     

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.     

Expression and functional characterization of the cancer-related serine protease, human tissue kallikrein 14

Human tissue kallikrein 14 (KLK14) is a novel extracellular serine protease. Clinical data link KLK14 expression to several diseases, primarily cancer; however, little is known of its (patho)-physiological role. To functionally characterize KLK14, we expressed and purified recombinant KLK14 in mature and proenzyme forms and determined its expression pattern, specificity, regulation, and in vitro substrates. By using our novel immunoassay, the normal and/or diseased skin, breast, prostate, and ovary contained the highest concentration of KLK14. Serum KLK14 levels were significantly elevated in prostate cancer patients compared with healthy males. KLK14 displayed trypsin-like specificity with high selectivity for P1-Arg over Lys. KLK14 activity could be regulated as follows: 1) by autolytic cleavage leading to enzymatic inactivation; 2) by the inhibitory serpins alpha1-antitrypsin, alpha2-antiplasmin, antithrombin III, and alpha1-antichymotrypsin with second order rate constants (k(+2)/Ki) of 49.8, 23.8, 1.48, and 0.224 microM(-1) min(-1), respectively, as well as plasminogen activator inhibitor-1; and 3) by citrate and zinc ions, which exerted stimulatory and inhibitory effects on KLK14 activity, respectively. We also expanded the in vitro target repertoire of KLK14 to include collagens I-IV, fibronectin, laminin, kininogen, fibrinogen, plasminogen, vitronectin, and insulin-like growth factor-binding proteins 2 and 3. Our results indicate that KLK14 may be implicated in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage. These findings may have clinical implications for the management of cancer and other disorders in which KLK14 activity is elevated.     

Identification and characterization of C6orf37, a novel candidate human retinal disease gene on chromosome 6q14

We have identified a novel human gene, chromosome 6 open reading frame 37 (C6orf37), that is expressed in the retina and maps to human chromosome 6q14, a genomic region that harbors multiple retinal disease loci. The cDNA sequence contains an open reading frame of 1314 bp that encodes a 437-amino acid protein with a predicted molecular mass of 49.2 kDa. Northern blot analysis indicates that this gene is widely expressed, with preferential expression observed in the retina compared to other ocular tissues. The C6orf37 protein shares homology with putative proteins in R. norvegicus, M. musculus, D. melanogaster, and C. elegans, suggesting evolutionary conservation of function. Additional sequence analysis predicts that the C6orf37 gene product is a soluble, globular cytoplasmic protein containing several conserved phosphorylation sites. Furthermore, we have defined the genomic structure of this gene, which will enable its analysis as a candidate gene for chromosome 6q-associated inherited retinal disorders.     

Structure and expression of a cluster of human hematopoietic serine protease genes found on chromosome 14q11.2

We previously identified a cluster of hematopoietic serine protease genes on chromosome 14 at band q11.2. This cluster contains the cathepsin G gene and the two related cathepsin G-like genes CGL-1 and CGL-2. The CGL-1 gene is identical with the cytotoxic T cell serine protease CSP-B (also called SECT, and in mice, CCP1, granzyme B, or CTLA-1). In this report, we determined that CGL-2 is identical with a recently described gene called h-CCPX. The coding sequences of CG, CGL-1, and CGL-2 are 65-75% identical at the DNA level. The intervening sequences are much less conserved, except for introns 3 of the CGL-1 and CGL-2 genes, which are 93% identical. Each of the genes has the same overall organization, with 5 exons and 4 introns, very short 5' untranslated regions, and identical splice phases for all of the introns. Cathepsin G is expressed at high levels in promyelocytes/promonocytes, and CGL-1/CSP-B is expressed at high levels in activated cytolytic T cells, lymphokine-activated killer (LAK), and natural killer (NK) cells. CGL-2/h-CCPX is expressed at much lower levels in activated peripheral blood lymphocytes, LAK and NK cells. To begin to define the regulatory elements that target expression of each of these genes to their specific lineages at specific times, the 5' flanking region of each gene was sequenced. The 5' flanking regions are minimally related and have few conserved consensus elements. Further experiments will be required to determine the critical cis-acting regulatory sequences required for tissue- and development-specific expression of each of these genes.     

A dense comparative gene map between human chromosome 19q13.3-->q13.4 and a homologous segment of swine chromosome 6

The human chromosome (HSA)19q region has been shown to correspond to swine chromosome (SSC) 6q11-->q21 by bi-directional chromosomal painting and gene mapping. However, since the precise correspondence has not been determined, 26 genes localized in HSA19q13.3-->q13.4 were assigned to the SSC6 region mainly by radiation hybrid (RH) mapping, and additionally, by somatic cell hybrid panel (SCHP) mapping, and fluorescent in situ hybridization (FISH). Out of the 26 genes, 24 were assigned to a swine RH map with LOD scores greater than 6 (threshold of significance). The most likely order of the 24 genes along SSC6 was calculated by CarthaGene, revealing that the order is essentially the same as that in HSA19q13.3-->q13.4. For AURKC and RPS5 giving LOD scores not greater than 6, SCHP mapping and FISH were additionally performed; SCHP mapping assigned AURKC and RPS5 to SSC6q22-->q23 and SSC6q21, respectively, which is consistent with the observation of FISH. Consequently, all the genes (26 genes) examined in the present study were shown to localize in SSC6q12-->q23, and the order of the genes along the chromosomes was shown to be essentially the same in swine and human, though several intrachromosomal rearrangements were observed between the species.     

A retrotransposon-derived gene, PEG10, is a novel imprinted gene located on human chromosome 7q21

A novel paternally expressed imprinted gene, PEG10 (Paternally Expressed 10), was identified on human chromosome 7q21. PEG10 is located near the SGCE (Sarcoglycan epsilon) gene, whose mouse homologue was recently shown to be imprinted. Therefore, it is highly possible that a new imprinted gene cluster exists on human chromosome 7q21. Analysis of two predicted open reading frames (ORF1 and ORF2) revealed that ORF1 and ORF2 have homology to the gag and pol proteins of some vertebrate retrotransposons, respectively. These data suggest that PEG10 is derived from a retrotransposon that was previously integrated into the mammalian genome. PEG10 is likely to be essential for understanding how exogenous genes become imprinted.     

Characterization of two novel KRAB-domain-containing zinc finger genes, ZNF460 and ZNF461, on human chromosome 19q13.1-->q13.4

This study reports the cloning and characterization of two novel human zinc finger protein cDNAs (ZNF460 and ZNF461) from a fetal brain cDNA library. The ZNF460 cDNA is 3,135 bp in length encoding a 562-amino-acid polypeptide and the ZNF461 cDNA is 2,548 bp encoding a 563-amino-acid protein. Both of the proteins contain a KRAB A+B box and eleven C2H2 type zinc finger motifs. ZNF461 shows high similarity with the rat GIOT-1 gene (GIOT1). The ZNF460 gene mapped to 19q13.4 with 3 exons, and ZNF461 mapped to 19q13.1 with 6 exons. Both of the two genes are ubiquitously expressed in normal human tissues and the abundance of the ZNF460 mRNA is relatively low.     

Cloning a new human gene from chromosome 21q22.3 encoding a glutamic acid-rich protein expressed in heart and skeletal muscle

The identification and functional characterization of genes on chromosome 21 is a necessary step to understand the pathogenesis of the various phenotypic anomalies that affect Down syndrome patients. Using direct cDNA selection we have identified a new gene, SH3BGR, that maps to 21q22.3, proximal to HMG14, and is differentially expressed in heart and skeletal muscle. SH3BGR encodes a novel protein that is characterized by the presence of a proline-rich region containing the consensus sequence for a SH3-binding domain and by an acidic carboxyl-terminal region containing a glutamic acid-rich domain predicted to assume a coiled coil. The presence of two functional domains involved in protein-protein interactions suggests that SH3BGR could be part of a multimeric complex. Its overexpression might alter specific functions of muscular tissue and therefore take part in the pathophysiology of muscular hypotonia in Down syndrome. Received: 12 August 1996 / Revised: 22 October 1996     

Purification and partial characterization of a myofibril-bound serine protease from ostrich skeletal muscle

A myofibril-bound serine protease (MBSP) was partially purified from ostrich (Struthio camelus) skeletal muscle. MBSP was dissociated from the myofibrillar fraction by ethylene glycol treatment at pH 8.5, followed by partial purification via Toyopearl Super Q 650 S and p-aminobenzamidine column chromatographies. Ostrich MBSP revealed a major protein band of approximately 21 kDa on SDS-PAGE, showing proteolytic activity after casein zymography. Optima pH and temperature of ostrich MBSP were 8 and 40 °C, respectively. Substrate specificity analysis revealed that the enzyme cleaved synthetic fluorogenic substrates at the carboxyl side of arginine residues. Kinetic parameters (K_m and V_max values) were calculated from Lineweaver–Burk plots. The kinetic characteristics of ostrich MBSP were compared to values obtained for commercial bovine trypsin in this study, as well as those obtained for MBSP from mouse and various fish species. The results suggest that ostrich MBSP is a tryptic-like serine protease. Ostrich MBSP exhibited low sequence identity to commercial bovine trypsin (44%), MBSP from lizard fish skeletal muscle (33%) and trypsinogen from ostrich pancreas (22%).     

Identification of a myofibril-bound serine protease and its endogenous inhibitor in mouse skeletal muscle

Myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. A proteolytic activity associated to myofibrils was found in mouse skeletal muscle, as show electrophoretic patterns, and denominated by us, as protease M. During incubation of whole myofibrils at 37 degrees C, myosin heavy chain, alpha actinin, actin and troponin T suffered degradation. These effects were inhibited selectively by serine protease inhibitors (soybean trypsin inhibitor, di-isopropyl phosphofluoridate, phenylmethanesulfonyl fluoride). Using myofibrils as protease M source, azocaseinolytic activity was also detected. Endogenous inhibitor and various compounds effects on protease M activity were also quantified by trichloroacetic acid soluble products formation, using radiolabeled myofibrils. An endogenous trypsin inhibitor isolated from the muscle cytoplasmic fraction could inhibit protease M activity on myofibrillar proteins and on azocasein. While K(+) increased protease M activity, the presence of Ca(2+) did not show any effect. Data presented in this study suggest that reported protease M may be implicated in myofibrillar degradation in vivo and isolated endogenous inhibitor may provide a mechanism to control its action in mouse skeletal muscle.     

Mapping of the human BAX gene to chromosome 19q13.3–q13.4 and isolation of a novel alternatively spliced transcript, BAXδ

The BAX gene is a member of the Bcl-2 gene family; it encodes a 21-kDa protein whose association with Bcl-2 is believed to play a critical role in regulating apoptosis. Through analysis of human—hamster somatic cell hybrid DNA and by in situ hybridization to metaphase chromosomes, we have determined that the human BAX gene is located in the q13.3–q13.4 region of human chromosome 19. We have also isolated a BAX cDNA clone in which that part of the mRNA encoded by exon 3 is absent. The skipping of exon 3 and the resultant splicing of exons 2 and 4 maintains the original reading frame and predicts the existence of an interstitially truncated form of the major Bax protein (Baxα), termed Baxδ. Unlike two previously described variant forms of Baxα (Baxß and Baxτ), Baxδ retains the functionally critical C-terminal membrane anchor region as well as the Bcl-2 homology 1 and 2 (BH1 and BH2) domains.     

Identification of a novel prostate cancer susceptibility variant in the KLK3 gene transcript

Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P?=?3.9?×?10(-22)). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P?=?1.9?×?10(-34)). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.