The hemoglobin ␣-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 g/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells. Endopeptidase EC 3.4.24.15 (ep24.15; also referred to as thimet oligopeptidase) and endopeptidase EC 3. 4.24.16 (ep24.16; also referred to as neurolysin) were initially detected in and purified from rat brain homogenates (1, 2). The cloned rat brain ep24.16 (3) showed 80% similarity and 63% identity with the previously cloned rat testis ep24.15 (4). Both peptidases share most of their natural substrates, including bradykinin, neurotensin, opioids, angiotensin I, and gonadotrophinreleasing hormone (5, 6

It has been shown that lysosomal cysteine proteinases, specially cathepsin B, has been implicated in a variety of diseases involving tissue remodeling states, such as inflammation, parasite infection, and tumor metastasis, by degradation of extracellular matrix components. Recently, we have shown that heparin and heparan sulfate bind to papain specifically; this interaction induces an increase of its ␣-helix content and stabilizes the enzyme structure even at alkaline pH (Almeida, P. C., Nantes, I. L., Rizzi, C. C. A., Jú dice, W. A. S., Chagas, J. R., Juliano, L., Nader, H. B., and Tersariol, I. L. S. (1999) J. Biol. Chem. 274, 30433-30438). In the present work, a combination of circular dichroism analysis, affinity chromatography, cathepsin B mutants, and fluorogenic substrate assays were used to characterize the interaction of human cathepsin B with glycosaminoglycans. The nature of the cathepsin B-glycosaminoglycans interaction was sensitive to the charge and type of polysaccharide. Like papain, heparin and heparan sulfate bind cathepsin B specifically, and this interaction reduces the loss of cathepsin B ␣-helix content at alkaline pH. Our data show that the coupling of cathepsin B with heparin or heparan sulfate can potentiate the endopeptidase activity of the cathepsin B, increasing 5-fold the half-life (t1 ⁄2 ) of the enzyme at alkaline pH. Most of these effects are related to the interaction of heparin and heparan sulfate with His 111 residue of the cathepsin B occluding loop. These results strongly suggest that heparan sulfate may be an important binding site for cathepsin B at cell surface, reporting a novel physiological role for heparan sulfate proteoglycans.Heparan sulfate is an ubiquitous glycosaminoglycan of animal cells (1). These classes of compounds are heteropolysaccharides made up of repeating units of disaccharides, an uronic acid residue, either D-glucuronic acid or L-iduronic acid, and D-glucosamine with N-and 6-O-sulfates and N-acetyl substitutions (2). Heparan sulfate occurs at the cell surface and in extracellular matrix as proteoglycans. Most of cellular heparan sulfate derives from the syndecans and glypicans proteoglycans. The syndecan family are associated with the cell membranes via transmembrane core proteins (3, 4), and the glypican family is anchored by glycosilyl phosphatidylinositolanchor core proteins (5). Also, heparan sulfate proteoglycans are present in basement membranes performing the perlecan family (6).Heparan sulfate and heparin are particular among glycosaminoglycans in their ability to bind a large number of different proteins. Heparin-like glycosaminoglycans play a complex role in the extracellular matrix, regulating a wide variety of biological process, including hemostasis, inflammation, angiogenesis, growth factors, cell adhesion, and others (7). Proteolytic enzymes control many of these biological process. Several reports in the literature have demonstrated that heparin-like glycosaminoglycans can modulate the activity of some serine proteinases and their natu...

Trypanosoma cruzi activates the kinin pathway through the activity of its major cysteine proteinase, cruzipain. Because kininogen molecules may be displayed on cell surfaces by binding to glycosaminoglycans, we examined whether the ability of cruzipain to release kinins from high molecular weight kininogen (HK) is modulated by heparan sulfate (HS). Kinetic assays show that HS reduces the cysteine proteinase inhibitory activity (K i app ) of HK about 10-fold. Conversely, the catalytic efficiency of cruzipain on kinin-related synthetic fluorogenic substrates is enhanced up to 6-fold in the presence of HS. Analysis of the HK breakdown products generated by cruzipain indicated that HS changes the pattern of HK cleavage products. Direct measurements of bradykinin demonstrated an up to 35-fold increase in cruzipain-mediated kinin liberation in the presence of HS. Similarly, kinin release by living trypomastigotes increased up to 10-fold in the presence of HS. These studies suggest that the efficiency of T. cruzi to initiate kinin release is potently enhanced by the mutual interactions between cruzipain, HK, and heparan sulfate proteoglycans.The plasma kallikrein-kinin system is a paradigm of a tightly controlled pro-inflammatory proteolytic cascade activated by vascular injury (1). Vasoactive peptides structurally related to bradykinin (generally termed as "kinins") are derived from enzymatic excision from an internal segment (D4 domain) of kininogens. These peptides are implicated in a broad range of pathophysiological responses, e.g. edema formation, vasodilatation, and pain. Although the nonapeptide bradykinin is released by the action of plasma kallikrein on high molecular weight kininogen (HK), 1 lysyl-bradykinin is liberated from extravascular low molecular weight kininogen (LK) or HK by the activity of tissue kallikreins (2). In inflammatory conditions, oxidized forms of kininogens may be cleaved by the concerted action of neutrophil elastase and mast cell tryptase, liberating Met-Lys-bradykinin (3). Once liberated, kinins activate local endothelial or smooth muscle cells through the constitutively expressed B 2 kinin receptor (4) or alternatively through the B 1 kinin receptor that is up-regulated during inflammation (5). The effect of kinin stimulation on its receptor(s) is tightly regulated by the action of kinin-degrading peptidases (kininases), such as the angiotensin-converting enzyme and neutral endopeptidase (1). HK comprises six major domains, and the C-terminal domains (D5 H and D6 H ) mediate plasma contact phase activation; they are not present in LK (6). The other domains, D1-D4, are shared with LK. Domains 1-3 are structures homologous to the cysteine-proteinase inhibitors, cystatins (7), and the bradykinin-containing segment is domain 4. Recent efforts to define the structural basis of HK interaction with endothelial cells have focused on two binding sites. One site is represented by 27 amino acids located in the D3 domain (8), hence overlapping with one of the cystatin-like domains. The second b...

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Kinetic data for the hydrolysis by human tissue kallikrein of fluorogenic peptides with o-aminobenzoyl-Phe-Arg (Abz-FR) as the acyl group and different leaving groups demonstrate that interactions with the S'1, S'2 and S'3 subsites are important for cleavage efficiency. In addition, studies on the hydrolysis of fluorogenic peptides with the human kininogen sequence spanning the scissile Met-Lys bond [Abz-M-I-S-L-M-K-R-P-N-(2,4-dinitrophenyl)ethylenediamine] and analogues with different residues at positions P'1, P'2 and P'3 showed that (a) the presence of a proline residue at P'3 and the interactions with the tissue kallikrein-binding sites S2 to S'2 are determinants of Met-Lys bond cleavage and (b) residues P3, P4 and/or P5 arc important for cleavage efficiency. The substitution of phenylalanine for methionine or arginine in substrates with scissile Met-Lys or Arg-Xaa bonds demonstrated that lysyl-bradykinin-releasing tissue kallikreins also have a primary specificity for phenylalanine. The replacement of arginine by phenylalanine in (D)P-F-R-p-nitroanilide (pNA) produced an efficient and specific chromogenic substrate, (D)P-F-F-pNA, for the lysyl-bradykinin-releasing tissue kallikreins as it is resistant to plasma kallikrein and other arginine hydrolases.