Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors.

We have cloned and characterized a cDNA encoding a new human serine proteinase, testisin, that is abundantly expressed only in the testis and is lost in testicular tumors. The testisin cDNA was identified by homology cloning using degenerate primers directed at conserved sequence motifs within the catalytic regions of serine proteinases. It is 1073 nucleotides long, including 942 nucleotides of open reading frame and a 113-nucleotide 3' untranslated sequence. Northern and dot blot analyses of RNA from a range of normal human tissues revealed a 1.4-kb mRNA species that was present only in testis, which was not detected in eight of eight testicular tumors. Testisin cDNA is predicted to encode a protein of 314 amino acids, which consists of a 19-amino acid (aa) signal peptide, a 22-aa proregion, and a 273-aa catalytic domain, including a unique 17-aa COOH-terminal hydrophobic extension that is predicted to function as a membrane anchor. The deduced amino acid sequence of testisin shows 44% identity to prostasin and contains features that are typical of serine proteinases with trypsin-like substrate specificity. Antipeptide antibodies directed against the testisin polypeptide detected an immunoreactive testisin protein of Mr 35,000-39,000 in cell lysates from COS-7 cells that were transiently transfected with testisin cDNA. Immunostaining of normal testicular tissue showed that testisin was expressed in the cytoplasm and on the plasma membrane of premeiotic germ cells. No staining was detected in eight of eight germ cell-derived testicular tumors. In addition, the testisin gene was localized by fluorescence in situ hybridization to the short arm of human chromosome 16 (16p13.3), a region that has been associated with allellic imbalance and loss of heterozygosity in sporadic testicular tumors. These findings demonstrate a new cell surface serine proteinase, loss of which may have a direct or indirect role in the progression of testicular tumors of germ cell origin.

Localization, expression and genomic structure of the gene encoding the human serine protease testisin.

Testisin is a recently identified human serine protease expressed by premeiotic testicular germ cells and is a candidate tumor suppressor for testicular cancer. Here, we report the characterization of the gene encoding testisin, designated PRSS21, and its localization on the short arm of human chromosome 16 (16p13.3) between the microsatellite marker D16S246 and the radiation hybrid breakpoint CY23HA. We have further refined the localization to cosmid 406D6 in this interval and have established that the gene is approximately 4. 5 kb in length, and contains six exons and five intervening introns. The structure of PRSS21 is very similar to the human prostasin gene (PRSS8) which maps nearby on 16p11.2, suggesting that these genes may have evolved through gene duplication. Sequence analysis showed that the two known isoforms of testisin are generated by alternative pre-mRNA splicing. A major transcription initiation site was identified 97 nucleotides upstream of the testisin translation start and conforms to a consensus initiator element. The region surrounding the transcription initiation site lacks a TATA consensus sequence, but contains a CCAAT sequence and includes a CpG island. The 5'-flanking region contains several consensus response elements including Sp1, AP1 and several testis-specific elements. Analysis of testisin gene expression in tumor cell lines shows that testisin is not expressed in testicular tumor cells but is aberrantly expressed in some tumor cell lines of non-testis origin. These data provide the basis for identifying potential genetic alterations of PRSS21 that may underlie both testicular abnormalities and tumorigenesis.

Urokinase binding and catabolism by Hep G2 cells is plasminogen activator inhibitor-1 dependent, analogous to interactions of tissue-type plasminogen activator with these cells.

The adherent human hepatoma cell line Hep G2 exhibits receptor mediated endocytosis and catabolism of tissue-type plasminogen activator.plasminogen activator inhibitor type-1 (t-PA.PAI-1) complexes formed when exogenous t-PA combines with endogenous PAI-1 in the extracellular matrix. To determine whether the other major PA, urokinase (u-PA), which also complexes with PAI-1, is metabolised via the same mechanism, 125I-labelled high (hmw) and low (lmw) molecular weight forms of u-PA were incubated with Hep G2 cells at 4 degrees C for 2 hr in the absence and presence of a 100-fold excess of unlabelled ligand in order to detect specific binding. Both hmw and lmw 125I-u-PA formed complexes with PAI-1 and these bound specifically and with high affinity (apparent Kd 3.9 and 4.1 nM, with Bmax 78 x 10(3) and 83 x 10(3) binding sites/cell respectively). Binding by each form of radiolabelled u-PA was inhibited in a dose-dependent fashion by unlabelled t-PA, hmw-u-PA, lmw-u-PA, and by monoclonal anti-PAI-1 antibody. At 37 degrees C, bound hmw and lmw 125I-u-PA.PAI-1 complexes were internalised and degraded rapidly. These findings indicate that the specificity of the previously described receptor which mediates PAI-1 dependent catabolism of t-PA by Hep G2 cells extends to complexes of u-PA with this inhibitor.

Localization of the mosaic transmembrane serine protease corin to heart myocytes.

Corin cDNA encodes an unusual mosaic type II transmembrane serine protease, which possesses, in addition to a trypsin-like serine protease domain, two frizzled domains, eight low-density lipoprotein (LDL) receptor domains, a scavenger receptor domain, as well as an intracellular cytoplasmic domain. In in vitro experiments, recombinant human corin has recently been shown to activate pro-atrial natriuretic peptide (ANP), a cardiac hormone essential for the regulation of blood pressure. Here we report the first characterization of corin protein expression in heart tissue. We generated antibodies to two different peptides derived from unique regions of the corin polypeptide, which detected immunoreactive corin protein of approximately 125-135 kDa in lysates from human heart tissues. Immunostaining of sections of human heart showed corin expression was specifically localized to the cross striations of cardiac myocytes, with a pattern of expression consistent with an integral membrane localization. Corin was not detected in sections of skeletal or smooth muscle. Corin has been suggested to be a candidate gene for the rare congenital heart disease, total anomalous pulmonary venous return (TAPVR) as the corin gene colocalizes to the TAPVR locus on human chromosome 4. However examination of corin protein expression in TAPVR heart tissue did not show evidence of abnormal corin expression. The demonstrated corin protein expression by heart myocytes supports its proposed role as the pro-ANP convertase, and thus a potentially critical mediator of major cardiovascular diseases including hypertension and congestive heart failure.

Organization and chromosomal localization of the murine Testisin gene encoding a serine protease temporally expressed during spermatogenesis.

The recently characterized human serine protease, Testisin, is expressed on premeiotic testicular germ cells and is a candidate type II tumor suppressor for testicular cancer. Here we report the cloning, characterization and expression of the gene encoding mouse Testisin, Prss21. The murine Testisin gene comprises six exons and five introns and spans approximately 5 kb of genomic DNA with an almost identical structure to the human Testisin gene, PRSS21. The gene was localized to murine chromosome 17 A3.3-B; a region syntenic with the location of PRSS21 on human chromosome 16p13.3. Northern blot analyses of RNA from a range of adult murine tissues demonstrated a 1.3 kb mRNA transcript present only in testis. The murine Testisin cDNA shares 65% identity with human Testisin cDNA and encodes a putative pre-pro-protein of 324 amino acids with 80% similarity to human Testisin. The predicted amino-acid sequence includes an N-terminal signal sequence of 27 amino acids, a 27 amino-acid pro-region, a 251 amino-acid catalytic domain typical of a serine protease with trypsin-like specificity, and a C-terminal hydrophobic extension which is predicted to function as a membrane anchor. Immunostaining for murine Testisin in mouse testis demonstrated specific staining in the cytoplasm and on the plasma membrane of round and elongating spermatids. Examination of murine Testisin mRNA expression in developing sperm confirmed that the onset of murine Testisin mRNA expression occurred at approximately day 18 after birth, corresponding to the appearance of spermatids in the testis, in contrast to the expression of human Testisin in spermatocytes. These data identify the murine ortholog to human Testisin and demonstrate that the murine Testisin gene is temporally regulated during murine spermatogenesis.