A Comparative Analysis of the Primary Sequences and Characteristics of Heparinases I, II, and III fromFlavobacterium heparinum

Godavarti, Ranga; Sasisekharan, Ram

doi:10.1006/bbrc.1996.1879

Cited by 37 publications

(47 citation statements)

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“…40 It is synthesized as a 65-kD latent enzyme that subsequently undergoes proteolytic cleavage, yielding a 50-and an 8-kD active heterodimer. 41 Lysosomes are the main storage site of active HPSE-1 because their acidic milieu (pH 5 to 6.5) is best suited to preserve its maximal activity. In addition to its intracellular housekeeping role in HS metabolism, active HPSE-1 is known to be transported by endosomes to the plasma membrane and extracellular space.…”

Section: Discussionmentioning

confidence: 99%

Glomerular Endothelial Glycocalyx Constitutes a Barrier to Protein Permeability

Singh

Satchell

Neal

et al. 2007

Journal of the American Society of Nephrology

243

222

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Glycocalyx, composed of glycoproteins including proteoglycans, coats the luminal surface of the glomerular capillaries. Human heparanase degrades heparan sulphate glycosaminoglycans and is upregulated in proteinuric states. In this study, we analyze the structure of the human glomerular endothelial cell glycocalyx in vitro and examine its functional relevance, especially after treatment with human heparanase. Electron microscopy of conditionally immortalized glomerular endothelial cells revealed a 200-nm thick glycocalyx over the plasma membrane, which was also demonstrated by confocal microscopy. Neuraminidase treatment removed the majority of glycocalyx, reduced trans-endothelial electrical resistance by 59%, and increased albumin flux by 207%. Heparinase III and human heparanase specifically cleaved heparan sulphate: this caused no change in trans-endothelial electrical resistance, but increased the albumin passage across the monolayers by 40% and 39%, respectively. Therefore, we have characterized the glomerular endothelial cell glycocalyx and have shown that it contributes to the barrier to flux of albumin across the cell layer. These results suggest an important role for this glycocalyx in the restriction of glomerular protein passage in vivo and suggest ways in which human heparanase levels may be linked to proteinuria in clinical disease.

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Section: Discussionmentioning

confidence: 99%

Glomerular Endothelial Glycocalyx Constitutes a Barrier to Protein Permeability

Singh

Satchell

Neal

et al. 2007

Journal of the American Society of Nephrology

243

222

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“…Approximately 400 g of the octasaccharide library was injected onto a CarboPac PA1 column (Dionex, Sunnyvale, CA) and SAX HPLC (Waters, Milford, MA) was performed at a flow rate of 1 ml/min using Solvent A (H 2 O, pH 3.5) and Solvent B (3 M NaCl, pH 3.5). A typical gradient consisted of: (1) 1-11 min, 0% B, (2) 11-110 min, 0 -100% B, and (3) 111-120 min, 0% B. Chromatograms were recorded by monitoring the UV absorbance at 232 nm [28]. Fractions corresponding to Octa/12 SO 3 were collected, lyophilized, and desalted.…”

Section: Isolation Of Heparin Octasaccharide Modified With 12 Sulfatementioning

confidence: 99%

“…Arixtra is susceptible to heparinase I cleavage, which yields an unsaturated disaccharide (containing the original reducing end) and a trisaccharide (containing the original non-reducing end). The enzymatically generated double-bond on the disaccharide has a strong absorbance at 232 nm [28], and thus the concentration of the disaccharide was determined and correlated 1:1 to the original concentration of Arixtra.…”

Section: Sample Preparation For Arixtramentioning

confidence: 99%

Potential inhibitors of chemokine function: Analysis of noncovalent complexes of cc chemokine and small polyanionic molecules by ESI FT-ICR mass spectrometry

Sweeney

Saad

et al. 2006

J. Am. Soc. Mass Spectrom.

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Chemokines play a critical role in inducing chemotaxis, extravasation, and activation of leukocytes both in routine immunosurveillance and autoimmune diseases. Traditionally, to disrupt chemokine function, strategies have focused on blockage of its interaction with the receptor. Recently, it has been demonstrated that binding to glycosaminoglycans (GAGs) is also required for the in vivo activity of many chemokines. Thus, interference with the GAG-binding of chemokines may offer an alternative, valid, anti-inflammatory strategy. However, the potential of using small polyanions to inhibit the interactions between chemokines and cell surface GAGs has not been fully explored. In this study, a mass spectrometry based filtration trapping assay was utilized to study the interactions between two CCR 2 ligands (MCP-1/CCL2 and MCP-3/CCL7) and a series of low molecular weight, polyanionic molecules. Findings were confirmed by using a hydrophobic trapping assay. The results indicated that Arixtra (fondaparinux sodium), sucrose octasulfate, and suramin were specific binders of the chemokines, while cyclodextrin sulfate, although the most highly sulfated molecule among the ones investigated, showed no binding. The binding stoichiometry of the small molecule ligand was determined from the measured molecular weight of the noncovalent complex. Furthermore, the dissociation constant between MCP-3 and Arixtra was determined by using electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry, which compared favorably with the result of the isothermal titration calorimetry (ITC) assay. The relative binding affinity of these ligands to MCP-3 was also determined using a competitive filtration trapping assay. (J Am Soc Mass Spectrom 2006, 17, 524 -535)

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“…Although all of them are eliminases that primarily act on the glycosidic bond between the uronic acid and glucosamine, releasing saturated or unsaturated disaccharide products, they show considerably a low degree of similarity in primary sequences and differ from each other in multiple aspects of chemical and activity properties (Lohse and Linhardt, 1992;Godavarti and Sasisekharan, 1996). Heparinase I (HepI; EC 4.2.2.7) is a heparin lyase with a molecular weight of 42.8 kDa and displays a preference toward highly sulfated regions of heparin as its main substrate (Desai et al, 1993a, b).…”

Section: Introductionmentioning

confidence: 99%

Structural basis of heparan sulfate-specific degradation by heparinase III

Dong¹,

Lu²,

McKeehan³

et al. 2012

Protein Cell

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Heparinase III (HepIII) is a 73-kDa polysaccharide lyase (PL) that degrades the heparan sulfate (HS) polysaccharides at sulfate-rare regions, which are important co-factors for a vast array of functional distinct proteins including the well-characterized antithrombin and the FGF/FGFR signal transduction system. It functions in cleaving metazoan heparan sulfate (HS) and providing carbon, nitrogen and sulfate sources for host microorganisms. It has long been used to deduce the structure of HS and heparin motifs; however, the structure of its own is unknown. Here we report the crystal structure of the HepIII from Bacteroides thetaiotaomicron at a resolution of 1.6 Å. The overall architecture of HepIII belongs to the (α/α)5 toroid subclass with an N-terminal toroid-like domain and a C-terminal β-sandwich domain. Analysis of this high-resolution structure allows us to identify a potential HS substrate binding site in a tunnel between the two domains. A tetrasaccharide substrate bound model suggests an elimination mechanism in the HS degradation. Asn260 and His464 neutralize the carboxylic group, whereas Tyr314 serves both as a general base in C-5 proton abstraction, and a general acid in a proton donation to reconstitute the terminal hydroxyl group, respectively. The structure of HepIII and the proposed reaction model provide a molecular basis for its potential practical utilization and the mechanism of its eliminative degradation for HS polysaccarides.

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A Comparative Analysis of the Primary Sequences and Characteristics of Heparinases I, II, and III fromFlavobacterium heparinum

Cited by 37 publications

References 0 publications

Glomerular Endothelial Glycocalyx Constitutes a Barrier to Protein Permeability

Glomerular Endothelial Glycocalyx Constitutes a Barrier to Protein Permeability

Potential inhibitors of chemokine function: Analysis of noncovalent complexes of cc chemokine and small polyanionic molecules by ESI FT-ICR mass spectrometry

Structural basis of heparan sulfate-specific degradation by heparinase III

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