Heparinases: cloning, expression and structural requirements for activity

Structural characteristics of heparin (Hep) and heparan sulfate (HS) have been determined using enzymes from Flavobacterium heparinum, a non-pathogenic soil bacterium. Upon induction with Hep/HS or their disaccharides as the sole source of carbon and nitrogen, the bacteria synthesize heparinase, and heparitinases I and II. This lyases cleaves heparin and HS by a -eliminative process in a random endolytic pattern. Heparitinase I cleaves exclusively Nacetyl or N-sulfo-glucosaminide-glucuronic acid linkage of HS/Hep without C6 sulfate substitution; Heparitinase II cleaves N-acetyl-6-sulfo or N-sulfo or N,6- sulfo glucosaminide-glucuronic/ iduronic acid linkage of HS/Hep without C3 sulfate substitution. Heparinase cleaves an -D-glucosamine N- and 6-sulfated (14) -L-iduronate 2-sulfated, present in heparin. This work aims to cloning and express heparinase and heparitinase I and study de kinetic behavior of heparinase. Recombinant enzymes were expressed in E. coli using the T7 polymerase pET expression system. The work shows that heparin-Ca2+- heparinase I complex is the true substrate for heparinase (KS = 1.4±0.1μM), whereas heparin Ca2+ free (KI=12±2μM) as an important pharmaceutical contaminant of commercial heparin, oversulfate chondroitin sulfate (OSCS) (Ki= 0.65μM) are competitive inhibitors. The pKa values of the prototropic groups of the active site were determined by measuring the pH-, solvent- and temperature-dependence upon kcat/KSA, kcat and KSA constants. The results show, at pHoptimium=6.67±0.05, a deprotonated histidine residue initiating the - elimination reaction by the abstraction of the C5 proton of the -L-iduronate 2- O-sulfate residue; and a tyrosin residue in a protonated form acting as a proton donor to the hexosamine leaving group. Mutations of histidine 165 and glutamine 163 residue were performed and kinetic analysis show specific activity maintenance (5,0 e-007 Abs/μg.s), suggesting that H165 and Q163 were not essencial for the heparinase activity.Características estruturais de heparina (Hep) e heparam sulfato (HS) têm sido determinadas usando enzima de Flavobacterium heparinum, uma bactéria de solo, não-patogênica. Sob indução com Hep/HS ou seus dissacarídeos como única fonte de carbono e nitrogênio, a bactéria sintetiza heparinase e heparitinases I e II. Essas liases clivam heparina e HS por um processo - eliminativo em um padrão endolítico aleatório. Heparitinase I cliva exclusivamente ligações -D-glucosamina N-acetilada ou N-sulfatada (14) β- D-glucuronato de HS/Hep sem haver substituição de sulfato em C6; Heparitinase II cliva ligações -D-glucosamina N-acetil-6-sulfato ou N-sulfato ou N,6-sulfato (14) β-D-glucuronato ou -L- iduronato de HS/Hep, sem haver substituição de sulfato em C3. Heparinase cliva uma -D-glucosamina N- e 6- sulfato (14) -L- iduronato 2-sulfato, presente em heparina. Este trabalho objetiva clonar e expressar heparinase e heparitinase I e estudar o comportamento cinético da heparinase. As enzimas recombinantes foram expressas em E. coli usando o sistema de expressão pET T7 polimerase. O trabalho mostrou que o complexo heparina-Ca2+-heparinase é o substrato verdadeiro para heparinase (KS = 1.4±0.1μM), enquanto que tanto a heparina livre de Ca2+ (KI= 12±2μM) como também um importante contaminante farmacêutico de heparina comercial, condroitim sulfato super sulfatado (OSCS) (KI= 0.65μM) são inibidores competitivos. Os valores de pKa dos grupos prototrópicos do sítio ativo foram determinados pela medida pH-, solvente- e temperatura-dependente sobre as contantes kcat/KS, kcat e KS. Os resultados mostram, em pHótimo=6.67±0.05, um resíduo de histidina desprotonada iniciando a reação de -eliminação pela abstração do próton de C5 do resíduo -L-iduronato 2-O-sulfato; e um resíduo de tirosina na forma protonada agindo como um doador de próton para o anel de hexosamina liberada. Mutações dos residues de histidina 165 e glutamina 163 foram realizadas e análises cinéticas mostraram a manutenção da atividade específica (5,0 e-007 Abs/μg.s), sugerindo que os resíduos H165 e Q163 não são essenciais para a atividade da heparinase.TEDEBV UNIFESP: Teses e dissertaçõe

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

Induction studies of endoglycosidases from Flavobacterium heparinum: heparinase III cloning

BV UNIFESP: Teses e dissertaçõe

On the catalytic mechanism of polysaccharide lyases: evidence of His and Tyr involvement in heparin lysis by heparinase I and the role of Ca 2+

The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but share common catalytic mechanisms. One, heparinase I (F. heparinum), is employed in the production of the major anticoagulant drug, low molecular weight heparin, and is a mainstay of cell surface proteoglycan analysis. We demonstrate that heparinase I specificity and efficiency depend on the cationic form of the substrate. Ca2+-heparin, in which alpha-L-iduronate-2-O-sulfate residues adopt C-1(4) conformation preferentially, is a substrate, while Na+-heparin is an inhibitor. His and Tyr residues are identified in the catalytic step and a model based on molecular dynamics and docking is proposed, in which deprotonated His203 initiates beta-elimination by abstracting the C5 proton of the alpha-L-iduonate-2-O-sulfate residue in the substrate, and protonated Tyr357 provides the donor to the hexosamine leaving group.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)National High Magnetic Field LaboratoryUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, Disciplina Biol Mol, BR-04044020 São Paulo, BrazilUniv Liverpool, Dept Struct & Chem Biol, Liverpool L69 7ZB, Merseyside, EnglandUniv Fed Rio Grande do Sul, Ctr Biotecnol, BR-91500970 Porto Alegre, RS, BrazilDiamond Light Source Ltd, Didcot OX11 ODE, Oxon, EnglandUniv Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, Ctr Ciencias Tecnol, BR-08780911 Mogi Das Cruzes, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, Disciplina Biol Mol, BR-04044020 São Paulo, BrazilCAPES: 172/2012Web of Scienc