Crystal structure of recombinant human tissue kallikrein at 2.0 A resolution.

Human tissue kallikrein, a trypsin-like serine protease involved in blood pressure regulation and inflammation processes, was expressed in a deglycosylated form at high levels in Pichia pastoris, purified, and crystallized. The crystal structure at 2.0 A resolution is described and compared with that of porcine kallikrein and of other trypsin-like proteases. The active and S1 sites (nomenclature of Schechter I, Berger A, 1967, Biochem Biophys Res Commun 27:157-162) are similar to those of porcine kallikrein. Compared to trypsin, the S1 site is enlarged owing to the insertion of an additional residue, cis-Pro 219. The replacement Tyr 228 --> Ala further enlarges the S1 pocket. However, the replacement of Gly 226 in trypsin with Ser in human tissue kallikrein restricts accessibility of substrates and inhibitors to Asp 189 at the base of the S1 pocket; there is a hydrogen bond between O delta1Asp189 and O gammaSer226. These changes in the architecture of the S1 site perturb the binding of inhibitors or substrates from the modes determined or inferred for trypsin. The crystal structure gives insight into the structural differences responsible for changes in specificity in human tissue kallikrein compared with other trypsin-like proteases, and into the structural basis for the unusual specificity of human tissue kallikrein in cleaving both an Arg-Ser and a Met-Lys peptide bond in its natural protein substrate, kininogen. A Zn+2-dependent, small-molecule competitive inhibitor of kallikrein (Ki = 3.3 microM) has been identified and the bound structure modeled to guide drug design.     

The cleavage of the Met-Lys bond in a bradykinin derivative by glandular kallikreins

The synthetic tridecapeptide Gly-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg was used as a model substrate for horse urinary and porcine pancreatic kallikreins. The Met-Lys bond is hydrolyzed selectively by both enzymes. Oxidation of the methionine residue to sulfoxide made the peptide resistant to both kallikreins. Substitution of either the methionine or lysine residues by norleucine led to peptides in which the Nle-Lys or the Met-Nle bonds, respectively, were susceptible to the urinary kallikrein. The esterolytic and Met-Lys bond-splitting activities of both enzymes were inhibited similarly by phenylmethanesulfonyl fluoride. Both activities of the pancreatic kallikrein were inhibited by the chloromethane derivative Ala-Leu-Lys-CH2Cl. Inhibition by benzamidine of Met-Lys hydrolysis by both kallikreins was observed.     

Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides

We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.     

Crystal structure and biochemical characterization of human kallikrein 6 reveals that a trypsin-like kallikrein is expressed in the central nervous system

The human kallikreins are a large multigene family of closely related serine-type proteases. In this regard, they are similar to the multigene kallikrein families characterized in mice and rats. There is a much more extensive body of knowledge regarding the function of mouse and rat kallikreins in comparison with the human kallikreins. Human kallikrein 6 has been proposed as the homologue to rat myelencephalon-specific protease, an arginine-specific degradative-type protease abundantly expressed in the central nervous system and implicated in demyelinating disease. We present the x-ray crystal structure of mature, active recombinant human kallikrein 6 at 1.75-A resolution. This high resolution model provides the first three-dimensional view of one of the human kallikreins and one of only a few structures of serine proteases predominantly expressed in the central nervous system. Enzymatic data are presented that support the identification of human kallikrein 6 as the functional homologue of rat myelencephalon-specific protease and are corroborated by a molecular phylogenetic analysis. Furthermore, the x-ray data provide support for the characterization of human kallikrein 6 as a degradative protease with structural features more similar to trypsin than the regulatory kallikreins.     

Crystal structure of a prostate kallikrein isolated from stallion seminal plasma: a homologue of human PSA

Prostate-specific kallikrein, a member of the gene family of serine proteases, was initially discovered in semen and is the most useful serum marker for prostate cancer diagnosis and prognosis. We report the crystal structure at 1.42A resolution of horse prostate kallikrein (HPK). This is the first structure of a serine protease purified from seminal plasma. HPK shares extensive sequence homology with human prostate-specific antigen (PSA), including a predicted chymotrypsin-like specificity, as suggested by the presence of a serine residue at position S1 of the specificity pocket. In contrast to other kallikreins, HPK shows a structurally distinct specificity pocket. Its entrance is blocked by the kallikrein loop, suggesting a possible protective or substrate-selective role for this loop. The HPK structure seems to be in an inactivated state and further processing might be required to allow the binding of substrate molecules. Crystal soaking experiments revealed a binding site for Zn(2+) and Hg(2+), two known PSA inhibitors.     

Nucleotide sequence of cloned cDNA for human pancreatic kallikrein

Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.     

Characterization of kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. A kallikrein-like enzyme from the venom of Crotalus ruber ruber (red rattlesnake) had been isolated and characterized by Mori and Sugihara. The enzyme was active upon the kallikrein substrates, Pro-Phe-Arg-MCA and z-Phe-Arg-MCA, and slightly hydrolyzed Boc-Val-Leu-Lys-MCA, and Boc-Phe-Ser-Arg-MCA. 2. Unlike thrombin, the newly isolated kallikrein-like enzyme did not cause formation of a fibrin clot when fibrinogen was mixed with the enzyme. 3. The B beta chain of fibrinogen was first split and A alpha chain was cleaved later. Pancreatic kallikrein hydrolyzed only the A alpha chain without affecting the B beta chain. 4. The kallikrein-like enzyme produced kallidin (Lys-bradykinin) by splitting the Met-Lys bond instead of producing bradykinin. 5. The kallikrein analog JSI-450 (Ac-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser-NH2) was also cleaved at the site of the Arg-Ser bond. 6. Its NH2-terminal amino acid sequence (Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-Arg-Pro-Phe-Leu-Val-Ala-Leu-Tyr- Asp-Ser-) is homologous to the rat pancreatic kallikrein and other snake venom proteases.     

Human kallikrein 4: enzymatic activity, inhibition, and degradation of extracellular matrix proteins

Human kallikrein 4 (hK4) is a member of the expanded family of human kallikreins, a group of 15 secreted proteases. While this protein has been associated with ovarian and prostate cancer prognosis, only limited functional information exists. Therefore, we have undertaken an investigation of its enzymatic properties regarding substrate preference, degradation of extracellular matrix proteins, and its inhibition by various inhibitors. We successfully expressed and purified active recombinant hK4 from supernatants of the Pichia pastoris expression system. This enzyme seems to cleave more efficiently after Arg compared to Lys at the P1 position and exhibits modest specificity for amino acids at positions P2 and P3. hK4 forms complexes with alpha1-antitrypsin, alpha2-antiplasmin and alpha2-macroglobulin. The protease mediates limited degradation of extracellular matrix proteins such as collagen I and IV, and more efficient degradation of the alpha-chain of fibrinogen. The cleavage of extracellular matrix proteins by hK4 suggests that this enzyme may play a role in tissue remodeling and cancer metastasis.     

Prostatic kallikreins: biochemistry and physiology

This review describes and compares the properties of seven individual kallikreins present in the prostate of four mammalian species. The first kallikrein discovered in prostate was the one of guinea-pig. That protein has kininogenase activity like classical kallikreins. The rat prostate expresses two different kallikreins, S3 and P1, whose physiological functions remain to be determined precisely. In man, prostate-specific antigen (PSA) is an abundant secretory protein. It is currently used as a prostate cancer marker. The human prostate may also contain renal/pancreatic kallikrein and human glandular kallikrein-1 (hGK-1). Arginine esterase secreted by dog prostate is probably the most abundant kallikrein. It has no known physiological substrate.     

The kallikrein world: an update on the human tissue kallikreins

Human tissue kallikreins (hKs) are attracting increased attention owing to their association with various forms of cancer and other diseases. Human tissue kallikrein genes represent the largest contiguous group of proteases within the human genome. There are many areas of kallikrein research that need to be further explored, including their tissue expression patterns, their regulation, identification of specific substrates, their participation in proteolytic cascades, and their clinical applicability as cancer biomarkers and therapeutic targets. In this review, we briefly describe the current status of kallikrein research and identify future avenues that will enhance our understanding of their function and involvement in human diseases.     

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.     

Tetrapeptide substrates for the discrimination among kallikreins and other trypsin-like serine proteinases

The three tetrapeptides Ac-Phe-Arg-Arg-Val-NH2 (I), Ac-Phe-Arg-Arg-Pro-NH2 (II) and Ac-Phe-Lys-Arg-Val-NH2 (III) were shown to form a most convenient substrate system for the discrimination of the serine proteinases listed below. Tissue kallikreins (porcine pancreatic, horse and human urinary) have the unique feature of cleaving well the Arg-Arg bond in peptide I (P'2 = Val), hardly splitting it in peptide II (P'2 = Pro). The kcat/Km for the hydrolysis of peptide II by horse urinary kallikrein was 600-fold lower than that for peptide I. Trypsin, plasma kallikreins (human and rat), tonin and rat urinary kallikrein were distinguished from each other by the sequence of the N-terminal fragments formed in the hydrolysis of peptides I and/or II. Differences in the cleavage sites in these peptides are explained by differences in the specificities of the proteinase subsite S2 and/or in their preference for Arg or Lys residues. The three tetrapeptides were not substrates for plasmin.     

Amino acid sequence of guinea pig prostate kallikrein

The primary structure of the major arginine esteropeptidase from guinea pig prostate has been deduced from automated Edman degradation of peptides generated by clostripain, cyanogen bromide, endoproteinase Lys-C, and Staphylococcus aureus V8 protease digestion of the protein. The esteropeptidase is a single polypeptide chain comprised of 239 amino acids and contains 2 apparent sites of carbohydrate attachment, Asn-78 and Asn-169. Both occur in consensus sequences for N-linked glycosylation sites. The esteropeptidase exhibits approximately 35% homology with trypsin including conservation of the catalytic residues and the aspartic acid which confers specificity toward basic amino acids. The sequence identity, however, extends to greater than 60% with the kallikrein family of serine proteases. In addition to the overall homology, the guinea pig enzyme displays a number of features characteristic of kallikreins including 10 conserved half-cystine residues, a C-terminal proline, and the "kallikrein loop". On the basis of this structural relatedness, the enzyme has been designed as guinea pig prostate kallikrein. In contrast to many of the kallikreins of other species and tissues, this enzyme does not contain any sites within the kallikrein loop sensitive to proteases that result in internal breaks in the polypeptide chain.     

Mapping of human plasma kallikrein active site by design of peptides based on modifications of a Kazal-type inhibitor reactive site

Human plasma kallikrein (huPK) is a proteinase that participates in several biological processes. Although various inhibitors control its activity, members of the Kazal family have not been identified as huPK inhibitors. In order to map the enzyme active site, we synthesized peptides based on the reactive site (PRILSPV) of a natural Kazal-type inhibitor found in Cayman plasma, which is not an huPK inhibitor. As expected, the leader peptide (Abz-SAPRILSPVQ-EDDnp) was not cleaved by huPK. Modifications to the leader peptide at P'1, P'3 and P'4 positions were made according to the sequence of a phage display-generated recombinant Kazal inhibitor (PYTLKWV) that presented huPK-binding ability. Novel peptides were identified as substrates for huPK and related enzymes. Both porcine pancreatic and human plasma kallikreins cleaved peptides at Arg or Lys bonds, whereas human pancreatic kallikrein cleaved bonds involving Arg or a pair of hydrophobic amino acid residues. Peptide hydrolysis by pancreatic kallikrein was not significantly altered by amino acid replacements. The peptide Abz-SAPRILSWVQ-EDDnp was the best substrate and a competitive inhibitor for huPK, indicating that Trp residue at the P'4 position is important for enzyme action.     

Characterization and purification of a kallikrein from human pancreatic juice and immunological comparison with other kallikreins.

A prekallikrein has been demonstrated in human pancreatic juice and the active enzyme has been purified from this material. The purification procedure included filtration on Sephadex G-100, chromatography on DEAE-cellulose and affinity chromatography on trypsin-inhibitor Sepharose. The purified kallikrein appeared to be homogeneous by polyacrylamide gel electrophoresis at pH 8.3 and by immunoelectrophoresis. Human pancreatic kallikrein is immunologically different from human plasma kallikrein and from pancreatic kallikreins of other species (hog, cat, rat and dog). Human pancreatic kallikrein has common antigenic determinants with human urinary and submandibular kallikreins but probably not with parotid kallikrein.     

S(1)' and S(2)' subsite specificities of human plasma kallikrein and tissue kallikrein 1 for the hydrolysis of peptides derived from the bradykinin domain of human kininogen

The S(1)' and S(2)' subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO(3)H(2))]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P(1)' and P(2)' positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS(390)S(391)RI-NH(2) was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S(390) and S(391) phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO(3)H(2))(391)RI-NH(2) was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg(9))-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S(1)' subsite, with lower specificity for the S(2)' subsite. Abz-MISLMKRPPGFSPFRSSRI-NH(2) was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO(3)H(2)) were poorly hydrolyzed. In conclusion, S(1)' and S(2)' subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.     

Hydrolysis by plasma kallikrein of fluorogenic peptides derived from prorenin processing site

Human plasma kallikrein (HPK) activates plasma prorenin to renin, and the physiological significance of this activation is still unknown. In this paper we investigated the efficiency and the cleavage pattern of the hydrolysis by HPK of the internally quenched fluorescent peptides (qf-peptides) derived from the amino acid sequence of human prorenin cleavage site. The peptide Abz-F-S-Q-P-M-K-R-L-T-L-G-N-T-T-Q-EDDnp (Abz=ortho-aminobenzoic acid, and EDDnp=N-[2,4-dinitrophenyl]-ethylene diamine), that corresponds to the amino acid sequence P(7) to P(7)' of human prorenin cleavage site, is hydrolyzed at the correct processing site (R-L bond) with k(cat)/K(m)=85 mM(-1) s(-1). Alanine was scanned in all positions from P(5) to P(5)' in order to investigate the substrate specificity requirements of HPK.The qf-peptides derived from the equivalent segment of rat prorenin, that has Lys-Lys as basic amino acid pair, and the peptide Abz-NVTSPVQ-EDDnp that contains the proposed cleavage site of rat prorenin have very low susceptibility to hydrolysis by rat plasma kallikrein. These data are according to the previously reported absence of rat plasma prorenin activation by rat plasma kallikrein (RPK), and with the view that prorenin activation in rat requires alternative enzymes and/or mechanism.All the obtained peptides described in this paper were also assayed with bovine trypsin that was taken as a reference protease because it is commonly used to activate prorenin.     

Human tissue kallikrein S1 subsite recognition of non-natural basic amino acids

We explored the unique substrate specificity of the primary S(1) subsite of human urinary kallikrein (hK1), which accepts both Phe and Arg, using internally quenched fluorescent peptides Abz-F-X-S-R-Q-EDDnp and Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp [Abz is o-aminobenzoic acid; EDDnp is N-(2,4-dinitrophenyl)ethylenediamine], which were based on the human kininogen sequence at the C-terminal region of bradykinin. Position X, which in natural sequence stands for Arg, received the following synthetic basic non-natural amino acids: 4-(aminomethyl)phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-(aminomethyl)-N-isopropylphenylalanine (Iaf), N(im)-(dimethyl)histidine [H(2Me)], 3-pyridylalanine (Pya), 4-piperidinylalanine (Ppa), 4-(aminomethyl)cyclohexylalanine (Ama), and 4-(aminocyclohexyl)alanine (Aca). Only Abz-F-Amf-S-R-Q-EDDnp and Abz-F-H(2Me)]-S-R-Q-EDDnp were efficiently hydrolyzed, and all others were resistant to hydrolysis. However, Abz-F-Ama-S-R-Q-EDDnp inhibited hK1 with a K(i) of 50 nM with high specificity compared to human plasma kallikrein, thrombin, plasmin, and trypsin. The Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp series were more susceptible to hK1, although the peptides with Gnf, Pya, and Ama were resistant to it. Unexpectedly, the peptides in which X is His, Lys, H(2Me), Amf, Iaf, Ppa, and Aca were cleaved at amino or at carboxyl sites of these amino acids, indicating that the S(1)' subsite has significant preference for basic residues. Human plasma kallikrein did not hydrolyze any peptide of this series except the natural sequence where X is Arg. In conclusion, the S(1) subsite of hK1 accepts amino acids with combined basic and aromatic side chain, although for the S(1)-P(1) interaction the preference is for aliphatic and basic side chains.