Roland A. Romijn

Structural Insights Into the Slit-Robo Complex.

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 resonance experiments and a mutational analysis of the interface confirm that Ig1 is the primary domain for binding Slit2. These structures provide molecular insight into Slit-Robo complex formation and will be important for the development of novel cancer therapeutics.

The β-Defensin Gallinacin-6 Is Expressed in the Chicken Digestive Tract and Has Antimicrobial Activity against Food-Borne Pathogens

ABSTRACT 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 Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Clostridium perfringens, and Escherichia coli. Treatment with 16 μg/ml synthetic Gal-6 resulted in a 3 log unit reduction in Clostridium perfringens survival within 60 min, indicating fast killing kinetics. Transmission electron microscopy examination of synthetic-Gal-6-treated Clostridium perfringens cells showed dose-dependent changes in morphology after 30 min, including intracellular granulation, cytoplasm retraction, irregular septum formation in dividing cells, and cell lysis. The high expression in the proximal digestive tract and broad antimicrobial activity suggest that chicken β-defensin gallinacin-6 plays an important role in chicken innate host defense.

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

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 dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombin-cleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors.

Factor B structure provides insights into activation of the central protease of the complement system

Factor B is the central protease of the complement system of immune defense. Here, we present the crystal structure of human factor B at 2.3-Å resolution, which reveals how the five-domain proenzyme is kept securely inactive. The canonical activation helix of the Von Willebrand factor A (VWA) domain is displaced by a helix from the preceding domain linker. The two helices conformationally link the scissile-activation peptide and the metal ion–dependent adhesion site required for binding of the ligand C3b. The data suggest that C3b binding displaces the three N-terminal control domains and reshuffles the two central helices. Reshuffling of the helices releases the scissile bond for final proteolytic activation and generates a new interface between the VWA domain and the serine protease domain. This allosteric mechanism is crucial for tight regulation of the complement-amplification step in the immune response.

Interaction of ß2-glycoprotein I with members of the low density lipoprotein receptor family

Summary.  The antiphospholipid syndrome (APS) is a non‐inflammatory autoimmune disease characterized by arterial and/or venous thrombosis and/or pregnancy morbidity in the presence of autoantibodies that recognize beta2‐glycoprotein I (β2GPI) bound to phospholipids. We have previously demonstrated that dimerization of β2GPI by autoantibodies induces platelet activation, involving the platelet receptor apolipoprotein E receptor 2’ (apoER2′) a receptor belonging to the low‐density lipoprotein receptor (LDL‐R) family. Here, we show that dimeric β2GPI, but not monomeric β2GPI, interacts with four other LDL‐R family members: the LDL‐R related protein (LRP), megalin, the LDL‐R and the very‐low density lipoprotein receptor (VLDL‐R). Interaction between dimeric β2GPI and LDL‐R, apoER2′ and VLDL‐R was best described with a one‐site binding model (half‐maximal binding; ∼20 nm for apoER2′ and VLDL‐R and ∼300 nm for LDL‐R), whereas the interaction between dimeric β2GPI and LRP or megalin was best described with a two‐site binding model, representing a high‐ (∼3 nm) and a low‐affinity site (∼0.2 μm). Binding to all receptors tested was unaffected by a tryptophane to serine (W316S) substitution in domain V of β2GPI, which is known to disrupt the phospholipid binding site of β2GPI. Also deletion of domain I or II left the interaction with the receptors unaffected. Deletion of domain V, however, significantly decreased the affinity for the receptors. In conclusion, our data show that dimeric β2GPI can interact with different LDL‐R family members. This interaction is dependent on a binding site within domain V of β2GPI, which does not overlap with the phospholipid‐binding site within domain V.

Staphylococcal SSL5 inhibits leukocyte activation by chemokines and anaphylatoxins

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. Furthermore, SSL5 increased binding of chemokines to cells independent of chemokine receptors through their common glycosaminoglycan-binding site. Importance of glycans was shown for both GPCR and chemokine binding. Thus, SSL5 is an important immunomodulatory protein of S aureus that targets several crucial, initial stages of leukocyte extravasation. It is therefore a potential new antiinflammatory compound for diseases associated with chemoattractants and their receptors and disorders characterized by excessive recruitment of leukocytes.

von Willebrand factor A1 domain can adequately substitute for A3 domain in recruitment of flowing platelets to collagen

Summary.  Background: Binding of von Willebrand factor (VWF) to platelet GPIbα and to collagen is attributed to VWF A1 and A3 domains, respectively. Objectives: Using VWF, VWF lacking A1 (ΔA1‐VWF) or A3 (ΔA3‐VWF) and VWF with defective A3 (H1786A‐VWF), in combination with recombinant A1 (residues 1262–1492) or A3 (residues 1671–1878), fused to glutathione‐S‐transferase (GST‐A1 and GST‐A3), we have re‐investigated the role of A1 in platelet recruitment to surfaces of collagen. Methods and Results: In flow, measurable binding of ΔA3‐VWF occurred to horse tendon, but also to human type III collagen. GST‐A1 and GST‐A3 both competed for binding of ΔA1‐VWF and ΔA3‐VWF to horse tendon collagen fibrils in static conditions and to human collagen III during plasmon surface resonance studies, substantiating overlapping binding sites on both collagens for A1 and A3. Heparin did not affect A3‐mediated binding of VWF and ΔA1‐VWF, but inhibited binding to horse tendon collagen of GST‐A1 and ΔA3‐VWF. Furthermore, A1‐mediated binding to type III collagen of ΔA3‐VWF binding was strongly salt‐sensitive. During perfusions at wall shear rate 2500 s−1 of calcein‐labeled platelets in reconstituted blood, ΔA3‐VWF and H1786A‐VWF triggered platelet binding to horse tendon collagen comparably and as potently as VWF, and to human type III collagen, only fivefold less potently, ΔA1‐VWF being inactive. Additional flow‐controlled interaction studies with ΔA3‐VWF, H1786A‐VWF, the collagen‐VWF antagonist saratin, heparin and the VWF neutralizing antibody 82D6A3 confirmed that H1786A‐VWF binds to collagen exclusively via A1. Conclusion: Hence, in shear forces the VWF A1 domain can assume the role of A3 to trigger substantial platelet recruitment to human collagen fibres.

A Consensus Tetrapeptide Selected by Phage Display Adopts the Conformation of a Dominant Discontinuous Epitope of a Monoclonal Anti-VWF Antibody That Inhibits the von Willebrand Factor-Collagen Interaction

Monoclonal antibody (mAb) 82D6A3 is an anti-von Willebrand factor (VWF) mAb directed against the A3-domain of VWF that inhibits the VWF binding to fibrillar collagens type I and III in vitro and in vivo. To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg963, Pro981, Asp1009, Arg1016, Ser1020, Met1022, and His1023 are part of the epitope of mAb 82D6A3. Inspection of residues Ser1020, Arg1016, Pro981, and Trp982 in the three-dimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser1020 and Arg1016 in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp982. In conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.

Assessment of the antiviral properties of recombinant porcine SP-D against various influenza A viruses in vitro.

The emergence of influenza viruses resistant to existing classes of antiviral drugs raises concern and there is a need for novel antiviral agents that could be used therapeutically or prophylacticaly. Surfactant protein D (SP-D) belongs to the family of C-type lectins which are important effector molecules of the innate immune system with activity against bacteria and viruses, including influenza viruses. In the present study we evaluated the potential of recombinant porcine SP-D as an antiviral agent against influenza A viruses (IAVs) in vitro. To determine the range of antiviral activity, thirty IAVs of the subtypes H1N1, H3N2 and H5N1 that originated from birds, pigs and humans were selected and tested for their sensitivity to recombinant SP-D. Using these viruses it was shown by hemagglutination inhibition assay, that recombinant porcine SP-D was more potent than recombinant human SP-D and that especially higher order oligomeric forms of SP-D had the strongest antiviral activity. Porcine SP-D was active against a broad range of IAV strains and neutralized a variety of H1N1 and H3N2 IAVs, including 2009 pandemic H1N1 viruses. Using tissue sections of ferret and human trachea, we demonstrated that recombinant porcine SP-D prevented attachment of human seasonal H1N1 and H3N2 virus to receptors on epithelial cells of the upper respiratory tract. It was concluded that recombinant porcine SP-D holds promise as a novel antiviral agent against influenza and further development and evaluation in vivo seems warranted.

Chicken lung lectin is a functional C-type lectin and inhibits haemagglutination by influenza A virus.

Many proteins of the calcium-dependent (C-type) lectin family have been shown to play an important role in innate immunity. They can bind to a broad range of carbohydrates, which enables them to interact with ligands present on the surface of micro-organisms. We previously reported the finding of a new putative chicken lectin, which was predominantly localized to the respiratory tract, and thus termed chicken lung lectin (cLL). In order to investigate the biochemical and biophysical properties of cLL, the recombinant protein was expressed, affinity purified and characterized. Recombinant cLL was expressed as four differently sized peptides, which is most likely due to post-translational modification. Crosslinking of the protein led to the formation of two high-molecular weight products, indicating that cLL forms trimeric and possibly even multimeric subunits. cLL was shown to have lectin activity, preferentially binding to alpha-mannose in a calcium-dependent manner. Furthermore, cLL was shown to inhibit the haemagglutination-activity of human isolates of influenza A virus, subtype H3N2 and H1N1. These result show that cLL is a true C-type lectin with a very distinct sugar specificity, and that this chicken lectin could play an important role in innate immunity.