Modulation of the Plasma Kallikrein-Kinin System Proteins Performed by Heparan Sulfate Proteoglycans

Human plasma kallikrein-kinin system proteins are related to inflammation through bradykinin. In the proximity of its target cells, high molecular weight kininogen (H-kininogen) is the substrate of plasma kallikrein, which releases bradykinin from H-kininogen. Heparan sulfate proteoglycans (HSPGs) play a critical role in either recruiting kinin precursors from the plasma, or in the assembly of kallikrein-kinin system components on the cell surface. Furthermore, HSPGs mediate the endocytosis and activation of H-kininogen and plasma prekallikrein. In the presence of HSPGs (Chinese hamster ovary cell, CHO-K1, wild type cells) both heparin and heparan sulfate strongly inhibit the H-kininogen interaction with the cell membrane. H-kininogen is internalized in endosomal acidic vesicles in CHO-K1 but not in CHO-745 cells (mutant cells deficient in glycosaminoglycan biosynthesis). The endocytosis process is lipid raft-mediated and is dependent on caveolae. Both types of CHO cells do not internalize bradykinin-free H-kininogen. At pH 7.35, bradykinin is released from H-kininogen on the surface of CHO-745 cells only by serine proteaseshowever, in CHO-K1 cells either serine or cysteine proteases are found to be involved. The CHO-K1 cell lysate contains different kininogenases. Plasma prekallikrein endocytosis in CHO-K1 cells is independent of H-kininogen, and also prekallikrein is not internalized by CHO-745 cells. Plasma prekallikrein cleavage/activation is independent of glycosaminoglycans but plasma kallikrein formation is more specific on H-kininogen assembled on the cell surface through glycosaminoglycans. In this mini-review, the importance of HSPGs in the regulation of plasma kallikrein-kinin system proteins is shown.Fundacdo de Amparo a Pesquisa do Estado de Sao Paulo, FAPESPConselho Nacional de Desenvolvimento Cientifico e TecnologicoCAPESUniv Fed Sao Paulo, Dept Bioquim, Escola Paulista Med, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Escola Paulista Med, Sao Paulo, BrazilFAPESP: 15/03964-6FAPESP: 16/14827-2CNPqCAPESWeb of Scienc

The role of Tyr(605) and Ala(607) of thimet oligopeptidase and Tyr(606) and Gly(608) of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series AbzGFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X = Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr(605) and Ala(607) in TOP and at Tyr(606) and Gly(608) in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. the kinetic parameters for the hydrolysis of substrates with systematic variations at position P-1 showed that Tyr(605) and Tyr(606) of TOP and NEL respectively, played a role in subsite S-1. Ala(607) of TOP and Gly(608) of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P-1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Ty,105 was an important anchor for its interaction with wild-type TOP. the hydroxy groups of Tyr 605 and Tyr.. did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-k(cat)/K-m dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilInst Butantan, Lab Especial Toxinol Aplicada, CAT, CEPID, BR-05467010 São Paulo, BrazilUniv São Paulo, Inst Ciencias Biomed, Dept Biol Celular & Desenvolvimento, Programa Biol Celular, BR-05508900 São Paulo, BrazilUniv São Paulo, Lab Neurociencias, BR-03071000 São Paulo, BrazilUniv Mogi das Cruzes, CIIB, BR-08780911 Mogi Das Cruzes, SP, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

The renal and hepatic distribution of Bence Jones proteins depends on glycosylation: A scintigraphic study in rats

The aim of the present study was to evaluate renal and liver distribution of two monoclonal immunoglobulin light chains. the chains were purified individually from the urine of patients with multiple myeloma and characterized as lambda light chains with a molecular mass of 28 kDa. They were named BJg (high amount of galactose residues exposed) and BJs (sialic acid residues exposed) on the basis of carbohydrate content. A scintigraphic study was performed on male Wistar rats weighing 250 g for 60 min after iv administration of 1 mg of each protein (7.4 MBq), as the intact proteins and also after carbohydrate oxidation. Images were obtained with a Siemens gamma camera with a high-resolution collimator and processed with a MicroDelta system. Hepatic and renal distribution were established and are reported as percent of injected dose. Liver uptake of BJg was significantly higher than liver uptake of BJs (94.3 vs 81.4%) (P<0.05). This contributed to its greater removal from the intravascular compartment, and consequently lower kidney accumulation of BJg in comparison to BJs (5.7 vs 18.6%) (P<0.05). After carbohydrate oxidation, there was a decrease in hepatic accumulation of both proteins and consequently a higher renal overload. the tissue distribution of periodate-treated BJg was similar to that of native BJs: 82.7 vs 81.4% in the liver and 17.3 vs 18.6% in the kidneys. These observations indicate the important role of sugar residues of Bence Jones proteins for their recognition by specific membrane receptors, which leads to differential tissue accumulation and possible toxicity.UNIV São Paulo,FAC MED,LAB FISIOPATOL RENAL,BR-01246903 São Paulo,SP,BRAZILUNIV São Paulo,CTR MED NUCL,BR-05403010 São Paulo,SP,BRAZILUNIV São Paulo,HOSP CLIN,SERV RADISISOTOPOS,BR-05403000 São Paulo,SP,BRAZILUniversidade Federal de São Paulo,MOL BIOL LAB,BR-04024900 São Paulo,SP,BRAZILUniversidade Federal de São Paulo,MOL BIOL LAB,BR-04024900 São Paulo,SP,BRAZILWeb of Scienc

Low molecular weight heparins: Structural differentiation by spectroscopic and multivariate approaches

Various branded low molecular weight heparins (LMWHs) have been used for the treatment and prevention of thrombotic for over 20 years. With the introduction of generic LMWHs and the recent events involving heparin contamination, a great deal of effort is being expended in investigating ways of monitoring and regulating this class of complex drugs. in this paper, we present the characterization of different forms of LMWHs, as well as the comparison of 5 enoxaparin copies from different manufactures. the data suggests that, while some of these drugs are structurally comparable, specific analytical methods as well as biological and pharmacological tests may be used to address their similarity, quality and potential interchangeability. the proposed approach may also be useful in comparing biosimilar and branded LMWHs. (C) 2011 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, SP, BrazilUniv Liverpool, Sch Biol Sci, Liverpool L69 7ZB, Merseyside, EnglandLoyola Univ, Med Ctr, Dept Pathol, Maywood, IL 60153 USAUniv Fed Parana, Lab Quim Carboidratos, Dept Bioquim & Biol Mol, BR-81531980 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, SP, BrazilWeb of Scienc

A New Approach for Heparin Standardization: Combination of Scanning UV Spectroscopy, Nuclear Magnetic Resonance and Principal Component Analysis

The year 2007 was marked by widespread adverse clinical responses to heparin use, leading to a global recall of potentially affected heparin batches in 2008. Several analytical methods have since been developed to detect impurities in heparin preparations; however, many are costly and dependent on instrumentation with only limited accessibility. A method based on a simple UV-scanning assay, combined with principal component analysis (PCA), was developed to detect impurities, such as glycosaminoglycans, other complex polysaccharides and aromatic compounds, in heparin preparations. Results were confirmed by NMR spectroscopy. This approach provides an additional, sensitive tool to determine heparin purity and safety, even when NMR spectroscopy failed, requiring only standard laboratory equipment and computing facilities

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k2 = 21±1 s−1) was much higher than the HNE deacylation step (k3 = 0.57±0.05 s−1). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k1 2.4-fold and reducing k−1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k2 value, whereas the k3 value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs

Heparin Oligosaccharides Have Antiarrhythmic Effect by Accelerating the Sodium-Calcium Exchanger

Background: Blockage of the Na+/Ca2+ exchanger (NCX) is used to determine the role of NCX in arrhythmogenesis. Trisulfated heparin disaccharide (TD) and Low Molecular Weight Heparins (LMWHs) can directly interact with the NCX and accelerate its activity.Objective: In this work, we investigated the antiarrhythmic effect of heparin oligosaccharides related to the NCX activity.Methods: The effects of heparin oligosaccharides were tested on the NCX current (patch clamping) and intracellular calcium transient in rat cardiomyocytes. The effects of heparin oligosaccharides were further investigated in arrhythmia induced in isolated rat atria and rats in vivo.Results: The intracellular Ca2+ concentration decreases upon treatment with either enoxaparin or ardeparin. These drugs abolished arrhythmia induction in isolated atria. The NCX antagonist KB-R7943 abolished the enoxaparin or ardeparin antiarrhythmic effects in isolated atria. In the in vivo measurements, injection of TD 15 min both before coronary occlusion or immediately after reperfusion, significantly prevented the occurrence of reperfusion-induced arrhythmias (ventricular arrhythmia and total AV block) and reduced the lethality rate. The patch clamping experiments showed that, mechanistically, TD increases the forward mode NCX current.Conclusion: Together, the data shows that heparin oligosaccharides may constitute a new class of antiarrhythmic drug that acts by accelerating the forward mode NCX under calcium overload

Fibroblast and prostate tumor cell cross-talk: Fibroblast differentiation, TGF-beta, and extracellular matrix down-regulation

Growth and survival of tumors at a site of metastasis involve interactions with stromal cells in the surrounding environment. Stromal cells aid tumor cell growth by producing cytokines as well as by modifying the environment surrounding the tumor through modulation of the extracellular matrix (ECM). Small leucine-rich proteoglycans (SLRPs) are biologically active components of the ECM which can be altered in the stroma surrounding tumors. the influence tumor cells have on stromal cells has been well elucidated. However, little is understood about the effect metastatic cancer cells have on the cell biology and behavior of the local stromal cells. Our data reveal a significant downregulation in the expression of ECM components such as collagens I, II, III, and IV, and the SLRPs, decorin, biglycan, lumican, and fibromodulin in stromal cells when grown in the presence of two metastatic prostate cancer cell lines PO and DU145. Interestingly, TGF-beta down-regulation was observed in stromal cells, as well as actin depolymerization and increased vimentin and alpha 5 beta 1 integrin expression. MT1-MMP expression was upregulated and localized in stromal cell protrusions which extended into the ECM. Moreover, enhanced stromal cell migration was observed after crosstalk with metastatic prostate tumor cells. Xenografting metastatic prostate cancer cells together with activated stromal cells led to increased tumorigenicity of the prostate cancer cells. Our findings suggest that metastatic prostate cancer cells create a metastatic niche by altering the phenotype of local stromal cells, leading to changes in the ECM. (C) 2010 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Bioquim, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, Escola Paulista Med, BR-04044020 São Paulo, BrazilFAPESP: 2007/59801-1Web of Scienc