Inosine, not adenosine, initiates endothelial glycocalyx degradation in cardiac ischemia and hypoxia.

Ischemia/reperfusion and hypoxia/reoxygenation of the heart both induce shedding of the coronary endothelial glycocalyx. The processes leading from an oxygen deficit to shedding are unknown. An involvement of resident perivascular cardiac mast cells has been proposed. We hypothesized that either adenosine or inosine or both, generated by nucleotide catabolism, attain the concentrations in the interstitial space sufficient to stimulate A3 receptors of mast cells during both myocardial ischemia/reperfusion and hypoxia/reoxygenation. Isolated hearts of guinea pigs were subjected to either normoxic perfusion (hemoglobin-free Krebs-Henseleit buffer equilibrated with 95% oxygen), 20 minutes hypoxic perfusion (buffer equilibrated with 21% oxygen) followed by 20 minutes reoxygenation, or 20 minutes stopped-flow ischemia followed by 20 minutes normoxic reperfusion (n = 7 each). Coronary venous effluent was collected separately from so-called transudate, a mixture of interstitial fluid and lymphatic fluid appearing on the epicardial surface. Adenosine and inosine were determined in both fluid compartments using high-performance liquid chromatography. Damage to the glycocalyx was evident after ischemia/reperfusion and hypoxia/reoxygenation. Adenosine concentrations rose to a level of 1 µM in coronary effluent during hypoxic perfusion, but remained one order of magnitude lower in the interstitial fluid. There was only a small rise in the level during postischemic perfusion. In contrast, inosine peaked at over 10 µM in interstitial fluid during hypoxia and also during reperfusion, while effluent levels remained relatively unchanged at lower levels. We conclude that only inosine attains levels in the interstitial fluid of hypoxic and postischemic hearts that are sufficient to explain the activation of mast cells via stimulation of A3-type receptors.

Shedding of the coronary endothelial glycocalyx: effects of hypoxia/reoxygenation vs ischaemia/reperfusion.

BACKGROUND: Vascular endothelium is covered by a glycocalyx. Damage to the glycocalyx after systemic inflammation or ischaemia/reperfusion contributes to increased vascular permeability and leucocyte adhesion. The underlying mechanisms leading to ischaemia/reperfusion-induced glycocalyx shedding are incompletely understood, in terms of lack of oxygen, absence of flow, or return of oxygen. METHODS: Isolated guinea pig hearts perfused with Krebs-Henseleit buffer at 37°C underwent 20 min of either stopped-flow ischaemia or hypoxic perfusion with subsequent reperfusion/reoxygenation (n = 6 each). Hearts perfused with normoxic buffer served as time controls. Epicardial transudate was collected to assess coronary net fluid filtration, colloid extravasation, and histamine release by mast cells. Syndecan-1 and heparan sulphate were measured in coronary effluent, together with lactate, purines, and the release of mast-cell tryptase ß. Additional hearts were perfusion-fixed to visualize the glycocalyx. RESULTS: Both ischaemia and hypoxia with reperfusion/reoxygenation resulted in significant increases in net fluid filtration (P < 0.05) and release of syndecan-1 and heparan sulphate in coronary effluent. These effects were already seen with the onset of hypoxic perfusion. Histamine was released during hypoxia and reoxygenation and also reperfusion, as was tryptase ß, and high concentrations of adenosine (>1 µmol litre⁻¹, hypoxia group) and inosine (> 7 µmol litre⁻¹, ischaemia group) were measured in effluent (P < 0.05). Damage to the coronary glycocalyx was evident upon electron microscopy. CONCLUSIONS: Both ischaemic and hypoxic hypoxia initiate glycocalyx degradation, promoting an increase in permeability. A contributing mechanism could be purine-mediated degranulation of resident mast cells, with liberated tryptase ß acting as potential 'sheddase'.

Sevoflurane preserves the endothelial glycocalyx against ischaemia-reperfusion injury.

BACKGROUND: Healthy vascular endothelium is coated by the glycocalyx, important in multiple endothelial functions, but destroyed by ischaemia-reperfusion. The impact of volatile anaesthetics on this fragile structure has not been investigated. We evaluated the effect of cardiac pre- and post-conditioning with sevoflurane on integrity of the endothelial glycocalyx in conjunction with coronary vascular function. METHODS: Isolated guinea pig hearts perfused with Krebs-Henseleit buffer underwent 20 min stopped-flow ischaemia (37 degrees C), either without or with 1 MAC sevoflurane. This was applied for 15 min before, for 20 min after, or both before and after ischaemia. Transudate was collected for assessing coronary net fluid extravasation and histamine release by mast cells. Coronary release of syndecan-1 and heparan sulphate was measured. In additional experiments with and without continuous sevoflurane, cathepsin B and tryptase beta-like protease activity were measured in effluent. Hearts were perfusion-fixed to visualize the endothelial glycocalyx. RESULTS: Ischaemia led to a significant (P<0.05) increase by 70% in transudate formation during reperfusion only in hearts without sevoflurane. This was accompanied by significant (P<0.05) increases in heparan sulphate (four-fold) and syndecan release (6.5-fold), with electron microscopy revealing massive degradation of glycocalyx. After ischaemia, histamine was released into transudate, and cathepsin B activity increased in effluent (P<0.05). Sevoflurane application attenuated all these changes, except for histamine release. CONCLUSIONS: Sevoflurane protects the endothelial glycocalyx from ischaemia-reperfusion-induced degradation, with both preconditioning and rapid post-conditioning being successful. The mechanism seems to involve attenuation of lysosomal cathepsin B release and to be independent from tissue mast cell degranulation.

Protease-activated receptor 2 mediates the proinflammatory effects of synovial mast cells.

OBJECTIVE: Mast cells are hypothesized to play a role in the pathogenesis of rheumatoid arthritis (RA) by mechanisms requiring elucidation. Tryptase released from these cells can activate protease-activated receptor 2 (PAR-2), which was recently shown to have proinflammatory actions. The purpose of this study was to examine the relationship between synovial mast cells and PAR-2. Mast cell proximity to PAR-2-expressing cells was investigated in RA synovium. In murine studies, we assessed the capacity of mast cell tryptase to mediate synovial proinflammatory responses via PAR-2 and whether degranulating mast cells induced synovial hyperemia by PAR-2 activation. METHODS: RA synovial tissue was examined by immunohistochemistry. PAR-2(+/+) and PAR-2(-/-) C57BL/6J mice were used to investigate the PAR-2 dependence of compound 48/80-induced synovial hyperemia, as measured by laser Doppler imaging, and joint swelling and hyperemic responses to recombinant human beta-tryptase. RESULTS: Mast cells and synovial lining cells staining for PAR-2 were colocalized in RA articular tissue. Compound 48/80 administration resulted in vasodilatation in PAR-2(+/+) mice but not in PAR-2(-/-) mice, which showed a vasoconstrictor response. Eliminating the 5-hydroxytryptamine-mediated component of this response with methysergide unveiled an enhanced PAR-2-mediated vasodilatation to compound 48/80 in PAR-2(+/+) mice and ablated the vasoconstrictor response in PAR-2(-/-) mice. Treatment with beta-tryptase resulted in dose-dependent knee joint swelling and synovial vasodilatation in PAR-2(+/+) mice but not PAR-2(-/-) mice. CONCLUSION: This in vivo study is the first to explore the relationship between synovial mast cells and PAR-2. Our results support the hypothesis that mast cells contribute to the pathogenesis of inflammatory arthritis through PAR-2 activation via release of mast cell tryptase.

Mast cell tryptase beta as a target in allergic inflammation: an evolving story.

Tryptases comprise a group of trypsin-like serine proteases that are highly and selectively expressed in mast cells and to a lesser extent in basophils. Among them interest has been focused on tryptase beta, primarily because it was the first tryptase identified and because it is the predominant protease and protein component of mast cells. Subsequent studies have provided convincing evidence that tryptase beta is not only a clinically useful marker of mast cells and their activation but that it contributes to the pathogenesis of allergic inflammatory disorders, most notably asthma. The pathogenetic relevance together with the apparent lack of overt physiological functions has caused considerable interest in beta-tryptase as a potential therapeutic target. Meanwhile diverse tryptase inhibitors have been synthesized whose design in part was fostered by the structural analysis of the enzymatically active beta tryptase tetramer. Various compounds have been studied both in animal models and in man, providing proof of principle that tryptase inhibitors have therapeutic potential in asthma. Here we review the rationale to develop tryptase inhibitors and the approaches pursued, and also try to pinpoint some of the problems that hamper the development of clinically applicable drugs.

Development of testicular inflammation in the rat involves activation of proteinase-activated receptor-2.

Mast cells are involved in early events crucial to inflammation and autoimmune disease. Recently, proteinase-activated receptor-2 (PAR(2)), a G-protein coupled receptor important to injury responses, was shown to be activated by mast cell tryptase. To investigate whether mast cells and PAR(2) are involved in the development and/or aggravation of testicular inflammation, we studied acute and chronic inflammatory models in the rat. In normal testes, PAR(2) was detected immunohistochemically in macrophages, in peritubular cells (PTCs) and in spermatid acrosomes. In experimentally induced autoimmune orchitis (EAO), PAR(2) was strongly upregulated in macrophages and peritubular-like cells, forming concentric layers around granulomas. Mast cells increased 10-fold in number, were more widely distributed throughout the interstitial tissue, and were partially degranulated. Isolated PTCs expressed functional PAR(2), responded to PAR(2) activation by phosphorylating extracellular signal-regulated kinases 1/2 (ERK1/2) and activating protein kinase c, and increased intracellular Ca(2+) concentrations as well as monocyte chemoattractant protein-1 (MCP-1), transforming growth factor beta(2) (TGFbeta(2)), and cyclooxygenase-2 (COX-2) mRNA expression. Expression of these inflammatory mediators, together with iNOS, also increased significantly in testes 50 days after EAO. In vivo, expression of cytokines and inflammatory mediators was upregulated after injection of recombinant tryptase (MCP-1, TGFbeta(2), and COX-2) and a specific PAR(2) peptide agonist (MCP-1, TGFbeta(2)) in the testis after 5 h. These results suggest that PAR(2) activation elicited on PTCs by mast cell tryptase contributes to acute testicular inflammation and that this pathogenetic mechanism may also play a role in autoimmune orchitis.

Agonists of proteinase-activated receptor-2 modulate human neutrophil cytokine secretion, expression of cell adhesion molecules, and migration within 3-D collagen lattices.

Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist-stimulated human neutrophils show significantly enhanced migration in 3-D collagen lattices. PAR2 agonist stimulation also induced down-regulation of L-selectin display and up-regulation of membrane-activated complex-1 very late antigen-4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)-1beta, IL-8, and IL-6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up-regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.

Mast cell tryptases and airway remodeling.

On the basis of their amino acid sequences, tryptases are just another group of serine proteinases related to trypsin that happen to be expressed and stored in mast cells rather than the pancreas. On the basis of their biochemical and biological features, however, tryptases show little family likeness to trypsin and most other trypsin-like proteases. The intriguing discrepancies have been explained by the crystal structure of the tryptase tetramer. It is now clear how tryptases, by forming tetramers, have gained the ability to prevail enzymatically active in tissues, but, at the cost of an unusual narrow substrate specificity. The tryptase tetramer thus became both a (neuro)peptidase and a long-lasting initiator that orchestrates responses by the cleavage of a few key proteins, the activation of other proteases with broader specificity, and the stimulation of cellular responses. With the support of these performers, tryptase drives a variety of processes contributing to chronic inflammation and tissue remodeling, the diversity of which is still emerging.

Bivalent inhibition of human beta-tryptase.

BACKGROUND: Human beta-tryptase is a mast cell specific trypsin-like serine protease that is thought to play a key role in the pathogenesis of diverse allergic and inflammatory disorders like asthma and psoriasis. The recently resolved crystal structure revealed that the enzymatically active tetramer consists of four quasi-identical monomers. The spatial display of the four identical active sites represents an ideal basis for the rational design of bivalent inhibitors. RESULTS: Based on modeling experiments homobivalent inhibitors were constructed using (i) 6A,6D-dideoxy-6A,6D-diamino-beta-cyclodextrin as a rigid template to bridge the space between the two pairs of identical active sites and (ii) 3-(aminomethyl)benzene as a headgroup to occupy the arginine/lysine specific S1 subsites. A comparative analysis of the inhibitory potencies of synthetic constructs that differ in size and type of the spacer between headgroup and template revealed that the construct contained two 3-(aminomethyl)benzenesulfonyl-glycine groups linked to the 6A,6D-diamino groups of beta-cyclodextrin as an almost ideal bivalent inhibitor with a cooperativity factor of 1.9 vs. the ideal value of 2. The bivalent binding mode is supported by the inhibitor/tetramer ratio of 2:1 required for inactivation of tryptase and by X-ray analysis of the inhibitor/tryptase complex. CONCLUSION: The results obtained with the rigid cyclodextrin template underlined the importance of a minimal loss of conformational entropy in bivalent binding, but also showed the limitations imposed by such rigid core molecules in terms of optimal occupancy of binding sites and thus of enthalpic strains in bidentate binding modes. The main advantage of bivalent inhibitors is their high selectivity for the target enzyme that can be achieved utilizing the principle of multivalency.

Matrix metalloproteinases (MMPs) in breast cancer cell lines of different tumorigenicity.

In the present study we investigated the presence, amount and activity of matrix metalloproteinases (MMPs)-1, -2, -3, -7, -8, -9, -10, -11 and -13 and TIMP-1 in three well-defined breast cancer cell lines with different biological behaviour; i.e. poorly-invasive MCF-7 cells, invasively growing MDA-MB-231 cells and invasive and highly-metastatic MDA-MB-435 cells. The parallel immunocytochemical determination of the degree of cellular differentiation, as monitored by the immunocytochemical expression of cytokeratins (CK), confirmed differences in the tumor cell differentiation. Thereby, MCF-7 cells expressed more glandular CKs than MDA-MB-231 cells, while MDA-MB-435 cells were only labelled by pancytokeratin markers, but neither by glandular nor by squamous epithelial CKs. Conditioned media were analyzed for the presence of MMPs and TIMP-1 using Western blot with specific polyclonal antibodies and for gelatinolytic and caseinolytic activity by zymography. In addition, the cellular pool of several MMPs was investigated by immunocytochemistry. An enhanced cytoplasmatic staining for MMP-3 and -9, MMP-1, -10 and -11 was seen in the highly metastatic cells at almost equal levels, while MMP-2 revealed only a minor intracellular staining in all three cell lines. Western blots of conditioned media showed enhanced amounts of MMP-1, -3, -7, -10 and -11 in media of the two metastatic cell lines. Casein zymography correlated with the results of the MMP-1 Western blots. By means of gelatin zymography, MMP-2 and -9 were detectable in cell culture supematants of all the three cell lines, while gelatinolytic activity was elevated in the media of the more malignant MDA-MB-435 cells. Separate addition of EDTA or Pefa bloc SL partially inhibited the gelatinoltic activity indicating the presence of metallo- and serine proteinases, respectively; combined application of both inhibitors resulted in a complete suppression of activity. We provide evidence that the deviation expression in secretion of various MMPs in breast cancer cell lines of different tumorigenicity correlates with the biological behaviour of these cells, ie. the more malignant cells synthesize more MMPs than the less malignant ones. In addition, the secretion of MMP-1, -3, -7, -10 and -11 was enhanced in the malignant MDA-MB-231 and -435 cells when compared to the corresponding intracellular pool. This analysis confirms previous results obtained in a keratinocyte tumor cell model and provides evidence for a more general biological association between MMP-expression and tumor cell growth.

Epoxysuccinyl peptide-derived affinity labels for cathepsin B.

Extracellular cysteine proteases, in particular cathepsin B, have been implicated in a variety of pathological processes. Selectively targeting labels of this enzyme are important tools to gain more detailed understanding of its specific roles. Starting from our recently developed irreversible epoxysuccinyl-based inhibitor (R-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH, R=OMe), we have synthesized two affinity labels, R=NH-(CH(2))(6)-NH-rhodamine B and R=NH-(CH(2))(6)-NH-biotin. Using MCF-7 cells, the labeled inhibitors were shown to be virtually non-cell-permeant. Moreover, affinity blot analysis with the biotinylated inhibitor allowed a highly sensitive and selective non-radioactive detection of active cathepsin B.

Mast cells in an angiosarcoma complicating xeroderma pigmentosum in a 13-year-old girl.

A cutaneous angiosarcoma, a rare tumor that occurs almost exclusively in sun-exposed skin of individuals older than 50 years, developed in an adolescent with Xeroderma pigmentosum (XP). As this is the second report of a child with angiosarcoma and XP, ultraviolet-induced DNA damage may be involved in the pathogenesis of this tumor. Strongly increased numbers of mast cells were found, particularly in the peripheral tumor area, which may reflect with the requirement of mast cells for the growth of vascular structures or a role for mast cells in the antitumor immune response.

Beta-cyclodextrin/epoxysuccinyl peptide conjugates: a new drug targeting system for tumor cells.

Beta-cyclodextrin is known to form inclusion complexes with hydrophobic drugs. Several tumor cell lines are known to secrete and/or contain membrane-associated cathepsin B which is possibly involved in invasion and metastasis. Based on these information, our recently developed endo-epoxysuccinyl peptide inhibitor MeO-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH for cathepsin B was conjugated with beta-cyclodextrin to obtain a site-directed drug carrier system. Furthermore, the conjugate, was shown to form an inclusion complex with the cytotoxic drug methotrexate.

The human mast cell tryptase tetramer: a fascinating riddle solved by structure.

Tryptases, the predominant proteins of human mast cells, have been implicated as pathogenetic mediators of allergic and inflammatory conditions, most notably asthma. Until recently, the fascinating properties that distinguish tryptases among the serine proteinases, particularly their activity as a heparin-stabilized tetramer, resistance to most proteinaceous inhibitors, and preference for peptidergic over macromolecular substrates presented a riddle. This review solves this riddle with the help of the crystal structure of the human beta(2)-tryptase tetramer, but also indicates controversies between the unique quaternary architecture and some experimental data.

Kinetic and thermodynamic analysis of leech-derived tryptase inhibitor interaction with bovine tryptase and bovine trypsin.

The interaction of leech-derived tryptase inhibitor (LDTI) with bovine liver capsule tryptase (BLCT) and bovine trypsin has been studied using both thermodynamic and kinetic approaches. Several differences were detected: (i) the equilibrium affinity of LDTI for BLCT (Ka = 8.9 x 10(5) M(-1)) is about 600-fold lower than that for bovine trypsin (Ka = 5.1 x 10(8) M(-1)); (ii) LDTI behaves as a purely non-competitive inhibitor of BLCT, while it is a purely competitive inhibitor of bovine trypsin. These functional data are compared with those previously reported for the LDTI binding to human tryptase, where tight inhibition occurs at two of the four active sites of the tetramer (Ka = 7.1 x 10(8) M(-1)). Amino acid sequence alignment of BLCT, human betaII-tryptase and bovine trypsin allows us to infer some possible structural basis for the observed functional differences.

The structure of the human betaII-tryptase tetramer: fo(u)r better or worse.

Tryptases, the predominant serine proteinases of human mast cells, have recently been implicated as mediators in the pathogenesis of allergic and inflammatory conditions, most notably asthma. Their distinguishing features, their activity as a heparin-stabilized tetramer and resistance to most proteinaceous inhibitors, are perfectly explained by the 3-A crystal structure of human betaII-tryptase in complex with 4-amidinophenylpyruvic acid. The tetramer consists of four quasiequivalent monomers arranged in a flat frame-like structure. The active centers are directed toward a central pore whose narrow openings of approximately 40 A x 15 A govern the interaction with macromolecular substrates and inhibitors. The tryptase monomer exhibits the overall fold of trypsin-like serine proteinases but differs considerably in the conformation of six surface loops arranged around the active site. These loops border and shape the active site cleft to a large extent and form all contacts with neighboring monomers via two distinct interfaces. The smaller of these interfaces, which is exclusively hydrophobic, can be stabilized by the binding of heparin chains to elongated patches of positively charged residues on adjacent monomers or, alternatively, by high salt concentrations in vitro. On tetramer dissociation, the monomers are likely to undergo transformation into a zymogen-like conformation that is favored and stabilized by intramonomer interactions. The structure thus provides an improved understanding of the unique properties of the biologically active tryptase tetramer in solution and will be an incentive for the rational design of mono- and multifunctional tryptase inhibitors.

Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma.

Granzymes are granule-stored lymphocyte serine proteases that are implicated in T- and natural killer cell-mediated cytotoxic defense reactions after target cell recognition. A fifth human granzyme (granzyme 3, lymphocyte tryptase-2), renamed as granzyme K (gene name GZMK), has recently been cloned from lymphocyte tissue. For its further characterization we successfully generated catalytically active enzyme in milligram quantities per liter of Escherichia coli culture. The natural proform of granzyme K with the amino-terminal propeptide Met-Glu was expressed as inclusion bodies and converted to its active enzyme by cathepsin C after refolding of precursor molecules. Recombinant granzyme K cleaves synthetic thiobenzyl ester substrates after Lys and Arg with k(cat)/K(m) values of 3.7 x 10(4) and 4.4 x 10(4) M(-1) s(-1), respectively. Granzyme K activity was shown to be inhibited by the synthetic compounds Phe-Pro-Arg-chloromethyl ketone, phenylmethylsulfonyl fluoride, PefablocSC, and benzamidine, by the Kunitz-type inhibitor aprotinin and by human blood plasma. The plasma-derived inter-alpha-trypsin inhibitor complex, its bikunin subunit, and the second carboxyl-terminal Kunitz-type domain of bikunin were identified as genuine physiologic inhibitors with K(i) values of 64, 50, and 22 nM, respectively. Inter-alpha-trypsin inhibitor and free bikunin have the potential to neutralize extracellular granzyme K activity after T cell degranulation and may thus control unspecific damage of bystander cells at sites of inflammatory reactions.

[Tryptase, a marker for the activation and localization of mast cells]. / Tryptaseryptase, ein Marker für die Aktivierung und Lokalisation von Mastzellen.

The serine protease tryptase (ECNr. 3.4. 21.59), which is almost exclusively expressed in mast cells, is released by mast cell degranulation in an enzymatically active form together with other mediators, e.g. histamine, into the extracellular space and the circulation. The capability of the enzyme to directly stimulate several cell types as well as to cleave polypeptide hormones and to activate pro-enzymes suggests a role for tryptase in inflammatory and tissue-remodeling processes. Therefore, in the skin, a role of tryptase is suggested not only in mastocytosis and immediate type hypersensitivity reactions, but also in other inflammatory diseases, degenerative or neoplastic conditions as well as in wound healing, where an accumulation and/or activation of mast cells is found. Extracellular tryptase may be superior to histamine as a parameter for the onset and course of immediate type reactions and as an indicator for the activation of mast cells in other conditions. Its absence during histamine-liberating reactions may suggest basophil activation. In addition, tryptase has been shown to be a sensitive and specific marker for the localization of mast cells in tissues.

LEKTI, a novel 15-domain type of human serine proteinase inhibitor.

Proteinase inhibitors are important negative regulators of proteinase action in vivo. We have succeeded in isolating two previously unknown polypeptides (HF6478 and HF7665) from human blood filtrate that are parts of a larger precursor protein containing two typical Kazal-type serine proteinase inhibitor motifs. The entire precursor protein, as deduced from the nucleotide sequence of the cloned cDNA, exhibits 15 potential inhibitory domains, including the Kazal-type domains, HF6478, HF7665, and 11 additional similar domains. An inhibitory effect of HF7665 on trypsin activity is demonstrated. Because all of the 13 HF6478- and HF7665-related domains share partial homology to the typical Kazal-type domain but lack one of the three conserved disulfide bonds, they may represent a novel type of serine proteinase inhibitor. The gene encoding the multidomain proteinase inhibitor, which we have termed LEKTI, was localized on human chromosome 5q31-32. As shown by reverse transcriptase-polymerase chain reaction and Northern blot analysis, it is expressed in the thymus, vaginal epithelium, Bartholin's glands, oral mucosa, tonsils, and the parathyroid glands. From these results, we assume that LEKTI may play a role in anti-inflammatory and/or antimicrobial protection of mucous epithelia.

A carboxypeptidase inhibitor from the medical leech Hirudo medicinalis. Isolation, sequence analysis, cDNA cloning, recombinant expression, and characterization.

A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptase-polymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metallocarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like carboxypeptidases, with equilibrium dissociation constants Ki of 0.2-0.4 x 10(-9) M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.