Kallikrein-like Serine Protease Research Articles

Co-option of the same ancestral gene family gave rise to mammalian and reptilian toxins

BackgroundEvolution can occur with surprising predictability when organisms face similar ecological challenges. For most traits, it is difficult to ascertain whether this occurs due to constraints imposed by the number of possible phenotypic solutions or because of parallel responses by shared genetic and regulatory architecture. Exceptionally, oral venoms are a tractable model of trait evolution, being largely composed of proteinaceous toxins that have evolved in many tetrapods, ranging from reptiles to mammals. Given the diversity of venomous lineages, they are believed to have evolved convergently, even though biochemically similar toxins occur in all taxa.ResultsHere, we investigate whether ancestral genes harbouring similar biochemical activity may have primed venom evolution, focusing on the origins of kallikrein-like serine proteases that form the core of most vertebrate oral venoms. Using syntenic relationships between genes flanking known toxins, we traced the origin of kallikreins to a single locus containing one or more nearby paralogous kallikrein-like clusters. Additionally, phylogenetic analysis of vertebrate serine proteases revealed that kallikrein-like toxins in mammals and reptiles are genetically distinct from non-toxin ones.ConclusionsGiven the shared regulatory and genetic machinery, these findings suggest that tetrapod venoms evolved by co-option of proteins that were likely already present in saliva. We term such genes ‘toxipotent’—in the case of salivary kallikreins they already had potent vasodilatory activity that was weaponized by venomous lineages. Furthermore, the ubiquitous distribution of kallikreins across vertebrates suggests that the evolution of envenomation may be more common than previously recognized, blurring the line between venomous and non-venomous animals.

Venom phenotypes of the Rock Rattlesnake (Crotalus lepidus) and the Ridge-nosed Rattlesnake (Crotalus willardi) from México and the United States

Although the Mexican Highlands has the highest diversity of small-bodied rattlesnakes in the world, studies on the species found throughout this region have been relatively scarce. This has led to challenges with examining venom phenotypic characteristics, as well as species misidentifications and misclassifications. In the current study we investigated venom variation among four subspecies of Crotalus lepidus (C. l. klaluberi, C. l. lepidus, C. l. maculosus, C. l. morulus) and four subspecies of C. willardi (C. w. amabilis, C. w. obscurus, C. w. silus, and C. w. willardi) that inhabit regions of southwestern United States and central México. SDS-PAGE patterns show the presence of many of the major compounds found in other rattlesnake venoms, although minor variations in protein banding patterns and intensity are recognizable. Most notably, PI-metalloproteinase (SVMP) bands appear to be very faint to absent in northern C. l. lepidus and C. l. klauberi subspecies, but are fairly prominent in all other C. lepidus and C. willardi subspecies. Enzyme activity assays revealed that C. lepidus subspecies exhibit higher SVMP and thrombin-like activities when compared to C. willardi subspecies. Significant differences between subspecies were also observed for kallikrein-like serine protease, L-amino acid oxidase, and phosphodiesterase activities, although these differences appear to be random and fail to follow a geographical or phylogenetic trend. The same relationship was also observed for fibrinogenolytic and coagulation assays. Toxicity assays conducted on lab mice (Mus musculus), house geckos (Hemidactylus frenatus), and house crickets (Acheta domestica) revealed varying toxicities between subspecies, with C. l klauberi being the most toxic towards mice (LD50 = 1.36 μg/g) and house geckos (LD50 = 0.17 μg/g), and C. w. silus being most toxic to house crickets (LD50 = 1.94 μg/g). These results provide additional evidence that geographical isolation, natural selection, and adaptive evolution in response to diets may be driving forces contributing to population-level variation in venom composition.

Magnetic-particle-based, ultrasensitive chemiluminescence enzyme immunoassay for free prostate-specific antigen

We report a magnetic-particle (MMP)-based chemiluminescence enzyme immunoassay (CLEIA) for free prostate-specific antigen (f-PSA) in human serum. In this method, the f-PSA is sandwiched between the anti-PSA antibody coated MMPs and alkaline phosphatase (ALP)-labeled anti-f-PSA antibody. The signal produced by the emitted photons from the chemiluminescent substrate (4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2′-adamantane)) is directly proportional to the amount of f-PSA in a sample. The present MMP-based assay can detect f-PSA in the range of 0.1–30ngmL−1 with the detection limit of 0.1ngmL−1. The linear detection range could match the concentration range within the “diagnostic gray zone” of serum f-PSA levels (4–10ngmL−1). The detection limit was sufficient for measuring clinically relevant f-PSA levels (>4ngmL−1). Furthermore, the method was highly selective; it was unaffected by cross-reaction with human glandular kallikrein-2, a kallikrein-like serine protease that is 80% similar to f-PSA. The proposed method was finally applied to determine f-PSA in 40 samples of human sera. Results obtained using the method showed high correlation with those obtained using a commercially available microplate CLEIA kit (correlation coefficient, 0.9821). This strategy shows great potential application in the fabrication of diagnostic kits for determining f-PSA in serum.

Molecular basis for prey relocation in viperid snakes.

BackgroundVertebrate predators use a broad arsenal of behaviors and weaponry for overcoming fractious and potentially dangerous prey. A unique array of predatory strategies occur among snakes, ranging from mechanical modes of constriction and jaw-holding in non-venomous snakes, to a chemical means, venom, for quickly dispatching prey. However, even among venomous snakes, different prey handling strategies are utilized, varying from the strike-and-hold behaviors exhibited by highly toxic elapid snakes to the rapid strike-and-release envenomation seen in viperid snakes. For vipers, this mode of envenomation represents a minimal risk predatory strategy by permitting little contact with or retaliation from prey, but it adds the additional task of relocating envenomated prey which has wandered from the attack site. This task is further confounded by trails of other unstruck conspecific or heterospecific prey. Despite decades of behavioral study, researchers still do not know the molecular mechanism which allows for prey relocation.ResultsDuring behavioral discrimination trials (vomeronasal responsiveness) to euthanized mice injected with size-fractionated venom, Crotalus atrox responded significantly to only one protein peak. Assays for enzymes common in rattlesnake venoms, such as exonuclease, L-amino acid oxidase, metalloproteinase, thrombin-like and kallikrein-like serine proteases and phospholipase A2, showed that vomeronasal responsiveness was not dependent on enzymatic activity. Using mass spectrometry and N-terminal sequencing, we identified the proteins responsible for envenomated prey discrimination as the non-enzymatic disintegrins crotatroxin 1 and 2. Our results demonstrate a novel and critical biological role for venom disintegrins far beyond their well-established role in disruption of cell-cell and cell-extracellular matrix interactions.ConclusionsThese findings reveal the evolutionary significance of free disintegrins in venoms as the molecular mechanism in vipers allowing for effective relocation of envenomated prey. The presence of free disintegrins in turn has led to evolution of a major behavioral adaptation (strike-and-release), characteristic of only rattlesnakes and other vipers, which exploits and refines the efficiency of a pre-existing chemical means of predation and a highly sensitive olfaction system. This system of a predator chemically tagging prey represents a novel trend in the coevolution of predator-prey relationships.

An immune responsive complement factor D/adipsin and kallikrein-like serine protease (PoDAK) from the olive flounder Paralichthys olivaceus

The cDNA encoding of a complement factor D/adipsin and kallikrein-like serine protease, designated PoDAK, was isolated from the olive flounder Paralichthys olivaceus. PoDAK cDNA encodes a polypeptide with 277 amino acids containing conserved catalytic triad residues of serine proteases. The amino acid sequence of PoDAK showed high similarity to the kallikrein-like protein of medaka, mammalian adipsin/complement factor D and tissue kallikrein homolog, KT-14 of trout, complement factor D of zebrafish, and shared 31.6–36.8% homology with complement factor D/adipsin known from other species, including mammals. Phylogenetic analysis revealed that PoDAK clustered with the kallikrein-like protein of medaka and mammalian adipsin/complement factor D and tissue kallikrein homolog KT-14 of trout. The expression of PoDAK mRNA was high in the gills and heart, moderate in muscle, liver, intestine, stomach, kidney, and spleen of healthy flounder, and increased in the kidney, liver, and spleen of flounder challenged by the viral hemorrhagic septicemia virus (VHSV) or Streptococcus iniae. In situ hybridization confirmed that PoDAK mRNA is localized in the kidney and heart of individuals infected with VHSV. Further investigations are needed to clarify the function of PoDAK in vivo and in vitro.

Testing New PSA Subforms to Enhance the Accuracy of Predicting Cancer Risk and Disease Outcome in Prostate Cancer

Featured Article: Lilja H, Christensson A, Dahlen U, et al. Prostate-Specific Antigen in Serum Occurs Predominantly in Complex with α1-Antichymotrypsin. Clin Chem 1991;37:1618–25.1 Prostate-specific antigen (PSA),2 a kallikrein-like serine protease, is one of the most abundant proteins secreted by the prostate into seminal fluid (1). In the early 1990s, when the study reported in the paper presented here was initiated, serum testing for PSA was coming into widespread use as a means of testing for prostate cancer. Nevertheless, several aspects of PSA in serum remained puzzling; notably, different assays yielded up to 4-fold differences in measured serum PSA concentrations, despite standardization against PSA from seminal fluid. Before our investigation, PSA in seminal fluid was known to occur as active monomeric peptidase (1), but the nature of PSA in blood remained unclear. We speculated that release of catalytic PSA into an abundance of protease inhibitors in blood could prompt PSA to form inhibitor complexes, similar to the way the digestive enzyme trypsin from the pancreas forms enzyme-inhibitor complexes with α1-antitrypsin in blood. A search for interactions between PSA and a panel of common protease inhibitors in serum allowed us …

Human seminal proteinase and prostate-specific antigen are the same protein

Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and compared. Both are glycoproteins of 32-34 kDa with protease activities. Based on some physicochemical,enzymatic and immunological properties,it is concluded that these proteins are in fact identical. The protein exhibits properties similar to kallikrein-like serine protease, trypsin,chymotrypsin and thiol acid protease. Tests of the activity of the enzyme against some potential natural and synthetic substrates showed that bovine serum albumin was more readily hydrolysed than casein. The results of this study should be useful in purifying and assaying this protein. Based on published studies and the present results,the broad proteolytic specificity of human seminal proteinase suggests a role for this protein in several physiological functions.

The Interaction among Protein C Inhibitor, Prostate-Specific Antigen, and the Semenogelin System

The kallikrein-like serine protease, prostate-specific antigen (PSA), is mixed in human seminal plasma with its protein substrates semenogelin (Sg) -I, Sg-II, and protein C inhibitor (PCI), which are produced in seminal vesicles. In the seminal plasma, PSA degrades Sg-I, and Sg-II, which are major components in insoluble coagula, and PCI inhibits PSA by forming a PSA-PCI complex. Digestion of seminal coagula with PSA releases PCI and PSA-PCI complex from the coagula into a soluble phase, suggesting the presence of active PCI within the coagula. PCI forms a ternary complex with PSA and Sg-II in the seminal plasma. The binding of Sg-II to PSA and PCI is influenced by pH, ionic strength, heparin, negatively charged dextran sulfate, divalent cations, and particularly by Zn 2 +. These observations suggest that binding of PCI to Sg in seminal vesicles regulates the PSA-catalyzed degradation of Sg in seminal plasma; the complex formation among PCI, PSA, and Sg is modulated by several factors in seminal plasma.

Seminal clotting and fibrinolytic balance: a possible physiological role in the male reproductive system.

Semen contains enzymes and inhibitors of the haemostatic system as well as the high molecular weight seminal vesicle (HMW-SV) proteins. The former may have roles in seminal clotting and in liquefaction through "fibrinolytic" activity, which may ultimately affect fertility. Although a limited number of studies have addressed the subject, the role of clotting and fibrinolytic factors in semen remains poorly understood. The liquefaction time and the distribution of components vary across split ejaculates. This may have an important bearing on the way clotting/fibrinolytic factors in semen are assessed. Semen contains tissue factor (TF, Thromboplastin, CD142), which originates from the prostate and is associated with prostasomes. The function of TF (and prostasomes) in semen is still a matter for speculation. Recently the presence of minute amounts of factor VII in semen has been demonstrated but its importance is uncertain. Semen also contains a thrombin-like enzyme, prothrombin fragments 1 and 2 (F1+2), D-dimer (DD) and thrombin-antithrombin (TAT) complexes. The presence of several fibrinolytic factors has been demonstrated in semen but few questions about their potential impact on semen quality have been raised. Factors found include tissue plasminogen activator (t-PA), urinary plasminogen activator (u-PA) and plasmin. There are also traces of fibrinogen, plasminogen, plasminogen activator inhibitor-1 (PAI-1), factor VIII coagulant activity (VIII:c) and fibrin monomers. The co-ordinate expression of both TF and PAI-1 by decidual cells of the endometrium is believed to be important in maintaining haemostasis during endovascular trophoblast invasion. Kallikrein-like serine protease inhibitors including prostate specific antigen (PSA) are known to be present in semen at high concentrations. In semen PSA is also found in a complex form with protein C inhibitor (PCI) with mutually inhibitory consequences. A better understanding of the spectrum of coagulating and liquefaction agents in semen to include classical haemostatic processes and the pathogenesis resulting from any imbalances between or within either system may provide the basis for the development of more selective and efficient agents affecting global fertility. Here we review aspects of male reproductive physiology in the light of recent findings concerning conventional clotting/fibrinolytic systems in human semen with a view to stimulating further research.

Molecular cloning and expression of a variant form of hippostasin/KLK11 in prostate

Hippostasin is a kallikrein-like serine protease, which has two alternatively spliced isoforms, brain-type and prostate-type. We previously reported alternative expression of hippostasin in prostate cancer cell lines. We studied the expression of a variant-form hippostasin (isoform 3) mRNA by RT-PCR. Localization of the isoform 3 protein was examined by immunohistochemistry. The enzymatic activity of the recombinant protein was measured with synthetic substrates. A novel isoform of hippostasin contains 25 additional amino acids in the catalytic triad of brain-type hippostasin. Its mRNA was expressed in normal prostate tissue, BPH, and prostate cancer cell lines. The protein was localized in the prostate secretory epithelium. The enzyme activity was similar to that of brain-type hippostasin, which has kallikrein-like activity. In this study, we have identified a third isoform of hippostasin, which was designated variant-form (isoform 3). Hippostasin isoform 3 may play a role in the prostate, including reproductive and/or tumorigenic functions.

Decreased cerebrospinal fluid levels of neurosin (KLK6), an aging-related protease, as a possible new risk factor for Alzheimer's disease.

Neurosin is a kallikrein-like serine protease expressed preferentially in the human brain. It is localized in senile plaques and neurofibrillary tangles in the brains of individuals with Alzheimer's disease (AD) and in Lewy bodies in patients with Parkinson's disease. Neurosin is present in the cerebrospinal fluid (CSF) as a proenzyme and does not show any enzymatic activity. We have developed a sandwich ELISA system using monoclonal and polyclonal antibodies against human neurosin and have measured neurosin levels in the CSF from AD and non-CNS disease patients. Both male and female patients with peripheral neuropathy showed statistically positive correlations between CSF neurosin concentrations and age (males, n = 52, r = 0.482, p < 0.005; females, n = 43, r = 0.365, p < 0.005). In contrast, such positive correlation was not observed in the CSF from patients with AD. Further, some such patients showed extremely low levels of CSF neurosin. Our results suggest that neurosin is an aging-related protease and that a decreased CSF concentration of neurosin may be a risk factor for developing AD.

Prostate-specific antigen (PSA/hK3): a further player in the field of breast cancer diagnostics?

Since its identification, much information has been obtained about prostate-specific antigen (PSA, or human glandular kallikrein 3 [hK3]), a kallikrein-like serine protease that is the most valuable tumour marker for the screening, diagnosis and management of human prostate carcinoma. Recently, it has become widely accepted that PSA is also present in many nonprostatic sources, casting doubts about the specificity of its tissue expression. Here we summarize the findings on the biomolecular expression of PSA in breast secretions, cells and tissues of healthy and diseased females. Although several studies have strongly suggested that the molecular forms of PSA seem to represent a potential tool for the risk assessment of breast cancer, recent reports have yielded conflicting results. Although several studies have suggested new biological function(s) for PSA in breast physiopathology, more studies are needed to enlist PSA unequivocally as an additional weapon in the anticancer armoury in breast cancer diagnostics.

Prostate-specific antigen synthesis and secretion by human placenta: a physiological kallikrein source during pregnancy.

Prostate-specific antigen (PSA), a kallikrein-like serine protease until recently thought to be prostate specific, has been demonstrated in various nonprostatic tissues and body fluids. PSA has been also found in human endometrium and amniotic fluids, even if the significance of this novel expression is unclear. In this study, we have demonstrated by multiple techniques that human placental tissue, obtained at delivery from normal full-term pregnancies, synthesizes and secretes PSA. RT-PCR showed the presence of PSA messenger ribonucleic acid; biochemical, chromatographic, and immunological studies revealed the expression of both free and complexed PSA forms; immunoelectron microscopy indicated the syncytiotrophoblast as the site of PSA synthesis and secretion. Moreover, in vitro experiments demonstrated that PSA production and secretion are up-regulated by 17beta-estradiol, a pregnancy-related steroid hormone. These results suggest that human placenta is a source of the PSA present in amniotic fluid and maternal serum during pregnancy.

Molecular Characterization of Zyme/Protease M/Neurosin (PRSS9), A Hormonally Regulated Kallikrein-like Serine Protease

The cDNA for the zyme/protease M/neurosin gene (HGMW-approved symbol PRSS9) has recently been identified. Zyme appears to play a role in Alzheimer disease as well as in breast cancer. In this paper, we describe the complete genomic organization of the zyme gene. Zyme spans 10.5 kb of genomic sequence on chromosome 19q13.3–q13.4. The gene consists of seven exons, the first two of which are untranslated. All splice junctions follow the GT/AG rule, and the intron phases are identical to those of many other genes belonging to the same family, i.e., the kallikreins, NES1, and neuropsin. Fine-mapping of the genomic locus indicates that zyme lies upstream of the NES1 gene and downstream from the PSA and KLK2 genes. Tissue expression studies indicate that zyme is expressed mainly in brain tissue, including spinal cord and cerebellum, in mammary gland, and in kidney and uterus. Zyme is regulated by steroid hormones in the breast carcinoma cell line BT-474. Estrogens and progestins, and to a lesser extent androgens, up-regulate the zyme gene in a dose-dependent manner.

Prostate-specific Antigen Expression in Normal Human Bone Marrow Cells

Improved procedures for measuring prostate-specific antigen (PSA) protein and mRNA have demonstrated that this kallikrein-like serine protease is present in many nonprostatic sources, indicating that PSA production/secretion is not tissue- or sex-specific, but rather is a steroid hormone-dependent phenomenon (1). Reports on PSA-positive cells in bone marrow (BM) and peripheral blood mononuclear cells are contradictory (2)(3)(4)(5)(6). The elucidation of this controversy might be of clinical utility to establish the nonspecificity of PSA as an indicator of micrometastases (7)(8). Belonging to a large study project on the extraprostatic expression of PSA, we …

Prostate-specific Antigen Expression in Neuroblastoma Cell Lines

We read with particular interest the article of Melegos et al. (1) on the prostate-specific antigen (PSA) immunoreactivity of cerebrospinal fluids, in which the authors report positive results for this kallikrein-like serine protease in ∼7% of patients affected by various neurological disorders, suggesting that PSA could originate from the brain tissue. In light of this hypothesis, we undertook to study the expression, subfraction distribution, and secretion of PSA from two neuroblastoma cell lines: the SK-N-BE cell line, established in culture from a bone marrow biopsy specimen (2); and the SK-N-MC cell line, derived from a metastatic tumor mass (3). Both cell lines have tumor-producing capacity as well as the morphological patterns of malignant neuronal cells (4). These cell lines, all Mycoplasma free, were cultured in Eagle’s minimum essential medium supplemented with nonessential amino acids, 15% fetal bovine serum, and …

Characterization of a ribonucleic acid transcript from the brook trout (Salvelinus fontinalis) ovary with structural similarities to mammalian adipsin/complement factor D and tissue kallikrein, and the effects of kallikrein-like serine proteases on follicle contraction.

A 2.4-kilobase (kb) clone (kallikrein trout #14; KT-14) was isolated from a brook trout ovulatory cDNA library. KT-14 contains an open reading frame (ORF) of 768 base pairs (bp), presumably encoding a protein of 255 amino acids. The KT-14 cDNA also contains a 711 -bp 5' untranslated region and a 793-bp region downstream of the ORF that includes a 66-bp sequence repeated 12 times. The amino acid sequence of the KT-14 ORF is 41 % identical to that of porcine complement factor D and 33% identical to that of porcine pancreatic kallikrein. On Northern blots of ovarian tissue, KT-14 hybridized with four transcripts of 1.8, 2.4, 2.9, and 3.2 kb. While the 3.2- and 2.4-kb transcripts were present in the ovary prior to meiotic maturation, they were significantly up-regulated at ovulation and at 12 h postovulation, respectively. Antibodies constructed against the recombinant KT-14 protein recognized one 30-kDa immunogenic protein in ovarian tissue and fluid. This immunogenic protein was significantly elevated in the tissue by ovulation. Using a follicle weight loss bioassay, we provide indirect evidence that mammalian kallikrein and related serine proteases can stimulate brook trout follicle contraction. Thus, one possible function of the KT-14 protein may be the regulation of oocyte expulsion at ovulation.

Renal epidermal growth factor precursor: proteolytic processing in an in vitro cell-free system

The enzymatic processing of the membrane-bound renal epidermal growth factor precursor (proEGF) could be an important step in the control of nephrogenic repair consecutive to kidney insult. The enzyme machinery responsible for that processing was examined in a cell-free system consisting of renal membranes isolated from kidney homogenates by differential centrifugation, and incubated in vitro. After a 24-h incubation at 37°C, 6–14% of membrane-bound proEGF was processed and soluble products with EGF immunoreactivity were released. As revealed by HPLC and Western blotting analysis, the products of proEGF proteolysis consisted of 6 kDa EGF (the molecular weight of mature EGF) and two polypeptides with molecular weights around 45 kDa. Interestingly the 45 kDa EGF forms, like the 6 kDa EGF, exhibited mitogenic activity toward growth-arrested NRK-52E renal cell line. The kinetic study of proEGF degradation gave data consistent with the 45 kDa product(s) being processing intermediate(s) between proEGF and 6 kDa EGF. The enzymatic activity responsible for proEGF nicking was inhibited by divalent heavy metal ions (Cu 2+ or Zn 2+) and several protease inhibitors (aprotinin, PMSF, leupeptin, soybean trypsin inhibitor), suggesting that proEGF is processed by kallikrein-like serine proteases present in the membrane preparations. Along with previous studies, the current observations suggest that renal kallikreins might play a role in renal tubular regeneration by promoting the release of soluble EGF in renal tissue.

Individual prostate-specific antigen (PSA) forms as prostate tumor markers

Prostate-specific antigen (PSA) is a kallikrein-like serine protease mainly expressed in the human prostate. It is responsible for the proteolysis of the gel-forming proteins in human semen. Two major extracellular protease inhibitors, alpha-1-antichymotrypsin (ACT) and alpha-2-macroglobulin (AMG) may inactivate PSA escaping from the prostate. The predominant immunodetected form of PSA in serum is complexed to ACT but PSA exists also in a free non-complexed form despite the large excess of inhibitors. The concentrations of PSA in serum are normally less than 4 μg/l, but elevated concentrations are found in a majority of patients with prostate cancer (CAP) and the analysis of PSA in serum has become invaluable in the detection and monitoring of patients with CAP. However, it is not an ideal tumor marker in the sense that there are CAP patients with normal PSA concentrations in serum and patients with benign hyperplasia of the prostate (BPH) with elevated PSA concentrations. Analysis of the various PSA forms in serum attracts much interest as there is a higher proportion of PSA in complex with ACT in patients with CAP than in those with BPH. Optimal combinations of monoclonal antibodies have been used to design sensitive noncross-reacting immunoassays for the detection of free PSA, PSA-ACT complexes and the detection of both free PSA and PSA complexes in an equimolar fashion (i.e. total PSA). Several studies have demonstrated that the analysis of the proportions of the free-to-total PSA in serum may increase the diagnostic specificity by 15–20% without significant loss in the sensitivity for detection of CAP.

Biochemical characteristics and recent biological knowledge on prostate-specific antigen.

Since its identification in seminal fluid in 1971, much new information has been obtained about the biology and expression of prostate-specific antigen (PSA). PSA is a glycoprotein composed of 93% amino acids and 7% carbohydrates, with a molecular weight of about 30,000 Da. Functionally and structurally PSA is a kallikrein-like serine protease, and its physiologic role is degradation of the major proteins of seminal coagulum (semenogelin I and II, fibronectin), which leads to semen liquefaction. The PSA gene is located on the 13q region of chromosome 19, and it has a high degree of homology (more than 80%) with genes of the human glandular kallikrein (hKGK1). PSA production and expression are preferentially but not exclusively associated to the normal, benign hyperplastic and cancerous tissues of the prostate. In fact, it has been demonstrated that PSA is also present in accessory male sex glands and breast cancer. It was recently reported that PSA was also present in milk of lactating women. Many factors may influence PSA synthesis and production, and among them the most important are androgen, retinoic acid and growth factor stimulation. Significant advances have been recently made as regards the molecular isoforms of PSA. In the seminal fluid PSA seems partially bound to a serpine (protein C inhibitor), whereas in serum it is predominantly associated to alpha-1-antichymotrypsin and in a small quantity to alpha-2-macroglobulin. These new findings will have implications for the clinical application of PSA as a tumor marker for prostate cancer.