Collagen in the subendothelium is suggested as a Major binding partner of von Willebrand factor (... more Collagen in the subendothelium is suggested as a Major binding partner of von Willebrand factor (vWF), thereby providing its function in primary hemostasis. The lack of the functionally Most active vWF high Molecular weight Multimers in von Willebrand disease (vWd) type 2A correlates with significantly decreased vWF binding to collagen type I and III, respectively, and offers a possible explanation for the bleeding symptoms in such patients. Consequently, one would expect a similar clinical phenotype in persons with an isolated defect of vWF collagen binding (vWF:CB) but with normal vWF Multimers. A Mutation in the vWF A3 domain that is regarded as the Major vWF:CB site [1], was recently identified in a family with Mother and daughter suffering from a significant bleeding tendency [2]. However, vWF:CB seemed normal in the vWF:CB assay. Only after identification of a Mutation in the vWF A3 domain and recombinant expression of the Mutant protein, a reduced VWF:CB was demonstrated by the collagen binding curve [2]. Here we report on three different novel Mutations in the A3 domain which do not correlate with bleeding symptoms, although they significantly decrease vWF collagen binding.
Von Willebrand factor (VWF), a multimeric plasma glycoprotein, mediates platelet adhesion to site... more Von Willebrand factor (VWF), a multimeric plasma glycoprotein, mediates platelet adhesion to sites of vascular damage. In this proces, the VWF-A3 domain binds to collagen in the vessel wall. This is followed by binding of the VWF-A1 domain to platelet receptor glycoprotein (Gp) Ibalpha. The investigations, using protein crystallography, site-directed mutagenesis and surface-plasmon-resonance, identified the collagen-binding site on the VWF- A3 domain. Based on the surface characteristics of the collagen-binding site, a hypothesis for the binding mode of a collagen triple helix on the VWF-A3 domain has been made. The identification of specific collagen sequences that interact with A3 is subject for future research. To investigate the binding of VWF to GpIbalpha, crystal structures of GpIbalpha, A1GpIbalpha, wt- and gain-of-function mutant R543Q-A1 have been solved. Based on these structures we hypothesized that the N- and C-terminal flanking peptides of the VWF-A1 domain shield one ...
Staphylococcus aureus secretes several virulence factors modulating immune responses. Staphylococ... more Staphylococcus aureus secretes several virulence factors modulating immune responses. Staphylococcal superantigen-like (SSL) proteins are a family of 14 exotoxins with homology to superantigens, but with generally unknown function. Recently, we showed that SSL5 binds to P-selectin glycoprotein ligand 1 dependently of sialyl Lewis X and inhibits P-selectin–dependent neutrophil rolling. Here, we show that SSL5 potently and specifically inhibits leukocyte activation by anaphylatoxins and all classes of chemokines. SSL5 inhibited calcium mobilization, actin polymerization, and chemotaxis induced by chemokines and anaphylatoxins but not by other chemoattractants. Antibody competition experiments showed that SSL5 targets several chemokine and anaphylatoxin receptors. In addition, transfection studies showed that SSL5 binds glycosylated N-termini of all G protein–coupled receptors (GPCRs) but only inhibits stimuli of protein nature that require the receptor N-terminus for activation. Furth...
Food-borne pathogens are responsible for most cases of food poisoning in developed countries and ... more Food-borne pathogens are responsible for most cases of food poisoning in developed countries and are often associated with poultry products, including chicken. Little is known about the role of β-defensins in the chicken digestive tract and their efficacy. In this study, the expression of chicken β-defensin gallinacin-6 (Gal-6) and its antimicrobial activity against food-borne pathogens were investigated. Reverse transcription-PCR analysis showed high expression of Gal-6 mRNA in the esophagus and crop, moderate expression in the glandular stomach, and low expression throughout the intestinal tract. Putative transcription factor binding sites for nuclear factor kappa beta, activator protein 1, and nuclear factor interleukin-6 were found in the Gal-6 gene upstream region, which suggests a possible inducible nature of the Gal-6 gene. In colony-counting assays, strong bactericidal and fungicidal activity was observed, including bactericidal activity against food-borne pathogens Campylob...
Proceedings of the National Academy of Sciences, 2007
Slits are large multidomain leucine-rich repeat (LRR)-containing proteins that provide crucial gu... more Slits are large multidomain leucine-rich repeat (LRR)-containing proteins that provide crucial guidance cues in neuronal and vascular development. More recently, Slits have been implicated in heart morphogenesis, angiogenesis, and tumor metastasis. Slits are ligands for the Robo (Roundabout) receptors, which belong to the Ig superfamily of transmembrane signaling molecules. The Slit-Robo interaction is mediated by the second LRR domain of Slit and the two N-terminal Ig domains of Robo, but the molecular details of this interaction and how it induces signaling remain unclear. Here we describe the crystal structures of the second LRR domain of human Slit2 (Slit2 D2), the first two Ig domains of its receptor Robo1 (Ig1–2), and the minimal complex between these proteins (Slit2 D2-Robo1 Ig1). Slit2 D2 binds with its concave surface to the side of Ig1 with electrostatic and hydrophobic contact regions mediated by residues that are conserved in other family members. Surface plasmon resonan...
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be p... more Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases...
Collagen in the subendothelium is suggested as a Major binding partner of von Willebrand factor (... more Collagen in the subendothelium is suggested as a Major binding partner of von Willebrand factor (vWF), thereby providing its function in primary hemostasis. The lack of the functionally Most active vWF high Molecular weight Multimers in von Willebrand disease (vWd) type 2A correlates with significantly decreased vWF binding to collagen type I and III, respectively, and offers a possible explanation for the bleeding symptoms in such patients. Consequently, one would expect a similar clinical phenotype in persons with an isolated defect of vWF collagen binding (vWF:CB) but with normal vWF Multimers. A Mutation in the vWF A3 domain that is regarded as the Major vWF:CB site [1], was recently identified in a family with Mother and daughter suffering from a significant bleeding tendency [2]. However, vWF:CB seemed normal in the vWF:CB assay. Only after identification of a Mutation in the vWF A3 domain and recombinant expression of the Mutant protein, a reduced VWF:CB was demonstrated by the collagen binding curve [2]. Here we report on three different novel Mutations in the A3 domain which do not correlate with bleeding symptoms, although they significantly decrease vWF collagen binding.
Von Willebrand factor (VWF), a multimeric plasma glycoprotein, mediates platelet adhesion to site... more Von Willebrand factor (VWF), a multimeric plasma glycoprotein, mediates platelet adhesion to sites of vascular damage. In this proces, the VWF-A3 domain binds to collagen in the vessel wall. This is followed by binding of the VWF-A1 domain to platelet receptor glycoprotein (Gp) Ibalpha. The investigations, using protein crystallography, site-directed mutagenesis and surface-plasmon-resonance, identified the collagen-binding site on the VWF- A3 domain. Based on the surface characteristics of the collagen-binding site, a hypothesis for the binding mode of a collagen triple helix on the VWF-A3 domain has been made. The identification of specific collagen sequences that interact with A3 is subject for future research. To investigate the binding of VWF to GpIbalpha, crystal structures of GpIbalpha, A1GpIbalpha, wt- and gain-of-function mutant R543Q-A1 have been solved. Based on these structures we hypothesized that the N- and C-terminal flanking peptides of the VWF-A1 domain shield one ...
Staphylococcus aureus secretes several virulence factors modulating immune responses. Staphylococ... more Staphylococcus aureus secretes several virulence factors modulating immune responses. Staphylococcal superantigen-like (SSL) proteins are a family of 14 exotoxins with homology to superantigens, but with generally unknown function. Recently, we showed that SSL5 binds to P-selectin glycoprotein ligand 1 dependently of sialyl Lewis X and inhibits P-selectin–dependent neutrophil rolling. Here, we show that SSL5 potently and specifically inhibits leukocyte activation by anaphylatoxins and all classes of chemokines. SSL5 inhibited calcium mobilization, actin polymerization, and chemotaxis induced by chemokines and anaphylatoxins but not by other chemoattractants. Antibody competition experiments showed that SSL5 targets several chemokine and anaphylatoxin receptors. In addition, transfection studies showed that SSL5 binds glycosylated N-termini of all G protein–coupled receptors (GPCRs) but only inhibits stimuli of protein nature that require the receptor N-terminus for activation. Furth...
Food-borne pathogens are responsible for most cases of food poisoning in developed countries and ... more Food-borne pathogens are responsible for most cases of food poisoning in developed countries and are often associated with poultry products, including chicken. Little is known about the role of β-defensins in the chicken digestive tract and their efficacy. In this study, the expression of chicken β-defensin gallinacin-6 (Gal-6) and its antimicrobial activity against food-borne pathogens were investigated. Reverse transcription-PCR analysis showed high expression of Gal-6 mRNA in the esophagus and crop, moderate expression in the glandular stomach, and low expression throughout the intestinal tract. Putative transcription factor binding sites for nuclear factor kappa beta, activator protein 1, and nuclear factor interleukin-6 were found in the Gal-6 gene upstream region, which suggests a possible inducible nature of the Gal-6 gene. In colony-counting assays, strong bactericidal and fungicidal activity was observed, including bactericidal activity against food-borne pathogens Campylob...
Proceedings of the National Academy of Sciences, 2007
Slits are large multidomain leucine-rich repeat (LRR)-containing proteins that provide crucial gu... more Slits are large multidomain leucine-rich repeat (LRR)-containing proteins that provide crucial guidance cues in neuronal and vascular development. More recently, Slits have been implicated in heart morphogenesis, angiogenesis, and tumor metastasis. Slits are ligands for the Robo (Roundabout) receptors, which belong to the Ig superfamily of transmembrane signaling molecules. The Slit-Robo interaction is mediated by the second LRR domain of Slit and the two N-terminal Ig domains of Robo, but the molecular details of this interaction and how it induces signaling remain unclear. Here we describe the crystal structures of the second LRR domain of human Slit2 (Slit2 D2), the first two Ig domains of its receptor Robo1 (Ig1–2), and the minimal complex between these proteins (Slit2 D2-Robo1 Ig1). Slit2 D2 binds with its concave surface to the side of Ig1 with electrostatic and hydrophobic contact regions mediated by residues that are conserved in other family members. Surface plasmon resonan...
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be p... more Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases...
Uploads
Papers by Roland Romijn