Inhibition profiles of human tissue kallikreins by serine protease inhibitors

Accumulated evidence has shown that human tissue kallikreins (hKs), a group of 15 homologous secreted serine proteases, are novel cancer biomarkers. We report here the inhibition profiles of selected hKs, including hK5, hK7, hK8, hK11, hK12, hK13, and hK14, by several common serine protease inhibitors (serpins) found in plasma. The association constants for the binding of serpins to kallikreins were determined and compared. Protein C inhibitor was found to be the fastest-binding serpin for most of these hKs. alpha2-Antiplasmin, alpha1-antichymotrypsin, and alpha1-antitrypsin also showed rapid inhibition of certain hKs. Kallistatin exhibited fast inhibition only with hK7. Our data demonstrate that these hKs are specifically regulated by certain serpins and their distinct inhibition profiles will be valuable aids in various aspects of kallikrein research.     

Development of recombinant inhibitors specific to human kallikrein 2 using phage-display selected substrates.

The reactive site loop of serpins undoubtedly defines in part their ability to inhibit a particular enzyme. Exchanges in the reactive loop of serpins might reassign the targets and modify the serpin-protease interaction kinetics. Based on this concept, we have developed a procedure to change the specificity of known serpins. First, reactive loops are very good substrates for the target enzymes. Therefore, we have used the phage-display technology to select from a pentapeptide phage library the best substrates for the human prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J. & Deperthes, D. (2002) Eur. J. Biochem. 269, 2747-2754]. Selected substrates were then transplanted into the reactive site loop of alpha1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease. Thus, we have developed some highly specific alpha1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity. These inhibitors might be useful to help elucidate the importance of hK2 in prostate cancer progression.     

Expression and functional characterization of the cancer-related serine protease, human tissue kallikrein 14

Human tissue kallikrein 14 (KLK14) is a novel extracellular serine protease. Clinical data link KLK14 expression to several diseases, primarily cancer; however, little is known of its (patho)-physiological role. To functionally characterize KLK14, we expressed and purified recombinant KLK14 in mature and proenzyme forms and determined its expression pattern, specificity, regulation, and in vitro substrates. By using our novel immunoassay, the normal and/or diseased skin, breast, prostate, and ovary contained the highest concentration of KLK14. Serum KLK14 levels were significantly elevated in prostate cancer patients compared with healthy males. KLK14 displayed trypsin-like specificity with high selectivity for P1-Arg over Lys. KLK14 activity could be regulated as follows: 1) by autolytic cleavage leading to enzymatic inactivation; 2) by the inhibitory serpins alpha1-antitrypsin, alpha2-antiplasmin, antithrombin III, and alpha1-antichymotrypsin with second order rate constants (k(+2)/Ki) of 49.8, 23.8, 1.48, and 0.224 microM(-1) min(-1), respectively, as well as plasminogen activator inhibitor-1; and 3) by citrate and zinc ions, which exerted stimulatory and inhibitory effects on KLK14 activity, respectively. We also expanded the in vitro target repertoire of KLK14 to include collagens I-IV, fibronectin, laminin, kininogen, fibrinogen, plasminogen, vitronectin, and insulin-like growth factor-binding proteins 2 and 3. Our results indicate that KLK14 may be implicated in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage. These findings may have clinical implications for the management of cancer and other disorders in which KLK14 activity is elevated.     

Mutant recombinant serpins as highly specific inhibitors of human kallikrein 14

The reactive center loop (RCL) of serpins plays an essential role in the inhibition mechanism acting as a substrate for their target proteases. Changes within the RCL sequence modulate the specificity and reactivity of the serpin molecule. Recently, we reported the construction of alpha1-antichymotrypsin (ACT) variants with high specificity towards human kallikrein 2 (hK2) [Cloutier SM, Kündig C, Felber LM, Fattah OM, Chagas JR, Gygi CM, Jichlinski P, Leisinger HJ & Deperthes D (2004) Eur J Biochem271, 607-613] by changing amino acids surrounding the scissile bond of the RCL and obtained specific inhibitors towards hK2. Based on this approach, we developed highly specific recombinant inhibitors of human kallikrein 14 (hK14), a protease correlated with increased aggressiveness of prostate and breast cancers. In addition to the RCL permutation with hK14 phage display-selected substrates E8 (LQRAI) and G9 (TVDYA) [Felber LM, Borgo?o CA, Cloutier SM, Kündig C, Kishi T, Chagas JR, Jichlinski P, Gygi CM, Leisinger HJ, Diamandis EP & Deperthes D (2005) Biol Chem386, 291-298], we studied the importance of the scaffold, serpins alpha1-antitrypsin (AAT) or ACT, to confer inhibitory specificity. All four resulting serpin variants ACT(E8), ACT(G9), AAT(E8) and AAT(G9) showed hK14 inhibitory activity and were able to form covalent complex with hK14. ACT inhibitors formed more stable complexes with hK14 than AAT variants. Whereas E8-based inhibitors demonstrated a rather relaxed specificity reacting with various proteases with trypsin-like activity including several human kallikreins, the two serpins variants containing the G9 sequence showed a very high selectivity for hK14. Such specific inhibitors might prove useful to elucidate the biological role of hK14 and/or its implication in cancer.     

The human kallikrein protein 5 (hK5) is enzymatically active,glycosylated and forms complexes with two protease inhibitors in ovarian cancer fluids

The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Binding of kallikreins to protease inhibitors is an important mechanism for regulating their enzymatic activity and may have potential clinical applications. Human kallikrein gene 5 (KLK5) is a member of this family and encodes for a secreted serine protease (hK5). This kallikrein was shown to be differentially expressed at the mRNA and protein levels in diverse malignancies. Our objective was to study the enzymatic activity and the interaction of recombinant hK5 protein with protease inhibitors. Recombinant hK5 protein was produced in yeast and mammalian expression systems and purified by chromatography. HPLC fractionation, followed by ELISA-type assays, immunoblotting and radiolabeling experiments were performed to detect the possible interactions between hK5 and proteinase inhibitors in serum. Enzymatic deglycosylation was performed to examine the glycosylation pattern of the protein. The enzymatic activity of hK5 was tested using trypsin and chymotrypsin-specific synthetic fluorogenic substrates. In serum and ascites fluid, in addition to the free ( approximately 40 kDa) form, hK5 forms complexes with alpha(1)-antitrypsin and alpha(2)-macroglobulin. These complexes were detected by hybrid ELISA-type assays using hK5-specific coating antibodies and inhibitor detection antibodies. The ability of hK5 to bind to these inhibitors was further verified in vitro. Spiking of serum samples with 125I-labeled hK5 results in the distribution of the protein in two higher molecular mass (bound) forms, in addition to the unbound form. The hK5 mature enzyme is active and shows trypsin, but not chymotrypsin-like, activity. The pro-form of hK5 is not active. Recombinant hK5 shows a higher than predicted molecular mass due to glycosylation. hK5 is partially complexed with alpha(1)-antitrypsin and alpha(2)-macroglobulin in serum and ascites fluid of ovarian cancer patients. The recombinant protein is glycosylated and its mature form shows trypsin-like activity.     

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.     

In vivo detection of aflatoxin-induced lipid free radicals in rat bile

Human kallikrein hK3 (prostate-specific antigen) is a chymotrypsin-like serine protease which is widely used in the diagnosis of prostate cancer. Assays of the enzymatic activity of hK3 in extracellular fluids have been limited by a lack of sensitive synthetic substrates. This report describes the design of a series of internally quenched fluorescent peptides containing an amino acid sequence based on preferential hK3 cleavage sites in semenogelins. Those were identified by 2-D gel electrophoresis analysis and N-terminal sequencing of semenogelin fragments generated by ex vivo proteolysis in freshly ejaculated semen. These peptides were cleaved by hK3 at the C-terminal of certain tyrosyl or glutaminyl residues with k(cat)/K(m) values of 15000-60000 M(-1) s(-1). The substrate Abz-SSIYSQTEEQ-EDDnp was cleaved at the Tyr-Ser bond with a specificity constant k(cat)/K(m) of 60000 M(-1) s(-1), making it the best substrate for hK3 described to date.     

Structural basis of the zinc inhibition of human tissue kallikrein 5

Human kallikrein 5 (hK5) is a member of the tissue kallikrein family of serine peptidases. It has trypsin-like substrate specificity, is inhibited by metal ions, and is abundantly expressed in human skin, where it is believed to play a central role in desquamation. To further understand the interaction of hK5 with substrates and metal ions, active recombinant hK5 was crystallized in complex with the tripeptidyl aldehyde inhibitor leupeptin, and structures at 2.3 A resolution were obtained with and without Zn2+. While the overall structure and the specificity of S1 pocket for basic side-chains were similar to that of hK4, a closely related family member, both differed in their interaction with Zn2+. Unlike hK4, the 75-loop of hK5 is not structured to bind a Zn2+. Instead, Zn2+ binds adjacent to the active site, becoming coordinated by the imidazole rings of His99 and His96 not present in hK4. This zinc binding is accompanied by a large shift in the backbone conformation of the 99-loop and by large movements of both His side-chains. Modeling studies show that in the absence of bound leupeptin, Zn2+ is likely further coordinated by the imidazolyl side-chain of the catalytic His57 which can, similar to equivalent His57 imidazole groups in the related rat kallikrein proteinase tonin and in an engineered metal-binding rat trypsin, rotate out of its triad position to provide the third co-ordination site of the bound Zn2+, rendering Zn2+-bound hK5 inactive. In solution, this mode of binding likely occurs in the presence of free and substrate saturated hK5, as kinetic analyses of Zn2+ inhibition indicate a non-competitive mechanism. Supporting the His57 re-orientation, Zn2+ does not fully inhibit hK5 hydrolysis of tripeptidyl substrates containing a P2-His residue. The P2 and His57 imidazole groups would lie next to each other in the enzyme-substrate complex, indicating that incomplete inhibition is due to competition between both imidazole groups for Zn2+. The His96-99-57 triad is thus suggested to be responsible for the Zn2+-mediated inhibition of hK5 catalysis.     

Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI)

LEKTI is a 120-kDa protein that plays an important role in skin development, as mutations affecting LEKTI synthesis underlie Netherton syndrome, an inherited skin disorder producing severe scaling. Its primary sequence indicates that the protein consists of 15 domains, all resembling a Kazal-type serine protease inhibitor. LEKTI and two serine proteases belonging to the human tissue kallikrein (hK) family (hK5 and hK7) are expressed in the granular layer of skin. In this study, we characterize the interaction of two recombinant LEKTI fragments containing three or four intact Kazal domains (domains 6-8 and 9-12) with recombinant rhK5, a trypsin-like protease, and recombinant rhK7, a chymotrypsin-like protease. Both fragments inhibited rhK5 similarly in binding and kinetic studies performed at pH 8.0, as well as pH 5.0, the pH of the stratum corneum where both LEKTI and proteases may function. Inhibition equilibrium constants (Ki) measured either directly in concentration-dependent studies or calculated from measured association (kass) and dissociation (kdis) rate constants were 1.2-5.5 nM at pH 8.0 and 10-20 nM at pH 5.0. At pH 8.0, kass and kdis values were 4.7 x 10(5) M(-1) s(-1) and 5.5 x 10(-4) s(-1), and at pH 5.0 they were 4.0 x 10(4) M(-1) s(-1) and 4.3 x 10(-4) s(-1), respectively. The low Ki and kdis values (t1/2 of 20-25 min) indicate tight and specific association. Only fragment 6-9' was a good inhibitor of rhK7, demonstrating a Ki of 11 nM at pH 8.0 in a reaction that was rapidly reversible. These results show that LEKTI, at least in fragment form, is a potent inhibitor of rhK5 and that this protease may be a target of LEKTI in human skin.     

Human kallikrein 6 activity is regulated via an autoproteolytic mechanism of activation/inactivation

Human kallikrein 6 (protease M/zyme/neurosin) is a serine protease that has been suggested to be a serum biomarker for ovarian cancer and may also be involved in pathologies of the CNS. The precursor form of human kallikrein 6 (pro-hK6) was overexpressed in Pichia pastoris and found to be autoprocessed to an active but unstable mature enzyme that subsequently yielded the inactive, self-cleavage product, hK6 (D81-K244). Site-directed mutagenesis was used to investigate the basis for the intrinsic catalytic activity and the activation mechanism of pro-hK6. A single substitution R80 --> Q stabilized the activity of the mature enzyme, while substitution of the active site serine (S197 --> A) resulted in complete loss of hK6 proteolytic activity and facilitated protein production. Our data suggest that the enzymatic activity of hK6 is regulated by an autoactivation/autoinactivation mechanism. Mature hK6 displayed a trypsin-like activity against synthetic substrates and human plasminogen was identified as a putative physiological substrate for hK6, as specific cleavage at the plasminogen internal bond S460-V461 resulted in the generation of angiostatin, an endogenous inhibitor of angiogenesis and metastatic growth.     

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.     

Inhibition of rat tissue kallikrein gene family members by rat kallikrein-binding protein and alpha 1-proteinase inhibitor.

The regulation of tissue kallikrein activity by plasma serine proteinase inhibitors (serpins) was investigated by measuring the association rate constants of six tissue-kallikrein family members isolated from the rat submandibular gland, with rat kallikrein-binding protein (rKBP) and alpha 1-proteinase inhibitor (alpha 1-PI). Both these serpins inhibited kallikreins rK2, rK7, rK8, rK9 and rK10 with association rate constants in the 10(3)-10(4) M-1.s-1 range, whereas only 'true' tissue kallikrein rK1 was not susceptible to alpha 1-PI. This results in slow inhibition of rK1 by plasma serpins, which could explain why this kallikrein is the only member of the gene family identified so far that induces a transient decrease in blood pressure when injected in minute amounts into the circulation.     

Biochemical characterization of human kallikrein 8 and its possible involvement in the degradation of extracellular matrix proteins

Human kallikrein 8 (KLK8) is a member of the human kallikrein gene family of serine proteases, and its protein, hK8, has recently been suggested to serve as a new ovarian cancer marker. To gain insights into the physiological role of hK8, the active recombinant enzyme was obtained in a pure state for biochemical and enzymatic characterizations. hK8 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. The protease degraded casein, fibronectin, gelatin, collagen type IV, fibrinogen, and high-molecular-weight kininogen. hK8 also converted human single-chain tissue-type plasminogen activator (65 kDa) to its two-chain form (32 and 33 kDa) by specifically cleaving the peptide bond Arg275-Ile276. This conversion resulted in a drastic increase in the activity of the activator toward the fluorogenic substrate Pyr-Gly-Arg-MCA and plasminogen in the absence of fibrin. Our findings suggest that hK8 may be implicated in ECM protein degradation in the area surrounding hK8-producing cells.     

Amidolytic activity of prostatic acid phosphatase on human semenogelins and semenogelin-derived synthetic substrates.

In addition to kallikrein hK3, a serine protease generally reported as PSA (prostate-specific antigen), at least two other enzymes in human seminal plasma also cleave synthetic peptidyl substrates derived from the sequence of human semenogelins. We have identified one of these as prostatic acid phosphatase (PAP), a major component of prostatic fluid whose physiological function is unclear. The other is a high Mr basic protein present at low concentrations in seminal plasma and that remains to be characterized. PAP was purified to homogeneity from freshly ejaculated seminal plasma. Its N-terminal sequence and its phosphatase properties (hydrolysis of para-nitrophenylphosphate at low pH) were determined, and its inhibition by sodium fluoride measured. Both purified and commercial PAP also had amidolytic activity on peptide substrates derived from the semenogelin sequence at neutral and slightly basic pH. The k(cat)/K(m) values were in the 10(2)-10(3) m(-1) x s(-1) range using fluorogenic semenogelin-derived substrates whose peptidyl moiety included cleavage sites that had been identified ex vivo. PAP cleavage sites differed from those of hK3 and were mainly at P1 = Gln residues or between residues bearing hydroxyl groups. PAP amidolytic activity was poorly inhibited by all currently used wide spectrum proteinase inhibitors. Only 3-4 dichloroisocoumarin and benzamidine inhibited purified PAP. Purified human semenogelin was cleaved by purified and commercial PAP at neutral pH; the two main cleavage sites were at Tyr292 and Ser170 (semenogelin I sequence), only the former has been identified ex vivo by analysis of seminal plasma.     

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.     

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.     

Substrate specificity of human kallikrein 6: salt and glycosaminoglycan activation effects

Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km 38,667 s(-1) mm(-1)). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Abeta amyloid peptide, reactive center loop of alpha1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1-4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.     

Interplay of human tissue kallikrein 4 (hK4) with the plasminogen activation system: hK4 regulates the structure and functions of the urokinase-type plasminogen activator receptor (uPAR)

The plasminogen activation system is involved in cancer progression and metastasis. Among other proteolytic factors, it includes the serine protease urokinase-type plasminogen activator (uPA) and its three-domain (D1D2D3) receptor uPAR (CD87), which focuses plasminogen activation to the cell surface. The function of uPAR is regulated in part through shedding of domain D1 by proteases, e.g., uPA itself or plasmin. Human tissue kallikrein 4 (hK4), which is highly expressed in prostate and ovarian tumor tissue, was previously shown to cleave and activate the pro-enzyme forms of prostate-specific antigen (PSA, tissue kallikrein hK3) and uPA. Here we demonstrate that uPAR is also a target for hK4, being cleaved in the D1-D2 linker sequence and, to a lesser extent, in its D3 juxtamembrane domain. hK4 may thus modulate the tumor-associated uPA/uPAR-system activity by either activating the pro-enzyme form of uPA or cleaving the cell surface-associated uPA receptor.     

Expression and enzymatic characterization of recombinant human kallikrein 14

Human kallikrein 14 (KLK14) is a member of the human kallikrein gene family of serine proteases, and its protein, hK14, has recently been suggested to serve as a new ovarian and breast cancer marker. To gain insights into hK14's physiological functions, the active recombinant enzyme was obtained in an enzymatically pure state for biochemical and enzymatic characterizations. We studied its substrate specificity and behavior to various protease inhibitors, and identified candidate physiological substrates. hK14 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. The protease degraded casein, fibronectin, gelatin, collagen type I, collagen type IV, fibrinogen, and high-molecular-weight kininogen. Furthermore, it rapidly hydrolyzed insulin-like growth factor binding protein-3 (IGFBP-3). These findings suggest that hK14 may be implicated in tumor progression in ovarian carcinoma.     

Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.