Large-scale propagation of recombinant adherent cells that secrete a stable form of human glandular kallikrein, hK2

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease that is expressed predominantly in the prostate epithelium and has 78% aa identity with prostate-specific antigen (PSA). hK2 has been recognized as a potential prostate cancer marker and has been demonstrated to be highly expressed in prostate cancer compared to benign prostatic tissue. Purification and characterization of hK2 have been impeded due to its lower expression in bodily fluids and tissues compared to PSA and its ability to autodegrade. Therefore, to study biochemical and biological characteristics of hK2, a stable and enzymatically inactive mutant form of hK2, hK2(A217V), was expressed in a hamster cell line, AV12-664 (AV12-hK2(A217V)). AV12-hK2(A217V) cells secreted prohK2(A217V) (phK2(A217V)) in the spent medium at approximately 2.5 microgram/ml. Since AV12-hK2(A217V) are adherent cells, it was necessary to develop an efficient system to propagate large numbers of cells to obtain significant quantities of phK2(A217V). In this paper, we compared ceramic core bioreactor and microcarrier beads as alternatives to static culture to propagate adherent cells. Considering production levels, ease of operation, cost effectiveness, and labor, microcarrier beads were found to be a better alternative. Our findings led to the development of a general protocol for large-scale propagation of adherent cells on microcarrier beads eliminating the need for propagating AV12-hK2(A217V) in culture flasks or bioreactors. Microcarrier beads coated with AV12-hK2(A217V) cells could be propagated in 1- or 3-liter spinner flasks and were passed from one spinner to the next in a manner analogous to static culture or could be frozen and later used as inoculum for subsequent spinners. Using this protocol, >40 liters of spent medium was harvested within 30 days, which in turn was used to purify phK2(A217V). phK2(A217V) purified from spent medium of cells grown either on microcarrier beads or in culture flasks were biochemically similar as indicated by HIC-HPLC profile followed by sequencing of relevant peaks.     

Enzymatic action of human glandular kallikrein 2 (hK2). Substrate specificity and regulation by Zn2+ and extracellular protease inhibitors.

Human glandular kallikrein 2 (hK2) is a serine protease expressed by the prostate gland with 80% identity in primary structure to prostate-specific antigen (PSA). Recently, hK2 was shown to activate the zymogen form of PSA (proPSA) in vitro and is likely to be the physiological activator of PSA in the prostate. hK2 is also able to activate urokinase and effectively cleave fibronectin. We studied the substrate specificity of hK2 and regulation of its activity by zinc and extracellular protease inhibitors present in the prostate and seminal plasma. The enzymatic activity and substrate specificity was studied by determining hK2 cleavage sites in the major gel proteins in semen, semenogelin I and II, and by measuring hydrolysis of various tripeptide aminomethylcoumarin substrates. HK2 cleaves substrates C-terminal of single or double arginines. Basic amino acids were also occasionally found at several other positions N-terminal of the cleavage site. Therefore, the substrate specificity of hK2 fits in well with that of a processor of protein precursors. Possible regulation mechanisms were studied by testing the ability of Zn2+ and different protease inhibitors to inhibit hK2 by kinetic measurements. Inhibitory constants were determined for the most effective inhibitors PCI and Zn2+. The high affinity of PCI for hK2 (kass = 2.0 x 10(5) M-1 x s-1) and the high concentrations of PCI (4 microM) and hK2 (0.2 microM) in seminal plasma make hK2 a very likely physiological target protease for PCI. hK2 is inhibited by Zn2+ at micromolar concentrations well below the 9 mM zinc concentration found in the prostate. The enzymatic activity of hK2 is likely to be reversibly regulated by Zn2+ in prostatic fluid. This regulation may be impaired in CAP and advanced metastatic cancer resulting in lack of control of the hK2 activity and a need for other means of control.     

Structural investigation of the alpha-1-antichymotrypsin: prostate-specific antigen complex by comparative model building.

Prostate-specific antigen (PSA), produced by prostate cells, provides an excellent serum marker for prostate cancer. It belongs to the human kallikrein family of enzymes, a second prostate-derived member of which is human glandular kallikrein-1 (hK2). Active PSA and hK2 are both 237-residue kallikrein-like proteases, based on sequence homology. An hK2 model structure based on the serine protease fold is presented and compared to PSA and six other serine proteases in order to analyze in depth the role of the surface-accessible loops surrounding the active site. The results show that PSA and hK2 share extensive structural similarity and that most amino acid replacements are centered on the loops surrounding the active site. Furthermore, the electrostatic potential surfaces are very similar for PSA and hK2. PSA interacts with at least two serine protease inhibitors (serpins): alpha-1-antichymotrypsin (ACT) and protein C inhibitor (PCI). Three-dimensional model structures of the uncleaved ACT molecule were developed based upon the recent X-ray structure of uncleaved antithrombin. The serpin was docked both to PSA and hK2. Amino acid replacements and electrostatic complementarities indicate that the overall orientation of the proteins in these complexes is reasonable. In order to investigate PSA's heparin interaction sites, electrostatic computations were carried out on PSA, hK2, protein C, ACT, and PCI. Two heparin binding sites are suggested on the PSA surface and could explain the enhanced complex formation between PSA and PCI, while inhibiting the formation of the ACT-PSA complex, PSA, hK2, and their preliminary complexes with ACT should facilitate the understanding and prediction of structural and functional properties for these important proteins also with respect to prostate diseases.     

Enzymatic profiling of human kallikrein 14 using phage-display substrate technology

The human KLK14 gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members. KLK14 , like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated that KLK14 gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin alpha-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.     

Improvement of expression level of keratinase Sfp2 from Streptomyces fradiae by site-directed mutagenesis of its N-terminal pro-sequence

The keratinase Sfp2, produced by Streptomyces fradiae var. k11, is a serine alkaline protease first synthesized as pre-pro-mature precursor, of which the N-terminal propeptide must be autocatalytically cleaved on the C-terminal of P1 amino acid to produce mature enzyme. Single amino acid substitutions were introduced at positions ?1 and ?2 to improve the expression level of mature Sfp2. The specific activity of L(?1)F mutant (48935 U/mg) was nine times that of wild-type Sfp2, whereas the mutants L(?1)D, L(?1)G, L(?1)H, K(?2)E, and K(?2)L had 2–52 % of the specific activity of wild-type. The yield of mature Sfp2 of L(?1)F mutant was estimated to be 800 μg/mg total protein and 112 mg/l culture supernatant, nine and twice that of wild-type, respectively. The L(?1)F mutant exhibited similar enzymatic properties to wild-type.     

Effects of mutations in poliovirus 3Dpol on RNA polymerase activity and on polyprotein cleavage.

A series of short insertion mutations was introduced into the poliovirus gene for 3Dpol at a number of different locations. When substituted for wild-type sequences in a full-length, infectious cDNA and tested for infectivity, all 3D mutants were nonviable. The mutant cDNAs were introduced into a bacterial plasmid designed to direct the expression of poliovirus 3CD, a viral protein composed of contiguous protease and RNA polymerase sequences. Bacteria transformed with these plasmids all expressed similar amounts of 3CD, and all mutant proteins cleaved themselves to generate wild-type 3Cpro and mutant 3Dpol polypeptides with approximately the same efficiency as wild-type 3CD. The released mutant 3Dpol proteins were all defective in RNA-dependent RNA polymerase activity in vitro. Uncleaved 3CD is a protease required for processing the viral capsid protein precursor, P1. In an in vitro assay of P1 cleavage activity, some of the mutant 3CD proteins expressed in Escherichia coli showed normal activity, while others were clearly inactive. Thus, alterations in the sequence and/or folding of different regions of the 3D protein have differential effects on its various activities.     

Mechanisms and kinetics of procathepsin D activation.

In vitro, procathepsin D is activated to pseudocathepsin D by incubation at low pH. To investigate the mechanism of this activation, recombinant human procathepsin D and two mutants were generated in a baculovirus expression system. One mutant carried a point mutation within the catalytic domain, which resulted in a catalytically inactive enzyme form (D77A). The other carried a point mutation within the propeptide, which prevented activation by processing at the 'autoproteolysis-site' (L26P). Neither mutant is capable of processing itself to form pseudocathepsin D, and L26P is not able to process D77A. Despite the inability of L26P to cleave either its own or a wild-type prosequence, it did exhibit activity against a synthetic peptide substrate. The ability of intact precursor (zymogen) to cleave a peptide, but not a protein substrate, offers new insights into the mechanism of inhibition by the propeptide. Mature cathepsin D can process the inactive D77A mutant to the pseudoform, demonstrating that processed species are capable of cleaving zymogen molecules in an intermolecular interaction. In addition, kinetic studies provide evidence for a two-phase mechanism for the conversion of procathepsin D to pseudocathepsin D, one phase where the first molecules of pseudocathepsin D are formed at a low rate and a second phase where the process is autocatalytically accelerated by newly formed pseudocathepsin D molecules. Finally, with the help of the mutants L26P and D77A it was observed that at least two additional proteinase activities, found in conditioned media from insect cell culture, are capable of activating procathepsin D by cleaving it within the proregion. This observation suggests that there are likely to be multiple proteinases in the extracellular matrix that are capable of activating procathepsin D, thereby triggering the second autocatalytic phase. This may also be important for solid tumors, where the presence of cathepsin D has been correlated with tumor growth and invasion.     

人体激肽释放酶2与前列腺癌

人体激肽释放酶2(human kallikrein 2,hK2)是一种主要在前列腺中表达的丝氨酸蛋白酶,近年作为前列腺癌的血清标记物受到广泛关注.随着对hK2结构特征、组织表达、生物学活性和调节,及其与前列腺癌病理过程的关系的研究更一步深入,hK2在前列腺癌诊断、病理分期及治疗中的潜在应用价值将越加瞩目.     

A sensitive proximity ligation assay for active PSA

Prostate-specific antigen (PSA) is a widely used marker for prostate cancer. The utility of PSA tests is limited by their inability to differentiate prostate cancer from non-malignant conditions such as benign prostatic hyperplasia and prostatitis. In circulation, PSA occurs in various complexed and free forms, and specific determination of some of these can be used to improve the diagnostic accuracy of PSA tests. We have previously identified peptides that specifically bind to enzymatically active PSA and using such a peptide we have developed an immunopeptidometric assay for this form of PSA. However, the sensitivity of that assay is too low to measure active PSA at clinically important levels. Recently a novel sensitive immunoassay for analysis of proteins, termed the proximity ligation assay, has been established. Here we describe a sensitive implementation of the proximity ligation assay, which utilizes a PSA-binding peptide and antibody as probes to detect active PSA. The assay has a sensitivity of 0.07 microg/l, which is approximately ten-fold lower than that of our previous assay. It does not cross-react with inactive proPSA or the highly similar kallikrein hK2. Our results show that a highly sensitive immunopeptidometric assay can be developed using proximity ligation. This principle should facilitate establishment of specific assays for active forms of other proteases.     

A novel protease homolog differentially expressed in breast and ovarian cancer.

BACKGROUND: Using differential display (DD), we discovered a new member of the serine protease family of protein-cleaving enzymes, named protease M. The gene is most closely related by sequence to the kallikreins, to prostate-specific antigen (PSA), and to trypsin. The diagnostic use of PSA in prostate cancer suggested that a related molecule might be a predictor for breast or ovarian cancer. This, in turn, led to studies designed to characterize the protein and to screen for its expression in cancer. MATERIALS AND METHODS: The isolation of protease M by DD, the cloning and sequencing of the cDNA, and the comparison of the predicted protein structure with related proteins are described, as are methods to produce recombinant proteins and polyclonal antibody preparations. Protease M expression was examined in mammary, prostate, and ovarian cancer, as well as normal, cells and tissues. Stable transfectants expressing the protease M gene were produced in mammary carcinoma cells. RESULTS: Protease M was localized by fluorescent in situ hybridization analysis to chromosome 19q13.3, in a region to which other kallikreins and PSA also map. The gene is expressed in the primary mammary carcinoma lines tested but not in the corresponding cell lines of metastatic origin. It is strongly expressed in ovarian cancer tissues and cell lines. The enzyme activity could not be established, because of difficulties in producing sufficient recombinant protein, a common problem with proteases. Transfectants were selected that overexpress the mRNA, but the protein levels remained very low. CONCLUSIONS: Protease M expression (mRNA) may be a useful marker in the detection of primary mammary carcinomas, as well as primary ovarian cancers. Other medical applications are also likely, based on sequence relatedness to trypsin and PSA.     

Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P'1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.     

Involvement of the C-terminal region of yeast proteinase B inhibitor 2 in its inhibitory action

Yeast proteinase B inhibitor 2 (YIB2), which is composed of 74 amino acid residues, is an unusual serine protease inhibitor, since it lacks disulfide bonds. To identify its reactive site for proteases, we constructed an expression system for a synthetic YIB2 gene and then attempted to change the inhibitory properties of YIB2 by amino acid replacements. The purified wild-type YIB2 inhibited the activity of subtilisin BPN', a protein homologous to yeast proteinase B, although its binding ability was not strong, and a time-dependent decrease in its inhibitory activity was observed, demonstrating that wild-type YIB2 behaves as a temporary inhibitor when subtilisin BPN' is the target protease. Since YIB2 exhibits sequence homology to the propeptide of subtilisin, which inhibits a cognate protease using its C-terminal region, we replaced the six C-termi nal residues of YIB2 with those of the propeptide of subtilisin BPN' to make the mutant YIB2m1. This mutant exhibited markedly increased inhibitory activity toward subtilisin BPN' without a time-dependent decrease in its inhibitory activity. Replacement of only the C-terminal Asn of YIB2 by Tyr, or deletion of the C-terminal Tyr of YIB2m1, inhibited subtilisin, but the ability of these mutants to bind subtilisin and their resistance to proteolytic attack were weaker than those of YIB2m1, indicating that the C-terminal residue contributes to the interaction with the protease to a greater extent than the preceding five residues and that the resistance of YIB2 to proteolyic attack is closely related to its ability to bind a protease. These results demonstrate that YIB2 is a unique protease inhibitor that involves its C-terminal region in the interaction with the protease.     

Novel peptide inhibitors of human kallikrein 2

Human kallikrein 2 (hK2) is a serine protease produced by the secretory epithelial cells in the prostate. Because hK2 activates several factors participating in proteolytic cascades that may mediate metastasis of prostate cancer, modulation of the activity of hK2 is a potential way of preventing tumor growth and metastasis. Furthermore, specific ligands for hK2 are potentially useful for targeting and imaging of prostate cancer and for assay development. We have used enzymatically active recombinant hK2 captured by a monoclonal antibody exposing the active site of the enzyme to screen phage display peptide libraries. Using libraries expressing 10 or 11 amino acids long linear peptides, we identified six different peptides binding to hK2. Three of these were shown to be specific and efficient inhibitors of the enzymatic activity of hK2 toward a peptide substrate. Furthermore, the peptides inhibited the activation of the proform of prostate-specific antigen by hK2. Amino acid substitution analyses revealed that motifs of six amino acids were required for the inhibitory activity. These peptides are potentially useful for treatment and targeting of prostate cancer.     

Disulfide-linked propeptides stabilize the structure of zymogen and mature pancreatic serine proteases.

Chymotrypsinogen and proelastase 2 are the only pancreatic proteases with propeptides that remain attached to the active enzyme via a disulfide bridge. It is likely, although not proven, that these propeptides are functionally important in the active enzymes, as well as in the zymogens. A mutant chymotrypsin was constructed to test this hypothesis, but it was demonstrated that the lack of the propeptide had no effect on the catalytic efficiency, substrate specificity, or folding of the protein [Venekei, I., et al. (1996) FEBS Lett. 379, 139-142]. In this paper, we investigate the role of the disulfide-linked propeptide in the conformational stability of chymotrypsin(ogen). We compare the stabilities of the wild-type and mutant proteins (lacking propeptide-enzyme interactions) in their zymogen (chymotrypsinogen) and active (chymotrypsin) forms. The mutants exhibited a substantially increased sensitivity to heat denaturation and guanidine hydrochloride unfolding, and a faster loss of activity at extremes of pH relative to those of their wild-type counterparts. From guanidine hydrochloride denaturation experiments, we determined that covalently linked propeptide provides about 24 kJ/mol of free energy of extra stabilization (DeltaDeltaG). In addition, the mutant chymotrypsinogen lacked the normal resistance to digestion by pepsin. This may also explain (besides keeping the zymogen inactive) the evolutionary conservation of the propeptide-enzyme interactions. Tryptophan fluorescence, circular dichroism, microcalorimetric, and activity measurements suggest that the propeptide of chymotrypsin restricts the relative mobility between the two domains of the molecule. In pancreatic serine proteases, such as trypsin, that lose the propeptide upon activation, this function appears to be accomplished via alternative interdomain contacts.     

Characterization of the enzymatic activity of human kallikrein 6: Autoactivation,substrate specificity,and regulation by inhibitors

Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer. To gain insights into the physiological role of this enzyme, we sought to determine its substrate specificity and its interactions with various inhibitors. We produced the proform of hK6 and showed that this enzyme was able to autoactivate, as well as proteolyse itself, leading to inactivation. Kinetic studies indicated that hK6 cleaved with much higher efficiency after Arg than Lys and with a preference for Ser or Pro in the P2 position. The efficient degradation of fibrinogen and collagen types I and IV by hK6 indicated that this kallikrein might play a role in tissue remodeling and/or tumor invasion and metastasis. We also demonstrated proteolysis of amyloid precursor protein by hK6 and determined the cleavage sites at the N-terminal end of the protein. Inhibition of hK6 was achieved via binding to different serpins, among which antithrombin III was the most efficient.     

The propeptide is not required to produce catalytically active neutral protease from Bacillus stearothermophilus

The thermolysin-like neutral protease from Bacillus stearothermophilus (TLP-ste) is usually produced extracellularly in Bacillus subtilis, where it is expressed as preproenzyme and subsequently processed in an autocatalytic, intramolecular process. To create the basis for the production of inactive mutants of TLP-ste, which cannot be processed in B. subtilis, we studied the expression of TLP-ste and its propeptide in cis and in trans in Escherichia coli. In contrast to thermolysin, subtilisin and alpha-lytic protease, which could be obtained only in the presence of the corresponding propeptides, TLP-ste could be produced as an active mature enzyme in E. coli in the absence of its prosequence. Surprisingly, however, a much more effective access to active mature protease was found when TLP-ste (devoid of its prosequence) was expressed as protein with an N-terminal His6 tag which accumulated in the form of inclusion bodies. Completely unexpected, the protein could be renatured from the inclusion bodies after solubilization in guanidine hydrochloride solutions in high yields. Purification to homogeneity was possible by affinity chromatography on Bacitracin silica as well as by immobilized metal ion affinity chromatography. By addition of separately expressed propeptide to the renaturation mixture yields of renaturation could not be increased significantly, confirming that the propeptide is not essential for proper folding of the enzyme or its stabilization during the folding process. Also in vivo, the expression levels of active mature TLP-ste in Escherichia coli did not significantly differ when the mature sequence was expressed alone or coexpressed with the prosequence in cis or in trans.     

Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain.

The heavy chain of tissue plasminogen activator (t-PA) consists of four domains [finger, epidermal-growth-factor (EGF)-like, kringle 1 and kringle 2] that are homologous to similar domains present in other proteins. To assess the contribution of each of the domains to the biological properties of the enzyme, site-directed mutagenesis was used to generate a set of mutants lacking sequences corresponding to the axons encoding the individual structural domains. The mutant proteins were assayed for their ability to hydrolyze artificial and natural substrates in the presence and absence of fibrin, to bind to lysine-Sepharose and to be inhibited by plasminogen activator inhibitor-1. All the deletion mutants exhibit levels of basal enzymatic activity very similar to that of wild-type t-PA assayed in the absence of fibrin. A mutant protein lacking the finger domain has a 2-fold higher affinity for plasminogen than wild-type t-PA, while the mutant that lacks both finger and EGF-like domains is less active at low concentrations of plasminogen. Mutants lacking both kringles neither bind to lysine-Sepharose nor are stimulated by fibrin. However, mutants containing only one kringle (either kringle 1 or kringle 2) behave indistinguishably from one another and from the wild-type protein. We conclude that kringle 1 and kringle 2 are equivalent in their ability to mediate stimulation of catalytic activity by fibrin.     

Corin Mutation R539C from Hypertensive Patients Impairs Zymogen Activation and Generates an Inactive Alternative Ectodomain Fragment

Corin is a cardiac transmembrane serine protease that regulates blood pressure by activating natriuretic peptides. Corin variants have been associated with African Americans with hypertension and heart disease. Here, we report a new mutation in exon 12 of the CORIN gene identified in a family of patients with hypertension. The mutation resulted in R539C substitution in the Fz2 (Frizzled-2) domain of the corin propeptide region. We expressed and characterized the corin R539C mutant in HEK293 cells. As determined by Western blot analysis, the R539C mutation did not alter corin expression in transfected cells but impaired corin zymogen activation. In a pro-atrial natriuretic peptide processing assay, the corin mutant had reduced activity and exhibited a dominant-negative effect on wild-type corin. In addition, the R539C mutation altered corin ectodomain shedding, producing an alternative ∼75-kDa fragment that was biologically inactive. Using protease inhibitors and the catalytically inactive corin mutant S985A, we showed that the ∼75-kDa fragment was generated by corin autocleavage. We constructed a series of mutants by replacing single or double Arg residues in the corin propeptide and identified Arg-530 in the Fz2 domain as the alternative autocleavage site. Our results show that the corin mutation R539C identified in hypertensive patients impairs corin zymogen activation and causes an alternative autocleavage that reduces corin activity. These data support that human CORIN gene mutations causing impaired corin activity may be an underlying mechanism in hypertension.     

Human tissue kallikrein 5 is a member of a proteolytic cascade pathway involved in seminal clot liquefaction and potentially in prostate cancer progression

Human tissue kallikreins (hKs) are a family of fifteen serine proteases. Several lines of evidence suggest that hKs participate in proteolytic cascade pathways. Human kallikrein 5 (hK5) has trypsin-like activity, is able to self-activate, and is co-expressed in various tissues with other hKs. In this study, we examined the ability of hK5 to activate other hKs. By using synthetic heptapeptides that encompass the activation site of each kallikrein and recombinant pro-hKs, we demonstrated that hK5 is able to activate pro-hK2 and pro-hK3. We then showed that, following their activation, hK5 can internally cleave and deactivate hK2 and hK3. Given the predominant expression of hK2 and hK3 in the prostate, we examined the pathophysiological role of hK5 in this tissue. We studied the regulation of hK5 activity by cations (Zn2+, Ca2+, Mg2+, Na2+, and K+) and citrate and showed that Zn can efficiently inhibit hK5 activity at levels well below its normal concentration in the prostate. We also show that hK5 can degrade semenogelins I and II, the major components of the seminal clot. Semenogelins can reverse the inhibition of hK5 by Zn2+, providing a novel regulatory mechanism of its serine protease activity. hK5 is also able to internally cleave insulin-like growth factor-binding proteins 1, 2, 3, 4, and 5, but not 6, suggesting that it might be involved in prostate cancer progression through growth factor regulation. Our results uncover a kallikrein proteolytic cascade pathway in the prostate that participates in seminal clot liquefaction and probably in prostate cancer progression.