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Molecular Cloning of the Human Kallikrein 15 Gene (KLK15)

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 15gene). 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 ofKLK15 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.

tumor marker for prostate cancer diagnosis and monitoring, is a member of the human kallikrein gene family of serine proteases (19,20). In addition to PSA, human glandular kallikrein 2 (hK2, encoded by the KLK2 gene) has been proposed as an adjuvant diagnostic marker for prostate cancer (21,22). Moreover, accumulating evidence indicates that other members of the expanded kallikrein gene family may be associated with malignancy (7). The normal epithelial cell-specific 1 gene (NES1; KLK10, according to the approved human tissue kallikrein gene nomenclature) was found to be a novel tumor suppressor, which is down-regulated during breast cancer progression (23). Other gene family members, including zyme (KLK6), neuropsin (KLK8), and human stratum corneum chymotyrptic enzyme (HSCCE; KLK7) were also found to be differentially expressed in certain types of malignancies (24 -26). The diagnostic usefulness of PSA in prostate cancer led us to speculate that other, related molecules might be valuable biomarkers for prostate cancer and other malignancies.
During our efforts to identify new kallikrein genes that might be involved in malignancy, we cloned a new gene, tentatively named kallikrein 15 gene (KLK15). Here, we describe the identification of the new gene, its genomic and mRNA structure, its location in relation to other known kallikreins, and its tissue expression pattern. The KLK15 gene has another three alternatively spliced forms, expressed in many tissues. Our preliminary data suggest that KLK15 is up-regulated in prostate cancer and that it is under steroid hormone regulation in the LNCaP prostate cancer cell line. Higher expression of KLK15 is associated with more aggressive (higher stage and higher grade) prostate tumors.

MATERIALS AND METHODS
Identification of the New Gene-We previously constructed the first contiguous map for the human kallikrein gene locus extending from the KLK1 gene (centromere) to the KLK14 gene (telomere) (7,8,11,12,27). Overlapping bacterial artificial chromosome (BAC) clones spanning this area were identified by screening of a human BAC library using different radiolabeled gene-specific probes. An area of ϳ300 kb of genomic sequence was established using different techniques, as described previously (11,27). By performing an EcoRI restriction analysis, we were able to orient the kallikrein locus along the EcoRI restriction map of chromosome 19q13 available from the Lawrence Livermore National Laboratory (LLNL). A BAC clone that extends more centromerically (BC 781134) was then identified. Contigs of linear genomic sequences from this clone are available from the LLNL. Initially, we have used these contig sequences to predict the presence of novel genes, using * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF242195.
Expressed Sequence Tag Searching-The predicted exons of the putative new gene were subjected to homology search using the BLASTN algorithm (28) from the National Center for Biotechnology Information web server against the human EST data base (dbEST). Clones with Ͼ95% homology were obtained from the IMAGE consortium (29) through Research Genetics Inc. (Huntsville, AL). The clones were propagated, purified as described elsewhere (30), and sequenced from both directions with an automated sequencer, using insert-flanking vector primers.
Prostate Cancer Cell Line and Hormonal Stimulation Experiments-The LNCaP prostate cancer cell line was purchased from the American Type Culture Collection (ATCC), Manassas, VA. Cells were cultured in RPMI media (Life Technologies, Inc., Gaithersburg, MD) supplemented with glutamine (200 mM), bovine insulin (10 mg/liter), fetal bovine serum (10%), antibiotics, and antimycotics, in plastic flasks, to near confluency. The cells were then aliquoted into 24-well tissue culture plates and cultured to 50% confluency. 24 h before the experiments, the culture media were changed into phenol red-free media containing 10% charcoal-stripped fetal bovine serum. For stimulation experiments, various steroid hormones dissolved in 100% ethanol were added into the culture media at a final concentration of 10 Ϫ8 M. Cells stimulated with 100% ethanol were included as controls. The cells were cultured for 24 h, then harvested for mRNA extraction.
RT-PCR for the KLK15 Gene-Total RNA was extracted from the LNCaP cell line or from prostate tissues using Trizol reagent (Life Technologies, Inc.) following the manufacturer's instructions. RNA concentration was determined spectrophotometrically. 2 g of total RNA was reverse-transcribed into first-strand cDNA using the Superscript preamplification system (Life Technologies, Inc.). The final volume was 20 l. Based on the combined information obtained from the predicted genomic structure of the new gene and the EST sequences (see below), two gene-specific primers were designed (KLK15-F1 and KLK15-R1) ( Table I), and PCR was carried out in a reaction mixture containing 1 l of cDNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 200 M dNTPs (deoxynucleoside triphosphates), 150 ng of primers, and 2.5 units of HotStar DNA polymerase (Qiagen Inc., Valencia, CA) on a PerkinElmer Life Sciences 9600 thermal cycler. The cycling conditions were 95°C for 15 min to activate the Taq DNA polymerase, followed by 35 cycles of 94°C for 30 s, 64°C for 30 s, 72°C for 1 min, and a final extension step at 72°C for 10 min. Equal amounts of PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. All primers for RT-PCR spanned at least two exons to avoid contamination by genomic DNA. To verify the identity of the PCR products, they were cloned into the pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The inserts were sequenced from both directions using vector-specific primers, with an automated DNA sequencer.
Tissue Expression-Total RNA isolated from 26 different human tissues was purchased from CLONTECH (Palo Alto, CA). We prepared cDNA as described above for the tissue culture experiments and used it for PCR reactions. Tissue cDNAs were amplified at various dilutions using two gene-specific primers (KLK15-F2 and KLK15-R1) ( Table I). Due to the high degree of homology between kallikreins, and to exclude nonspecific amplification, PCR products were cloned and sequenced.
Prostate Cancer Tissues-Prostate tissue samples were obtained from 29 patients who had undergone radical retropubic prostatectomy for prostatic adenocarcinoma at the Charite University Hospital, Berlin, Germany. The patients did not receive any hormonal therapy before surgery. The use of these tissues for research purposes was approved by the Ethics Committee of the Charite Hospital. Fresh prostate tissue samples were obtained from the cancerous and non-cancerous parts of the same prostates that had been removed. Small pieces of tissue were dissected immediately after removal of the prostate and stored in liquid nitrogen until analysis. Histological analysis from all the tissue pieces was performed as described previously (31) to ensure that the tissue was either malignant or benign. The tissues were pulverized with a hammer under liquid nitrogen, and RNA was extracted as described above, using Trizol reagent.
Statistical Analysis-Statistical analysis was performed with SAS software (SAS Institute, Cary, NC). The analysis of differences between KLK15 expression in non-cancerous versus cancerous tissues from the same patient was performed with the non-parametric McNemar test. The binomial distribution was used to compute the significance level. Prostate tumor KLK15 mRNA levels were qualitatively classified into two categories (KLK15-low and KLK15-high groups), and associations between KLK15 status and other variables were analyzed using the Fisher's exact test.
Structure Analysis-Multiple alignment was performed using the ClustalX software package and the multiple alignment program available from the Baylor College of Medicine (Houston, TX). Phylogenetic studies were performed using the Phylip software package. Distance matrix analysis was performed using the Neighbor-Joining/UPGMA program, and parsimony analysis was done using the Protpars program. Hydrophobicity study was performed using the Baylor College of Medicine search launcher. Signal peptide was predicted using the Sig-nalP server. Protein structure analysis was performed by the SAPS (structural analysis of protein sequence) program.

RESULTS
Cloning of the KLK15 Gene-A contiguous map for the human kallikrein gene locus extending from the KLK1 gene (centromere) to the KLK14 gene (telomere) was previously established (7,8,11,12,27). To investigate the presence of other kallikrein-like genes centromeric to KLK1, a BAC clone (BC 781134) was obtained as described under "Materials and Methods." According to the published genomic sequence of prostate specific antigen (PSA) and human renal kallikrein (KLK1) genes, we designed gene-specific primers for each of these genes (Table I) and developed polymerase chain reaction (PCR)-based amplification protocols, which allowed us to generate specific PCR products with genomic DNA as a template. PCR screening of the BAC clone by these gene-specific primers indicated that this clone is positive for KLK1 but negative to PSA, thus, confirming its location to be centromeric to PSA.
A putative new serine protease was predicted from the sequence of this clone by computer programs as described previously (12). This clone was digested, blotted on a membrane, and hybridized with gene-specific primers for the putative KLK15 gene (according to the predicted sequence), and positive fragments were subcloned and sequenced to verify the structure of the putative gene. This putative gene sequence was then compared using the BLASTN program against the human EST data base, and two EST clones were identified (GenBank accession numbers AW274270 and AW205420). These two clones were 99% identical to the last exon and the 3Ј-untrans- lated region of the gene, and the second EST ends with a stretch of 17 adenine (A) nucleotides that were not found in the genomic sequence, thus verifying the 3Ј-end of the gene and the position of the poly(A) tail.
To identify the full mRNA structure of the gene and to determine the exon/intron boundaries, PCR reactions were performed using primers located in different computer-predicted exons, using a panel of 26 human tissue cDNAs as templates. PCR products were sequenced. Two of these primers (KLK15-F1 and KLK15-R1) ( Table I) were able to amplify the full coding region of the gene from different tissues. Comparing the mRNA with the genomic structure indicated the presence of a gene formed from five coding exons with four intervening introns. Translation of the mRNA sequence in all possible reading frames revealed the presence of only one frame that gives an uninterrupted polypeptide chain, which also contains the highly conserved structural motifs of the kallikreins, as discussed below.
Structural Characterization of the KLK15 Gene-As shown in Fig. 1, the KLK15 gene is formed of five coding exons and four intervening introns, although, as with other kallikrein genes, the presence of further upstream untranslated exon(s) could not be ruled out (17,32,33). All of the exon/intron splice sites conform to the consensus sequence for eukaryotic splice sites (34). The gene further follows strictly the common structural features of other members of the human kallikrein multigene family, as described below. The predicted protein-coding region of the gene is formed of 771 bp, encoding a deduced 256-amino acid polypeptide with a predicted molecular mass of 28.1 kDa. The potential translation initiation codon matches the consensus Kozak sequence (35), moreover, there is a purine at position Ϫ3, which occurs in 97% of vertebrate mRNAs (36). It should also be noted that, like most other kallikrein-like genes, KLK15 does not have the consensus G nucleotide at position ϩ4.
Nucleotides 7764 -7769 (ATTAAA) (the numbers refer to our GenBank submission number AF242195) closely resemble a consensus polyadenylation signal (37) and are followed, after 17 nucleotides, by the poly(A) tail. No other potential polyadenylation signals were discernible in the 3Ј-untranslated region, suggesting that the above sequence is the actual polyadenylation signal. Although AATAAA is highly conserved, natural variants do occur, and the ATTAAA sequence is reported to occur as a natural polyadenylation variant in 12% of vertebrate mRNA sequences (38). The presence of glutamic acid (E) at position 203 suggests that KLK15 will likely possess a unique substrate specificity. PSA has a serine (S) residue in the corresponding position and has chymotryptic-like activity. Many other kallikreins usually have aspartate (D) in this position, indicating a trypsin-like activity (Fig. 2) (6).
Although the KLK15 protein sequence is unique, comparative analysis revealed that it has a considerable degree of homology with other members of the kallikrein multigene family. KLK15 shows 51% protein identity and 66% similarity with the trypsin-like serine protease (TLSP) and 49% and 48% identity with the neuropsin and KLK-L3 proteins, respectively. Hydrophobicity analysis revealed that the amino-terminal region is quite hydrophobic (Fig. 3), consistent with the possibility that this region harbors a signal sequence, analogous to other serine proteases. Computer analysis of the KLK15 protein sequence predicted a cleavage site between amino acids 16 and 17 (TAA-QD). Sequence alignment (Fig. 2) also revealed another potential cleavage site (Lys 21 ), at a site homologous to the activation site of other serine proteases (lysine (K) or arginine (R) is present in most cases) (39). Several evenly distributed hydrophobic regions throughout the KLK15 polypeptide are consistent with a globular protein, similar to other kallikreins and serine proteases. Thus, as is the case with other kallikreins, and although direct experimental evidence is lack-ing, KLK15 is presumably translated as an inactive 256-amino acid preproenzyme precursor. Prepro-KLK15 has 21 additional residues, which constitute the preregion (the signal peptide formed of 16 residues), and the propeptide (5 residues).
The dotted area in Fig. 2 indicates an 11-amino acid loop characteristic of the classical kallikreins (PSA, KLK1, and KLK2) but not found in KLK15 or other members of the kallikrein multigene family (10,11,13,14). However, KLK15 has a unique 8-amino acid loop (HNEPGTAG) at positions 148 -155, not found in any other kallikrein (Fig. 2). 29 "invariant" amino acids surrounding the active site of serine proteases have been described (40). Of these, 28 are conserved in KLK15. One of the unconserved amino acids (Ser 173 instead of Pro) is also found in prostase, KLK-L2, and KLK-L5 proteins, and   represents a conserved evolutionary change to a protein of the same group, according to protein evolution studies (41). Twelve cysteine residues are present in the putative mature KLK15 protein; 10 of them are conserved in all kallikreins and would be expected to form disulfide bridges. The other two (Cys 131 and Cys 243 ) are not found in PSA, KLK1, KLK2, or KLK-L4, however, they are found in similar positions in all other kallikrein genes and are expected to form an additional disulfide bond.
To predict the phylogenetic relatedness of the KLK15 protein with other serine proteases, the amino acid sequences were aligned together using the Unweighted Pair Group Method with Arithmetic mean (UPGMA) and the Neighbor-Joining distance matrix methods, and the Protpars parsimony method. All phylogenetic trees obtained agreed that other serine proteases (non-kallikreins) can be grouped together as a separate group, indicating that kallikreins represent a separate step in the evolution of serine proteases. KLK15 was grouped with the KLK-L3 and TLSP (Fig. 4) and the classical kallikreins (hK1, hK2, and PSA) are grouped together in all trees, suggesting that the separation between classical kallikreins and the kallikrein-like genes occurred early during evolution, consistent with suggestions of previous studies (13).
Splice Variants of the KLK15 Gene-PCR screening for KLK15 transcripts using gene-specific primers (KLK15-F2 and KLK15-R2) ( Table I) revealed the presence of three bands in most of the tissue cDNAs examined (Fig. 7). These bands were gel-purified, cloned, and sequenced. The upper band represents the classical form of the gene, and the lower band is splice variant 3 (Fig. 5). The middle band represents other two-splice variants. Restriction digestion of the PCR product of the middle band with StuI, followed by gel separation, purification, and sequencing, revealed that it is composed of splice variants 1 and 2, which have approximately the same length (splice variant 1 has exon 4 (137 bp) but is missing 118 bp from exon 3, whereas splice variant 2 has an additional 118 bp of exon 3 but missing exon 4. All splice variants are expected to encode for truncated protein products (Fig. 5).
Chromosomal Localization of the KLK15 Gene-Restriction analysis study of a number of overlapping BAC clones spanning the human kallikrein locus followed by comparison with the EcoRI restriction map of the area (available from the LLNL web site) enabled us to identify a BAC clone (BC 25479) that is telomerically adjacent to BC 781134 (which harbors the KLK15 gene). A BLASTN search of the sequences of the two clones showed that the ends of these clones are overlapping. By identifying the position of the KLK1, KLK3, and KLK15 genes along these clones, we were able to precisely define the relative location and the direction of transcription of these three genes. KLK1 is the most centromeric, and its direction of transcription is from telomere to centromere, followed by KLK15, which is more telomeric and transcribes in the same direction. The distance between the two genes is a length of 1501 bp. The KLK3 gene is more telomeric, located at a distance of 23,335 bp from the position of KLK15, and is transcribed in the opposite direction (Fig. 6). These results are consistent with previous reports where the distance between KLK3 and KLK1 was roughly estimated to be ϳ31 kb (6,27). Fig. 7, the KLK15 gene is expressed at highest levels in the thyroid gland. Lower levels of expression are also seen in the prostate, salivary, and adrenal glands and in the colon, testis, and kidney. To verify the RT-PCR specificity, representative PCR products were cloned and sequenced. Fig. 8 shows that the KLK15 gene is up-regulated by steroid hormones in the human LNCaP prostate cancer cell line.

Tissue Expression and Hormonal Regulation of the KLK15 Gene-As shown in
KLK15 Expression in Prostate Cancer-The expression of the KLK15 gene in normal and cancerous prostatic tissues was examined by RT-PCR. Actin was included as a control gene to ensure the quality and amount of the cDNA used. To examine the relative expression of the KLK15 gene in normal compared with malignant tissues, we analyzed 29 pairs of prostatic tissues. Each pair represented normal and cancerous tissue obtained from the same patient. The intensity of the PCR band was compared between each pair of normal/cancerous tissue to establish if the expression was higher or lower in cancer, compared with normal tissue. The results are summarized in Table  II. Thirteen out of 29 patients had significantly higher KLK15 expression in the cancer tissue and only three had the expression of KLK15 higher in non-cancer than to cancer tissues. Analysis by the McNemar test indicated that the differences between normal and cancerous tissues are statistically significant (p ϭ 0.021). Because of the small number of cases, the binomial distribution was used to compute the significance level. We have further qualitatively classified the prostate cancer patients in two groups: (a) KLK15 expression-positive (n ϭ 21) and (b) KLK15 expression-negative (or very low) (n ϭ 8).
When we compared the association of KLK15 expression with clinicopathological prognostic variables, we found that higher KLK15 expression was more frequent in patients with late stage disease and tumors of higher grade (Table III). DISCUSSION Kallikreins are a subgroup of serine proteases. The term "kallikrein" is usually utilized to describe an enzyme that acts upon a precursor molecule (kininogen) for release of a bioactive   peptide (kinin) (3,42). However, the generic term "tissue kallikrein" is not restricted to the functional definition of the enzyme. This term is now used to describe a group of enzymes with a highly conserved gene and protein structure, which also co-localizes in the same chromosomal locus. Among the three classical human kallikrein genes, only KLK1 encodes for a protein with potent kininogenase activity. The enzymes encoded by KLK2 and KLK3 genes have very weak kininogenase activity. The already cloned 14 members of the human kallikrein gene family have a number of similarities (7, 11) as follows: 1) All genes localize to the same chromosomal region (proximal19q13.4). 2) All genes encode for putative serine proteases with a conserved catalytic triad in the appropriate positions, i.e. histidine near the end of the second coding exon, aspartic acid in the middle of the third exon, and serine at the beginning of the fifth (last) exon. 3) All genes have five coding exons (some members contain one or more 5Ј-untranslated exons). 4) Coding exon sizes are similar or identical. 5) Intron phases are fully conserved. 6) All genes have significant sequence homologies at the DNA and amino acid levels (30 -80%). 7) Many of these genes are regulated by steroid hormones.
Figs. 2 and 9 show that the newly identified KLK15 gene shares all the above similarities and is thus suggested to be a new member of the human kallikrein multigene family. We named this gene KLK15 based on the recommendations for the new kallikrein gene nomenclature, as approved by the Human Gene Nomenclature Committee and available on the Web (43).
Many kallikrein genes are related to the pathogenesis of human diseases, depending on the tissue of their primary ex-pression. The KLK1 gene is involved in many disease processes, including inflammation (3), hypertension (44), renal nephritis and diabetic renal disease (45,46), The connections of HSCCE (KLK7) with skin diseases, including pathological keratinization and psoriasis, have already been reported (47,48). Little et al. suggested that zyme (KLK6) is amyloidogenic and plays a role in the development of Alzheimer's disease (14). There are other reports describing connection of neuropsin (KLK8) expression with diseases of the central nervous system, including epilepsy (49,50). Being primarily expressed in the thyroid, KLK15 may play an important role in the normal physiology and pathophysiology of this gland. Among all other discovered kallikreins, many are expressed in the thyroid but none at the highest levels in this tissue (7,11).
Our preliminary findings indicate that the KLK15 gene is up-regulated, at the mRNA level, in a subset of prostate cancers. The distributions of KLK15 qualitative expression status (high or low) between subgroups of patients differing by disease stage, tumor grade, and Gleason score indicated that high KLK15 expression was found more frequently in grade 3 tumors as well as in stage III and Gleason score Ͼ6 patients. These findings suggest that overexpression of KLK15 is associated with more aggressive forms of the disease and may be an indicator of poor prognosis. These findings require verification, because our patient population was relatively small (Table III).
There is now growing evidence that many kallikreins and kallikrein-like genes are related to malignancy. PSA is the best marker for prostate cancer so far (20). Recent reports suggest that hK2 (encoded by the KLK2 gene) could be another useful diagnostic marker for prostate cancer (21,51). NES1 (KLK10) H denotes histidine, D aspartic acid, and S serine. Roman numerals indicate intron phases. The intron phase refers to the location of the intron within the codon; I denotes that the intron occurs after the first nucleotide of the codon, II the intron occurs after the second nucleotide, and 0 the intron occurs between codons. The intron phases are conserved in all genes. Numbers inside boxes indicate exon lengths in base pairs. Names inside brackets represent the official nomenclature approved by the Human Gene Nomenclature Committee. Untranslated 3Ј-and 5Ј-regions and 5Ј-untranslated exons are not shown. appears to be a novel tumor suppressor gene (23). The zyme (KLK6) gene was shown to be differentially expressed in primary breast and ovarian tumors (24), and the human stratum corneum chymotryptic enzyme (HSCCE, KLK7) has been shown to be expressed at abnormally high levels in ovarian cancer (25). Another recently identified kallikrein-like gene, tentatively named the tumor-associated differentially expressed gene-14 (TADG-14/neuropsin) (KLK8) was found to be overexpressed in about 60% of ovarian cancer tissues (26). prostase/KLK-L1/(KLK4), another newly discovered kallikreinlike gene, is speculated to be linked to prostate cancer (13). Two newly discovered kallikreins, KLK-L4 (KLK13) and KLK-L5 (KLK12), were also found to be down-regulated in breast cancer (10). Thus, extensive new literature suggests multiple connections of various kallikrein genes to many forms of human cancer.
The existence of multiple alternatively spliced mRNA forms is frequent among the kallikreins. Distinct RNA species are transcribed from the PSA gene, in addition to the major 1.6-kb transcript (19,52,53). Also, Reigman et al. (54) reported the identification of two alternatively spliced forms of the human glandular kallikrein 2 (KLK2) gene. A novel transcript of the tissue kallikrein gene (KLK1) was also isolated from the colon (55). Neuropsin, a recently identified kallikrein-like gene, was found to have two alternatively spliced forms, in addition to the major form (26,56). KLK-L4 was also found to have different alternatively spliced forms (10). Because the splice variants of KLK15 have an identical 5Ј-sequence required for translation, secretion, and activation, it is possible to assume that they encode for a secreted protein (53). These proposals need experimental verification, and the role, if any, of these isoforms in tissue physiology and pathophysiology are worth examining in the future.
In conclusion, we characterized a new member of the human kallikrein gene family, KLK15, which maps to the human kallikrein locus (chromosome 19q13.4). This gene has three related splice forms in addition to the classical form. KLK15 is expressed in a variety of tissues but predominantly in the thyroid, it appears to be up-regulated in more aggressive forms of prostate cancer, and its expression is influenced by steroid hormones. Because a few other kallikreins are already used as valuable tumor markers, we speculate that KLK15 may also find similar clinical applications. This possibility, as well as the physiological function of this protein in various tissues need further investigation.