The human kallikrein-related peptidase 4 (KLK4) and the transcribed pseudogene KLKP1 are reported to be highly expressed in the prostate. When trying to clone transcripts of KLKP1, we partly failed. Instead, we identified an androgen-regulated transcript, KLK4T2, which appeared to be a splice variant of KLK4 that also contained exons of KLKP1. Expression analysis of KLK4, KLK4T2, and KLKP1 transcripts in prostate cancer cell lines showed high levels of KLKP1 transcripts in the nucleus and in unfractionated cell extract, whereas it was almost completely absent in the cytoplasmatic fraction. This was in contrast to KLK4 and KLK4T2, which displayed high to moderate levels in the cytoplasm. In patient cohorts we found significantly higher expression of both KLK4T2 and KLK4 in benign prostatic hyperplasia compared to both primary prostate cancer and bone metastasis. Analysis of tissue panels demonstrated the highest expression of KLK4T2 in the prostate, but in contrast to the classical KLK4, relatively high levels were also found in placenta. So far, the function of KLK4T2 is still to be explored, but the structure of the translation product indicated that it generates a 17.4 kDa intracellular protein with possible regulatory function.

The seminal vesicle secretions of guinea pig and rabbit were analyzed for semen coagulum proteins. Using SDS-PAGE we discovered a previously not fully recognized semen coagulum protein, Svp5, in the guinea pig and a single predominant component, SVP200, in the rabbit. Potential genes of these proteins were identified in genome databases by their homology with human and murine genes. The structure of their fullength transcripts was determined using seminal vesicle cDNA and sequencing primers based on genomic sequences. Homology searching indicated that both Svp5 and SVP200 were synthesized from composite genes that were the result of merger between two genes showing homology with human SEMG2 and PI3. For a deeper understanding of the evolution of the genes, we retrieved and analyzed genome sequences from the REST gene loci, encompassing genes of semen coagulum proteins and related rapidly evolving seminal vesicle-transcribed genes, of 14 rodents and 2 lagomorphs. The analysis showed that rodents of the suborders myomorpha, hystricomorpha, and castorimorpha had unique sets of REST genes, whereas sciuromorpha seemed to be lacking such genes. It also indicated a closer relationship between myomorpha and castorimorpha than to rodents of the two other analyzed suborders. In the lagomorph species, the pika appeared to be devoid of REST genes, whereas the rabbit had a single expressed REST gene, SVP200, and two pseudogenes. The structural similarity of semen coagulum proteins in rabbit and hystricomph species suggests that they are closely related. This was also supported by other similarities at their REST gene loci, e.g. the finding of a PI3-like gene in the rabbit that also had features in common with caltrin2 of hystricomorph rodents. The homologies indicate that hystricomorpha may have separated from myomorpha and castorimorpha before the separation of hystricomorpha from lagomorpha.

Ghiso, J. et al. “Amyloid Fibrils...' Proc. Natl. Acad. Sci. U.S.A., (1986) pp. 2974-2978. Tonelle, C. et al. "Partial Amino Acid . . . ' Biochem. & BioPHys. Research Communications (1979) pp. 613-619. Barett "The Cystatins:a new Class of peptidase . . . ' TIBS, (May 1987) pp. 193-196. Barrett, A.J. et al. Biochem. J. 201:189-198 (1982). Ghiso, J. et al. Proc. Natl. Acad. Sci. USA 83:2974-2978 (May 1986). Rijnders, A. W. M. et al. Biochemical and Biophysical Research Communications 132(2):548-554 (Oct. 1985). Lindahl, P. et al. Biochemistry 27:5074-5082 (1988). Barrett, A.J. et al. Methods in Enzymology 80:535-561 (1981). Abrahamson, et al. FEB 06165, 236(1):14-18, Aug. 1988. Gounaris, A. et al. Biochem J. 1984, 221, 445-452. Lofberg, H. et al. Scand, J. Clin. Lab. Invest. 39:619-626 (1979). Lofberg, H. et al. Biomedical Research 203):298–306 (1981). Primary Examiner-Garnette D. Draper Assistant Examiner-Sally P. Teng Attorney, Agent, or Firm-Ladas & Parry

Background: Factors affecting serum prostate-specific antigen (PSA) levels in men are clinically important, but apart from effects mediated through the androgen receptor, they are poorly understood.Objective: To investigate whether microRNA (miRNA) affects the synthesis and serum levels of PSA.Design, setting, and participants: Reporter assays with PSA and KLK2 3' untranslated regions (UTRs) to confirm posttranscriptional regulation was followed by high-throughput screening of the effect of 1129 miRNAs on PSA levels using reverse phase protein arrays (RPPAs) to identify individual regulatory miRNAs. The candidate miRNAs were investigated further in vitro by Western blot, immunofluorometrics, activity assays, quantitative reverse transcriptase polymerase chain reaction, reporter assays, and growth assays. Prostate levels of miR-183 were compared with PSA transcript and serum PSA levels in prostate cancer cohorts.Outcome measurements and statistical analysis: RankProd was used to evaluate the RPPAs, and the Student t test was used for the in vitro experiments. The Spearman and Cuzick tests were used in the patient material, and overall survival was analysed by Kaplan-Meier and log-rank analysis.Results and limitations: Gain-of-function screenings identified 32 miRNAs that increase PSA levels. One of these, miR-183, was found to bind the 3' UTR of PSA directly and increase both protein and messenger RNA levels. Prostatic levels of miR-183 and serum PSA showed correlation in a cohort of 74 men. In addition, miR-183 promotes cellular growth in vitro and correlates to clinical parameters such as World Health Organisation grade and clinical progression.Conclusions: The synthesis and serum levels of PSA are directly affected by miR-183 and may be a factor to consider when PSA values are evaluated in clinical settings.Patient summary: These findings offer novel insights into the regulation of prostate-specific antigen and may eventually affect clinical decision making in prostate cancer. (C) 2014 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Summary The human kallikrein locus consists of KLK1, the gene of major tissue kallikrein, and 14 genes of kallikrein-related peptidases (KLKs) located in tandem on chromosome 19q13.3-13.4. In this review, based on information retrieved from the literature or extracted from genome databases, it is hypothesised that the kallikrein locus is unique to mammals. The majority of genes are highly conserved, as demonstrated by the identification of 11 KLK genes in the opossum, a metatherian species. In contrast, a sublocus, encompassing KLK1-4, has gone through major transformations that have generated new genes, which in most cases are closely related to KLK1. In the primate lineage, this process created KLK3, the gene of the prostate cancer marker, prostate-specific antigen (PSA), whereas in the murine lineage it gave rise to 13 genes unique to the mouse and nine unique to the rat. The KLK proteases are effector molecules that emerged early in mammalian evolution and their importance in skin homeostasis and male reproductive function is undisputed and there are also accumulating evidence for a role of KLK proteases in the development of the brain. It is speculated that the KLK gene family arose as part of the process that generated distinguishing mammalian features, like skin with hair and sweat glands, and specialised anatomical attributes of the brain and the reproductive tract.

BACKGROUND:β-Microseminoprotein (MSMB) is an abundant protein in seminal plasma. Most of it is present as a free protein but a small part is bound to cysteine-rich secretory protein 3 (CRISP3) as a non-covalent complex. Even though their physiological function is unknown, both MSMB and CRISP3 have been ascribed roles in prostate carcinogenesis. Thus, several recent experimental studies indicate a tumor-suppressor role for MSMB. The present study was undertaken in order to evaluate, for the first time, the expression of MSMB and CRISP3 in ovaries and in ovarian tumors and to determine if their expression might indicate a role in ovarian tumor development.MATERIALS AND METHODS:Biopsies from prospectively collected samples from ovaries and benign, borderline and invasive ovarian tumors were analyzed for expression of MSMB and CRISP3 by immunohistochemistry. In patients with ovarian cancer the expression was compared to survival.RESULTS:Both MSMB and CRISP3 were strongly stained in ovarian epithelial cells and weakly stained in the stroma. In ovarian blood vessels, CRISP3 exhibited strong to medium staining, while MSMB was only weakly expressed. In benign and borderline tumors the staining pattern was similar to the one observed in the ovaries. In invasive neoplasms, the expression of MSMB in the tumor cells was significantly reduced. In univariate analysis, decreased expression of MSMB correlated to reduced survival. No correlation was found with stage, the strongest prognostic indicator for ovarian cancer, which supports an independent role of MSMB in ovarian carcinogenesis. For CRISP3, a staining pattern comparable to that for MSMB was observed in all groups, except the fact that decreased expression was not observed in invasive tumor cells.CONCLUSION:MSMB and CRISP3 were widely distributed in ovaries and in ovarian tumors; the expression of MSMB fits well with a tumor-suppressor function in ovarian carcinogenesis.

We have previously demonstrated that the genes of SCPs (semen coagulum proteins) and the WFDC (whey acidic protein four-disulfide core)-type protease inhibitor elafin are homologous in spite of lacking similarity between their protein products. This led to the discovery of a locus on human chromosome 20, encompassing genes of the SCPs, SEMG1 (semenogelin I) and SEMG2, and 14 genes containing the sequence motif that is characteristic of WFDC-type protease inhibitors. We have now identified additional genes at the locus that are similarly organized, but which give rise to proteins containing the motif of Kunitz-type protease inhibitors. Here, we discuss the evolution of genes encoding SCPs and describe mechanisms by which they and genes with Kunitz motifs might have evolved from genes with WFDC motifs. We can also demonstrate an expansion of the WFDC locus with 0.6 Mb in the cow. The region, which seems to be specific to ruminants, contains several genes and pseudogenes with Kunitz motifs, one of which is the much-studied BPTI (bovine pancreatic trypsin inhibitor).

BACKGROUND:We have previously identified a locus on human chromosome 20q13.1, encompassing related genes of postulated WFDC-type protease inhibitors and semen coagulum proteins. Three of the genes with WFDC motif also coded for the Kunitz-type protease inhibitor motif. In this report, we have reinvestigated the locus for homologous genes encoding Kunitz motif only. The identified genes have been analyzed with respect to structure, expression and function.RESULTS:We identified three novel genes; SPINT3, SPINT4 and SPINT5, and the structure of their transcripts were determined by sequencing of DNA generated by rapid amplification of cDNA ends. Each gene encodes a Kunitz domain preceded by a typical signal peptide sequence, which indicates that the proteins of 7.6, 8.7, and 9.7 kDa are secreted. Analysis of transcripts in 26 tissues showed that the genes predominantly are expressed in the epididymis. The recombinantly produced proteins could not inhibit the amidolytic activity of trypsin, chymotrypsin, plasmin, thrombin, coagulation factor Xa, elastase, urokinase and prostate specific antigen, whereas similarly made bovine pancreatic trypsin inhibitor (BPTI) had the same bioactivity as the protein isolated from bovine pancreas.CONCLUSIONS:The similar organization, chromosomal location and site of expression, suggests that the novel genes are homologous with the genes of WFDC-type protease inhibitors and semen coagulum proteins, despite the lack of similarity in primary structure of their protein products. Their restricted expression to the epididymis suggests that they could be important for male reproduction. The recombinantly produced proteins are presumably bioactive, as demonstrated with similarly made BPTI, but may have a narrower spectrum of inhibition, as indicated by the lacking activity against eight proteases with differing specificity. Another possibility is that they have lost the protease inhibiting properties, which is typical of Kunitz domains, in favor of hitherto unknown functions.

BACKGROUND:Beta-microseminoprotein, an abundant component in prostatic fluid, is encoded by the potential tumor suppressor gene MSMB. Some New World monkeys carry several copies of this gene, in contrast to most mammals, including humans, which have one only. Here we have investigated the background for the species difference by analyzing the chromosomal organization and expression of MSMB in the common marmoset (Callithrix jacchus).METHODS:Genes were identified in the Callithrix jacchus genome database using bioinformatics and transcripts were analyzed by RT-PCR and quantified by real time PCR in the presence of SYBR green.RESULTS:The common marmoset has five MSMB: one processed pseudogene and four functional genes. The latter encompass homologous genomic regions of 32-35 kb, containing the genes of 12-14 kb and conserved upstream and downstream regions of 14-19 kb and 3-4 kb. One gene, MSMB1, occupies the same position on the chromosome as the single human gene. On the same chromosome, but several Mb away, is another MSMB locus situated with MSMB2, MSMB3 and MSMB4 arranged in tandem. Measurements of transcripts demonstrated that all functional genes are expressed in the male genital tract, generating very high transcript levels in the prostate. The transcript levels in seminal vesicles and testis are two and four orders of magnitude lower. A single gene, MSMB3, accounts for more than 90% of MSMB transcripts in both the prostate and the seminal vesicles, whereas in the testis around half of the transcripts originate from MSMB2. These genes display rapid evolution with a skewed distribution of mutated nucleotides; in MSMB2 they affect nucleotides encoding the N-terminal Greek key domain, whereas in MSMB3 it is the C-terminal MSMB-unique domain that is affected.CONCLUSION:Callitrichide monkeys have four functional MSMB that are all expressed in the male genital tract, but the product from one gene, MSMB3, will predominate in seminal plasma. This gene and MSMB2, the predominating testicular gene, have accumulated mutations that affect different parts of the translation products, suggesting an ongoing molecular specialization that presumably yields functional differences in accessory sex glands and testis.

Beta-microseminoprotein (MSP) is one of the most abundant proteins secreted by the prostate gland. Because MSP is also synthesized in nonreproductive organs, the establishment of a solid relationship between the levels of MSP in serum and semen is crucial for future studies connecting MSP with aging or diseases of the prostate gland. We developed a specific, competitive, europium-based immunoassay to measure MSP in serum and seminal plasma. We also produced recombinant MSP in insect cells using baculo virus and purified it to homogeneity by a novel approach with ethanol extraction and gel filtration. The median values of MSP in 205 young men were 12 microg/L (2.5-97.5 percentile, 4.9-26 microg/L) in serum and 0.53 g/L (2.5-97.5 percentile, 0.13-2.0 g/L) or 1.8 mg (2.5-97.5 percentile, 0.32-6.6 mg) in seminal plasma. MSP in serum showed significant correlation to MSP in seminal plasma (r = .50, P < .001). Significant correlations were also found in seminal plasma between MSP and prostate-specific antigen (PSA) (r = .65, P < .001) and between MSP and Zn(2+) (r = .54, P < .001). The yield of recombinant MSP in culture medium was 35 mg/L or higher, and recovery following ethanol extraction was 80%-90%. MSP in serum reflects the prostate secretion of MSP, and correlations were also found in seminal plasma between MSP and PSA and Zn(2+). This suggests that MSP in serum can be used as a marker of prostate secretion, despite the contribution from extra prostatic tissues.

beta-Microseminoprotein (MSP) is one of the most abundant proteins in human seminal plasma and is secreted from the prostate gland. Its evolution can be traced from primates down to nonvertebrate species such as amphioxus, despite substantial differences in the primary structure. Most mammals are known to have one single MSP gene, but we have previously shown that the cotton-top tamarin and the common marmoset-two New World monkeys-carry several MSP genes. In this study we continue our characterization of MSP genes in the cotton-top tamarin by presenting the full nucleotide sequence of the three previously identified genes, mspA, mspE, and mspJ. A promoter analysis using the luciferase reporter showed that mspE is as transcriptionally active as the single human MSP gene, whereas mspA and mspJ display no activity with this assay. Two novel MSP genes were also identified, mspB and mspH, both of which are pseudogenes. MspB has a frameshift mutation in the third exon resulting in a new C-terminus and premature stop of translation. MspH has the features of a processed pseudogene, originating from a transcript of mspE. It is integrated into the genome together with another processed pseudogene originating from a transcript of the nucleoporin gene NUP88. The MSP genes described in this study probably arose by phylogenetically rather late duplication or retrotransposition, suggesting that they are confined to a limited number of New World monkeys.

Kallikrein 1 (KLK1), a key component of the kallikrein-kinin system, originates from a locus on the long arm of chromosome 19 that contains several related serine endopeptidases. The biological role of these kallikrein-related peptidases is not clear, but emerging evidence suggests that they might be important in several physiological systems, e.g., in male reproduction, skin homeostasis, tooth enamel formation and neural development and plasticity. The kallikrein locus has undergone some major evolutionary events. Most spectacular are relatively recent duplications of KLK1 that have created 13 and 9 functional genes that are unique to the mouse and the rat, respectively. Human paralogs are KLK2 and KLK3: the latter encoding the cancer biomarker prostate-specific antigen. In this review on kallikrein-related peptidases, the focus is on their evolution, their role in skin homeostasis and semen liquefaction, and their utility as cancer biomarkers.

The two cellular targets of interest in age-related macular degeneration (AMD) are the photoreceptors and the RPE. However, the mechanisms involved in AMD pathology are not yet fully understood. In the present report, we extend our previous studies on semenogelin proteins (Sgs) in normal human retina and compare these with the distribution in retinas from AMD donor eyes. Semenogelins I (SgI) and II (SgII) are the major structural protein components of semen coagulum, but have been recently found in non-genital tissues as well. Cryo and paraffin sections of human retina were processed for both immunofluorescence and DAB reaction with a specific antibody. The presence of SgI was analyzed in retina and RPE total lysates and SgI was detected by western blot in human retina and RPE. The intensity of immunoreactivity was significantly reduced in the AMD eyes. SgI is expressed in the normal human retina and in the retina of AMD donor eyes, where localization was detected in the photoreceptors and in a few ganglion cells. We find the distribution of SgI in the AMD retinas substantially lower than observed in normal retina. SgI localization to photoreceptors and the RPE suggests a possible function related to the ability of these cells to sequester zinc.

BACKGROUND:Hypoxia induces increased tumor growth by promoting angiogenic and glycolytic pathways. Tumors expressing hypoxia-inducible factor-1alpha (HIF-1alpha), an important transcriptional activator of oxygen-regulated genes, are resistant to chemotherapy and radiotherapy. The major challenge in prostate cancer therapy today is to gain a better understanding of the development of hormone-refractory tumors, which is often characterized by neuroendocrine differentiation. Here we studied the expression of HIF-1alpha and HIF-2alpha in neuroendocrine cells of the benign prostate and in prostate cancer.METHODS:Tissue sections from 30 patients who underwent radical prostatectomy and from 21 patients operated by transurethral resection of the prostate were selected for immunohistochemical analysis for expression of HIF-1alpha, HIF-2alpha, androgen receptor (AR), neuroendocrine markers (chromogranin A, synaptophysin), and two gene products downstream of HIF-1alpha: VEGF and GAPDH.RESULTS:Immunoreactive HIF-1alpha was detected in a subpopulation of AR-negative neuroendocrine cells in benign and malignant prostate tissue. Analysis of serial sections showed that the levels of expression of GAPDH and VEGF proteins are increased in AR-negative malignant neuroendocrine cells expressing HIF-1alpha. In situ-hybridization indicated that HIF-1alpha mRNA levels are not higher in neuroendocrine prostate cancer cells relative to corresponding non-neuroendocrine tumor cells. We also demonstrated induced stabilization of nuclear HIF-1alpha in LNCaP cells by hypoxia and long-term stimulation with interleukin-6. Focal HIF-2 expression was detected in benign neuroendocrine-like cells and in malignant prostatic cells.CONCLUSIONS:The expression of HIF-1alpha and HIF-2alpha in prostate cancer has been confirmed, but we also identified immunoreactive HIF-1alpha and downstream gene products in benign and malignant prostate neuroendocrine cells.

In this study we characterize a novel gene on human chromosome 9 and its translation product, PC3-secreted microprotein (PSMP). The gene contains three exons that encode a protein of 139 amino acid residues, including a predicted signal peptide of 36 residues. The molecule is homologous to beta-microseminoprotein (MSP), a protein of unknown function, secreted at high concentration by the prostate gland. These two proteins have only 23% sequence identity, but their common origin is revealed by a preserved pattern of Cys residues. In contrast to MSP, which shows poor conservation between species, PSMP is very conserved. High transcript levels were detected in the prostate cancer cell line PC-3. Antiserum raised against PSMP detected a protein with an apparent molecular mass of 18 kDa in culture medium conditioned by PC-3 cells, but in cell lysates the antiserum also recognized a molecular species of 16 kDa, suggesting that PSMP undergoes post-translational modification. Xenografted PC-3 cell tumors in athymic nude mice showed strong staining for both PSMP protein and mRNA. Studies on human prostate cancer specimens showed immunohistochemical staining of both tumor and benign glandular cells. Our results suggest that PSMP is an important protein with significance in prostate cancer.

During liquefaction of the ejaculate, the semen coagulum proteins semenogelin I (SEMG1) and semenogelin II (SEMG2) are degraded to low molecular mass fragments by kallikrein-related peptidase 3 (KLK3), also known as prostate-specific antigen. Semenogelin molecules initiate their own destruction by chelating Zn(2+) that normally would completely inhibit the proteolytic activity of KLK3. In a similar way, semenogelins might regulate the activity of kallikrein-related peptidases in the epididymis, something that might be of importance for the maturation of spermatozoa or generation of anti-bacterial peptides. Studies on the evolution of semen coagulum proteins have revealed that most of them carry an exon that displays a rapid and unusual evolution. As a consequence, homologous proteins in rodents and primates show almost no conservation in primary structure. Further studies on their evolution suggest that the progenitor of the semen coagulum proteins probably was a protease inhibitor that might have displayed antimicrobial activity. The semenogelin locus on chromosome 20 contains at least 17 homologous genes encoding probable protease inhibitors with homology to semen coagulum proteins. All of these are highly expressed in the epididymis where they, similar to the semenogelins, could affect the maturation of spermatozoa or display antibacterial properties.

The semen coagulum proteins have undergone substantial structural changes during evolution. In primates, these seminal vesicle-secreted proteins are known as semenogelin I (SEMG1) and semenogelin II (SEMG2). Previous studies on the common marmoset (Callithrix jacchus) showed that ejaculated semen from this New World monkey contains semenogelin, but it remained unclear whether it carries both genes or only SEMG1 and no SEMG2, like the closely related cotton-top tamarin (Saguinus oedipus). In this study we show that there are two genes, both expressed in the seminal vesicles. Surprisingly, the genes show an almost perfect sequence identity in a region of 1.25 kb, encompassing nearly half of the genes and containing exon 1, intron 1, and the first 0.9 kb of exon 2. The underlying molecular mechanism is most likely gene conversion, and a phylogenetic analysis suggests that SEMG1 is the most probable donor gene. The marmoset SEMG1 in this report differs from a previously reported cDNA by a lack of nucleotides encoding one repeat of 60 amino acids, suggesting that marmoset SEMG1 displays allelic size variation. This is similar to what was recently demonstrated in humans, but in marmosets the polymorphism was generated by a repeat duplication, whereas in humans it was a deletion. Together, these studies shed new light on the evolution of semenogelins and the mechanisms that have generated the structural diversity of semen coagulum proteins.