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.     

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.     

Organization and evolution of the glandular kallikrein locus in Mus musculus

The gene of tissue kallikrein and closely related genes constitute the glandular kallikrein (GK) gene family. The number of members varies between species, ranging from three human to 25 murine. Recently, the gene family was extended with 12 new members, KLK4-KLK15, that were identified adjacent to the classical GK genes on human chromosome 19. In this report, the structure and phylogeny of the mouse GK gene locus are described. A comparison of the human and murine loci shows that the locations of the tissue kallikrein gene and KLK4-KLK15 are conserved. The region between the tissue kallikrein gene and KLK15, devoid of genes in human, is expanded and contains 23 classical GK genes in mouse. Downstream of KLK15, where the genes encoding PSA and hK2 are located in human, mouse carries the pseudogene PsimGK25. Phylogenetic analyses show that classical GK genes emerged after the separation of the primate and rodent lineages, forming a subgroup within the newly extended GK family.     

Activation profiles of human kallikrein-related peptidases by proteases of the thrombostasis axis

The human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of KLK function. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, we have characterized the ability of proteases associated with thrombostasis to hydrolyze the pro-peptide sequences of the KLK family using a previously described pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. Pro-KLK6 and 14 can be activated by both plasmin and uPA, with plasmin being the best activator of pro-KLK6 identified to date. Pro-KLK11 and 12 can be activated by a broad-spectrum of thrombostasis proteases, with thrombin exhibiting a high degree of selectivity for pro-KLK12. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families.     

Evolutionary history of tissue kallikreins

The gene family of human kallikrein-related peptidases (KLKs) encodes proteins with diverse and pleiotropic functions in normal physiology as well as in disease states. Currently, the most widely known KLK is KLK3 or prostate-specific antigen (PSA) that has applications in clinical diagnosis and monitoring of prostate cancer. The KLK gene family encompasses the largest contiguous cluster of serine proteases in humans which is not interrupted by non-KLK genes. This exceptional and unique characteristic of KLKs makes them ideal for evolutionary studies aiming to infer the direction and timing of gene duplication events. Previous studies on the evolution of KLKs were restricted to mammals and the emergence of KLKs was suggested about 150 million years ago (mya). In order to elucidate the evolutionary history of KLKs, we performed comprehensive phylogenetic analyses of KLK homologous proteins in multiple genomes including those that have been completed recently. Interestingly, we were able to identify novel reptilian, avian and amphibian KLK members which allowed us to trace the emergence of KLKs 330 mya. We suggest that a series of duplication and mutation events gave rise to the KLK gene family. The prominent feature of the KLK family is that it consists of tandemly and uninterruptedly arrayed genes in all species under investigation. The chromosomal co-localization in a single cluster distinguishes KLKs from trypsin and other trypsin-like proteases which are spread in different genetic loci. All the defining features of the KLKs were further found to be conserved in the novel KLK protein sequences. The study of this unique family will further assist in selecting new model organisms for functional studies of proteolytic pathways involving KLKs.     

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.     

Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression

Kallikrein-related peptidase 4 (KLK4) is one of the 15 members of the human KLK family and a trypsin-like, prostate cancer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasma membrane primarily by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. Here we show, using Ca(2+) flux assays, that KLK4 signals via both PAR-1 and PAR-2 but not via PAR-4. Dose-response analysis over the enzyme concentration range 0.1-1000 nM indicated that KLK4-induced Ca(2+) mobilization via PAR-1 is more potent than via PAR-2, whereas KLK4 displayed greater efficacy via the latter PAR. We confirmed the specificity of KLK4 signaling via PAR-2 using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressing and small interfering RNA-mediated receptor knockdown cell lines. Consistently, confocal microscopy analyses indicated that KLK4 initiates loss of PAR-2 from the cell surface and receptor internalization. Immunohistochemical analysis indicated the co-expression of agonist and PAR-2 in primary prostate cancer and bone metastases, suggesting that KLK4 signaling via this receptor will have pathological relevance. These data provide insight into KLK4-mediated cell signaling and suggest that signals induced by this enzyme via PARs may be important in prostate cancer.     

Kallikrein-related peptidases in prostate, breast, and ovarian cancers: from pathobiology to clinical relevance

Tissue kallikrein (KLK1) and kallikrein-related peptidases (KLK2-15) comprise a family of 15 highly conserved secreted serine proteases with similar structural characteristics and a wide spectrum of functional properties. Both gene expression and protein activity of KLKs are rigorously controlled at various levels via diverse mechanisms, including extensive steroid hormone regulation, to exert their broad physiological role. Nevertheless, deregulated expression, secretion, and function of KLK family members has been observed in several pathological conditions and, particularly, in endocrine-related human malignancies, including those of the prostate, breast, and ovary. The cancer-related abnormal activity of KLKs upon substrates such as growth factors, cell adhesion molecules, cell surface receptors, and extracellular matrix proteins facilitate both tumorigenesis and disease progression to the advanced stages. The well-documented relationship between KLK status and the clinical outcome of cancer patients has led to their identification as promising diagnostic, prognostic, and treatment response monitoring biomarkers for these complex disease entities. The main objective of this review is to summarize the existing knowledge concerning the role of KLKs in prostate, breast, and ovarian cancers and to highlight their continually evolving biomarker capabilities that can provide significant benefits for the management of cancer patients.     

Distinctly different gene structure of KLK4/KLK-L1/prostase/ARM1 compared with other members of the kallikrein family: intracellular localization, alternative cDNA forms, and Regulation by multiple hormones

The tissue kallikreins (KLKs) form a family of serine proteases that are involved in processing of polypeptide precursors and have important roles in a variety of physiologic and pathological processes. Common features of all tissue kallikrein genes identified to date in various species include a similar genomic organization of five exons, a conserved triad of amino acids for serine protease catalytic activity, and a signal peptide sequence encoded in the first exon. Here, we show that KLK4/KLK-L1/prostase/ARM1 (hereafter called KLK4) is the first significantly divergent member of the kallikrein family. The exon predicted to code for a signal peptide is absent in KLK4, which is likely to affect the function of the encoded protein. Green fluorescent protein (GFP)-tagged KLK4 has a distinct perinuclear localization, suggesting that its primary function is inside the cell, in contrast to the other tissue kallikreins characterized so far that have major extracellular functions. There are at least two differentially spliced, truncated variants of KLK4 that are either exclusively or predominantly localized to the nucleus when labeled with GFP. Furthermore, KLK4 expression is regulated by multiple hormones in prostate cancer cells and is deregulated in the androgen-independent phase of prostate cancer. These findings demonstrate that KLK4 is a unique member of the kallikrein family that may have a role in the progression of prostate cancer.     

Evolution of the rat kallikrein gene family: Gene conversion leads to functional diversity

Summary Kallikrein-like simple serine proteases are encoded by closely related members of a gene family in several mammalian species. Molecular cloning and genomic Southern blot analysis after conventional and pulsed-field gel electrophoresis indicate that the rat kallikrein gene family comprises 15–20 members, probably closely linked at a single locus. Determination of the nucleotide sequences of the rGK-3,-4, and-6 genes here completes sequence data for a total of nine rat kallikrein family members. Comparison of the rat gene sequences to each other and to those of human and mouse kallikrein family genes reveals patterns of relatedness indicative of concerted evolution. Analysis of nucleotide sequence variants in kallikrein family members shows that most sequence variants are shared by multiple family members; the patterns of shared variants are complex and indicate multiple short gene conversions between family members. Sequence exchanges between family members generate novel assortments of variants in amino acid coding regions that may affect substrate specificity and thereby contribute to the diversity of enzyme activity. Furthermore, small sequence exchanges also may play a role in generating the diverse patterns of tissue-specific expression of rat family members. These analyses indicate an important role for gene conversion in the evolution of the functional diversity of these duplicated genes.     

Evolution of kallikrein-related peptidases in mammals and identification of a genetic locus encoding potential regulatory inhibitors

The human kallikrein locus on chromosome 19 consists of 15 genes encoding serine proteases. Here we review studies on their evolution, which demonstrate that there are several taxon-unique KLK1 paralogs in mouse, rat and horse, but not in primates and many other mammals. Furthermore, the duplication yielding KLK2 and prostate-specific antigen (KLK3) appears to be specific to primates, but a functional progenitor to KLK2 is expressed in the dog prostate. The linkage to a locus of possible regulatory protease inhibitors on chromosome 20 is discussed, as is the potential role of the kallikrein locus in innate immunity.     

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.     

Dissection of the fusarium I2 gene cluster in tomato reveals six homologs and one active gene copy.

The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici. The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome. A yeast artificial chromosome (YAC) clone covering approximately 750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone. Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F. o. lycopersici race 2. These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes. At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes. However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes. Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region. They consisted of deletions or duplications of one or more leucine-rich repeats. We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.     

The role of kallikrein-related peptidases in prostate cancer: potential involvement in an epithelial to mesenchymal transition

Several members of the kallikrein-related peptidase family of serine proteases have proteolytic activities that may affect cancer progression; however, the in vivo significance of these activities remains uncertain. We have demonstrated that expression of PSA or KLK4, but not KLK2, in PC-3 prostate cancer cells changed the cellular morphology from epithelial to spindle-shaped, markedly reduced E-cadherin expression, increased vimentin expression and increased cellular migration. These changes are indicative of an epithelial to mesenchymal transition (EMT), a process important in embryonic development and cancer progression. The potential novel role of kallikrein-related peptidases in this process is the focus of this brief review.     

Content and organization of the human Ig VH locus: definition of three new VH families and linkage to the Ig CH locus.

We present a detailed analysis of the content and organization of the human immunoglobulin VH locus. Human VH genes representing five distinct families were isolated, including novel members belonging to two out of three of the known VH gene families (VH1 and VH3) as well as members of three new families (VH4, VH5, and VH6). We report the nucleotide sequence of 21 novel human VH genes, many of which belong to the three new VH gene families. In addition, we provide a preliminary analysis of the organization of these gene segments over the full extent of the locus. We find that the five multi-segment families (VH1-5) have members interspersed over nearly the full 1500-2000 kb of the VH locus, and estimate that the entire heavy chain locus covers 2500 kb or less. Finally, we provide the first report of the physical linkage of the variable and constant loci of a human Ig gene family by demonstrating that the most proximal known human VH segments lie within 100 kb of the constant region locus.