Three genes expressing Kunitz domains in the epididymis are related to genes of WFDC-type protease inhibitors and semen coagulum proteins in spite of lacking similarity between their protein products

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>We identified three novel genes; <it>SPINT3, SPINT4 </it>and <it>SPINT5</it>, 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.</p> <p>Conclusions</p> <p>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.</p

Rapidly evolving marmoset MSMB genes are differently expressed in the male genital tract

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

Recovery of ζ-chain expression and changes in spontaneous IL-10 production after PSA-based vaccines in patients with prostate cancer

Circulating T lymphocytes of patients with prostate cancer have been reported to have functional deficits, including low or absent ζ-chain expression. To determine whether these functional impairments could be reversed by prostate specific antigen-based vaccination therapy, 10 patients treated with recombinant human prostate specific antigen plus GM-CSF and eight others receiving prostate specific antigen plus oil emulsion in two pilot clinical trials were evaluated prior to and after vaccination for several immunologic end points, including ζ-chain expression and cytokine production by circulating T cells as well as the frequency of T cells able to respond to prostate specific antigen in ELISPOT assays. The flow cytometry assay for ζ-chain expression was standardized to allow for a reliable comparison of pre- vs post-vaccination samples. Prior to therapy, the patients were found to have significantly lower ζ-chain expression in circulating CD3+ cells and a higher percentage of ζ-chain negative CD3+ and CD4+ cells than normal donors. The patients' peripheral blood mononuclear cells spontaneously produced more IL-10 ex vivo than those of normal controls. After vaccination, recovery of ζ-chain expression was observed in 50% of patients in both clinical trials. Also, spontaneous IL-10 secretion by peripheral blood mononuclear cells decreased following immunotherapy in patients treated with prostate specific antigen and GM-CSF. The frequency of prostate specific antigen-reactive T cells was detectable in 7 out of 18 patients vs 4 out of 18 patients prior to vaccination. Only one of 18 patients was a clinical responder. The vaccine had stimulatory effects on the patients' immune system, but post-vaccine immune recovery could not be correlated to progression-free survival in this small cohort of patients with prostate cancer

N-glycans of Human Protein C Inhibitor: Tissue-Specific Expression and Function

Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH2-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc3Hex5HexNAc4, consistent with a core fucosylated bi-antennary glycan with terminal Lewisx. A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH2-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M−1s−1 for the natural full-length PCI and a form lacking six amino acids at the NH2-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M−1s−1 for the corresponding PNGase F-treated forms. The 7–8-fold higher rate constants obtained when both the N-glycans and the NH2-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA

The structure of the semenogelin gene locus--nucleotide sequence of the intergenic and the flanking DNA

The sequence of 15.7 kb from the human semenogelin gene locus has been determined. Together with previously published sequences, this gives the structure of a 28-kb region encompassing the two semenogelin genes. The two transcription units are separated by 11616 bp intergenic DNA comprising more than 40% repetitive DNA sequences, predominantly located to a 4-kb block of L1 and Alu repeats. Two more blocks of L1 sequences are present in the DNA flanking the genes, so that approximately 20% of the completed sequence consists of long interspersed repeated sequences, so called LINES. A comparison of the SgI gene and the SgII gene suggests that they evolved by the duplication of an approximately 8 kb DNA segment about 61 million years ago, probably by a mechanism involving recombination between L1 elements

The cotton-top tamarin carries an extended semenogelin I gene but no semenogelin II gene

Previous studies have shown that the predominant proteins secreted by the seminal vesicles are transglutaminase substrates which have undergone major structural alterations during evolution. In man, they are known as semenogelin I and II; recently it was shown that, similar to man, several new world and old world monkeys carry two semenogelin genes as well, the exception being the cotton-top tamarin (Saguinus oedipus) with a single gene. This gene has now been cloned and identified as a semenogelin I gene, because of a higher number of conserved nucleotides in the human semenogelin I gene (89%) than in the human and the rhesus monkey semenogelin II genes (82%). Furthermore, the difference in sequence similarity indicates that the semenogelin II gene was deleted from the genome of a progenitor to the cotton-top tamarin after the duplication that yielded the two semenogelin genes seen in man. Like several other genes expressing seminal-vesicle-secreted transglutaminase substrates, the cotton-top tamarin semenogelin I gene consists of three exons of 97, 1816 and 146 bp. It codes for a signal peptide of 23 amino acid residues and the secreted protein of 592 amino acid residues. The molecular mass of 66 kDa is 32% larger than that of the human counterpart and, contrary to human semenogelin I, the cotton-top tamarin protein has the potential to be highly glycosylated as there are 14 sites with the consensus sequence for N-linked glycosylation. Approximately half of the primary structure consists of five nearly identical tandem repeats of 58 amino acid residues, that probably evolved relatively late

The cloning of a rapidly evolving seminal-vesicle-transcribed gene encoding the major clot-forming protein of mouse semen

Approximately 30 kb of the mouse genome, containing the gene for a major seminal vesicle transcript, has been cloned. The gene was identified by the similarity to members of a family with rapidly evolving genes that includes the gene encoding the major clot protein in rat semen, SVS II, and the human semenogelin genes. The nucleotide sequence of 16.9 kb was determined; this sequence encompasses the gene of 2215 bp plus 9-kb and 5.6-kb regions flanking the 5' and 3' ends of the gene. The transcription unit is divided into three exons, of which the first encodes the signal peptide, the second the secreted protein, while the third exon contains 3'-nontranslated nucleotides only. The transcript encodes a protein of 375 amino acid residues, including a signal peptide of 22 residues. The secreted polypeptide is a protein of Mr 38442 and is similar in sequence but smaller than the major clot-forming protein of rat semen, SVS II. It is highly charged at pH 7 and it has an isoelectric point of 10.68. The central part of the protein consists of tandem repeats that might serve as a substrate for transglutaminase

Characterization of the gene for prostate-specific antigen, a human glandular kallikrein

The gene for the human glandular kallikrein, prostate-specific antigen, has been cloned. The sequence of 7130 nucleotides encompassing the gene and 633 bp of 5' and 639 bp of 3' flanking DNA has been determined. The translation initiation site was slightly heterogeneous, yielding 5' non-translated leader sequences of 41 and 35 bp. The gene is divided into five exons, with introns located at positions identical with those found in other glandular kallikrein genes. The nucleotide sequence is very similar to that of the human kallikrein gene hGK-1, with 76 to 93% of the nucleotides being identical in the exons and 76 to 87% in the introns. The similarity also extends approximately 200 bp into the sequence flanking the 5' end of hGK-1 and several other, both human and rodent, glandular kallikrein genes

A locus on chromosome 20 encompassing genes that are highly expressed in the epididymis

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 Zn2+ 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