Comparison of the factor H heparin binding sites on SCR domains 7 and 20

White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded β-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.

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Antiheparin Antibody: Preparation And Use As A Functional Probe

10.1055/s-0038-1652531 ◽

1981 ◽

Author(s):

S N Gitel ◽

V M Medina ◽

S Wessler

Keyword(s):

Aqueous Solution ◽

Binding Sites ◽

Anticoagulant Activity ◽

Red Cells ◽

Heparin Binding ◽

Antibody Preparation ◽

Specific Antibodies ◽

Coagulation Proteins ◽

Heparin Complexes ◽

Presence And Absence

Antiheparin antibodies were raised in 5 of 6 rabbits immunized sequentially with covalent complexes of ovalbumin-heparin and bovine IgG-heparin. Confirmation of the presence of heparin-specific antibodies in the antisera was based on: (1) antisera precipitation of BSA-heparin but not BSA alone, (2) antisera inhibition of heparin anticoagulant activity without precipitating heparin, (3) antisera precipitation of heparin-heparin complexes, and (4) antibody removal from the antisera by heparin- Sepharose. The antisera had hemagglutination titers between 1:10,000 and 1:50,000 when tested against tanned red cells labeled with BSA-heparin complex; whereas no hemagglutination occurred with tanned erythrocytes tagged with BSA. Heparin-heparin oligomers, prepared by carbo- diimide coupling in aqueous solution and retaining anticoagulant activity, precipitated in the presence of the antiheparin antibodies. Data indicating that heparin has separate binding sites for antithrombin 111 and thrombin were obtained by quantitating the heparin oligomer- antibody precipitate in the presence and absence of these two purified, heparin-binding, coagulation proteins.

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Leptospiral Endostatin-Like Protein A Is a Bacterial Cell Surface Receptor for Human Plasminogen

Infection and Immunity ◽

10.1128/iai.01282-09 ◽

2010 ◽

Vol 78 (5) ◽

pp. 2053-2059 ◽

Cited By ~ 53

Author(s):

Ashutosh Verma ◽

Catherine A. Brissette ◽

Amy A. Bowman ◽

Samir T. Shah ◽

Peter F. Zipfel ◽

...

Keyword(s):

Binding Sites ◽

Protein A ◽

Aminocaproic Acid ◽

Factor H ◽

Cell Surface Receptor ◽

Dependent Manner ◽

Public Health Importance ◽

Amino Terminal ◽

Surface Receptor ◽

Human Plasminogen

ABSTRACT The spirochete Leptospira interrogans is a highly invasive pathogen of worldwide public health importance. Studies from our laboratories and another have demonstrated that L. interrogans can acquire host plasminogen on its surface. Exogenous plasminogen activators can then convert bound plasminogen into the functionally active protease plasmin. In this study, we extend upon those observations and report that leptospiral endostatin-like protein A (LenA) binds human plasminogen in a dose-dependent manner. LenA-plasminogen interactions were significantly inhibited by the lysine analog ξ-aminocaproic acid, suggesting that the lysine-binding sites on the amino-terminal kringle portion of the plasminogen molecule play a role in the binding. Previous studies have shown that LenA also binds complement regulator factor H and the extracellular matrix component laminin. Plasminogen competed with both factor H and laminin for binding to LenA, which suggests overlapping ligand-binding sites on the bacterial receptor. Finally, LenA-bound plasminogen could be converted to plasmin, which in turn degraded fibrinogen, suggesting that acquisition of host-derived plasmin by LenA may aid bacterial dissemination throughout host tissues.

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Mechanism of thrombin binding to endothelial cells

Blood ◽

10.1182/blood.v61.2.368.368 ◽

1983 ◽

Vol 61 (2) ◽

pp. 368-372 ◽

Cited By ~ 36

Author(s):

PT Bauer ◽

R Machovich ◽

P Aranyi ◽

KG Buki ◽

E Csonka ◽

...

Keyword(s):

Endothelial Cells ◽

Binding Sites ◽

Antithrombin Iii ◽

Affinity Binding ◽

Heparin Binding ◽

High Affinity Binding ◽

Aortic Endothelial Cells ◽

High Affinity ◽

Lysine Residues ◽

Two Populations

Abstract The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5′-phosphate (PLP2-thrombin) destroyed the high- affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. The results revealed that 3H- heparin also bound to cells. This binding was characterized by a Kd of 3 x 10(-7) M, approximately 10(6) sites/cell. Furthermore, thrombin bound to endothelial cells was released by antithrombin III. On the basis of these and other data in the literature, a model is proposed for the mechanism of the binding of thrombin to endothelial cells.

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Streptococcal β Protein Has Separate Binding Sites for Human Factor H and IgA-Fc

Journal of Biological Chemistry ◽

10.1074/jbc.m112072200 ◽

2002 ◽

Vol 277 (15) ◽

pp. 12642-12648 ◽

Cited By ~ 72

Author(s):

Thomas Areschoug ◽

Margaretha Stålhammar-Carlemalm ◽

Ingrid Karlsson ◽

Gunnar Lindahl

Keyword(s):

Binding Sites ◽

Human Factor ◽

Factor H ◽

Β Protein

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Reactive oxygen species mediate modification of glycocalyx during ischemia-reperfusion injury

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00796.2005 ◽

2006 ◽

Vol 290 (6) ◽

pp. H2247-H2256 ◽

Cited By ~ 138

Author(s):

Ivan Rubio-Gayosso ◽

Steven H. Platts ◽

Brian R. Duling

Keyword(s):

Reactive Oxygen Species ◽

Binding Sites ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Reactive Oxygen ◽

Control Site ◽

Oxygen Species ◽

Heparin Binding ◽

Heparin Binding Domain

The glycocalyx (Gcx) is a complex and poorly understood structure covering the luminal surface of endothelial cells. It is known to be a determinant of vascular rheology and permeability and may be a key control site for the vascular injuries caused by ischemia-reperfusion (I/R). We used intravital-microscopy to evaluate the effects of I/R injury on two properties of Gcx in mouse cremasteric microvessels: exclusion of macromolecules (anionic-dextrans) and intracapillary distribution of red blood cells (RBC). In this model, the Gcx is rapidly modified by I/R injury with an increase in 70-kDa anionic-dextran penetration without measurable effect on the penetration of 580-kDa anionic-dextran or on RBC exclusion. The effects of I/R injury appear to be mediated by the rapid production of reactive oxygen species (ROS) because they are ameliorated by the addition of exogenous superoxide dismutase-catalase. Intravenous application of allopurinol or heparin also inhibited the effects of I/R injury, and we interpret efficacy of allopurinol as evidence for a role for xanthine-oxidoreductase (XOR) in the response to I/R injury. Heparin, which is hypothesized to displace XOR from a heparin-binding domain in the Gcx, reduced the effects of I/R. The effects of I/R injury were also partially prevented or fully reversed by the intravascular infusion of exogenous hyaluronan. These data demonstrate: 1) the liability of Gcx during I/R injury; 2) the importance of locally produced ROS in the injury to Gcx; and 3) the potential importance of heparin-binding sites in modulating the ROS production. Our findings further highlight the relations between glycosaminoglycans and the pathophysiology of Gcx in vivo.

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