CRASPs are multifunctional surface proteins of Borrelia burgdorferi that bind two additional plasma proteins, FHR-1 and plasminogen

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Abstracts / Molecular Immunology 44 (2007) 3909–3994

age-related macular degeneration. This polymorphism is in the domain implicated in binding C-reactive protein (CRP) and reduced binding of fH-His compared to fH-Tyr has been reported. However, we observed no specific binding of fH to structurally and functionally intact natural human CRP, to CRP expressed in CHO cells, or to bacterial recombinant CRP (Calbiochem) immobilised by direct coating to microtitre plates from Ca2+ -containing buffer. In contrast, when plates were coated with urea-denatured CRP there was strong, dose-dependent binding of fH. The native structure of CRP is strongly stabilised by Ca2+ , suggesting that reported binding of fH may have reflected denaturation of CRP by use of Ca2+ -free buffers and prolonged coating times in the absence of Ca2+ . We coated plates with CRP in the presence or absence of Ca2+ and for 1, 2 or 3 days at 4 ◦ C. Similar amounts of CRP were immobilised in all conditions, but there was no binding of fH to plates coated in Ca2+ whereas with coating in the absence of Ca2+ there was weak binding of fH to plates coated for 1 day and increased binding to day 2 and day 3 plates; when the two fH variants were tested separately there was more binding of fH-Tyr than fH-His throughout. Denaturation of CRP during coating on plastic is thus required for binding of fH. Further experiments will establish whether fH binds native CRP immobilised by ligands such as cellular constituents, as occurs under pathophysiological conditions. doi:10.1016/j.molimm.2007.06.194 P116 CRASPs are multifunctional surface proteins of Borrelia burgdorferi that bind two additional plasma proteins, FHR-1 and plasminogen Katrin Haupt a , Reinhard Wallich b , Peter Kraiczy c , Volker Brade c , Christine Skerka a , Peter F. Zipfel a a

Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology—Hans-Kn¨oll-Institute, Jena, Germany b Department of Immunology, University of Heidelberg, Heidelberg, Germany c Institute for Medical Microbiology, University Hospital Frankfurt, Frankfurt am Main, Germany The spirochete Borrelia burgdorferi, the causative agent of Lyme disease, uses multiple strategies to inhibit host immune attack and to invade host tissues. B. burgdorferi expresses up to five distinct complement regulator-acquiring surface proteins (CRASP-1 to CRASP-5) that bind the human complement regulators Factor H and FHL-1, the splicing variant of Factor H. Here we identify two additional CRASP-binding plasma proteins, Factor H related protein 1 (FHR-1) and plasminogen, the precursor of the serine protease plasmin. Adsorption assays demonstrate acquisition of FHR-1 to spirochetes. Ligand blot analysis, ELISA and surface plasmon resonance studies show binding of FHR-1 to CRASP-3, CRASP-4 and CRASP-5. The FHR-1 protein competes with Factor H for binding to CRASP-3 and to CRASP-5, when both proteins are provided simultaneously. FHR-1 binds strongly to CRASP-4. Cofactor activity of

Factor H decreased when FHR-1 concentrations were increased. Plasminogen binds CRASP-1 via lysine residues as shown in ligand blot experiments and ELISA. Peptide spot analysis revealed two binding sites in CRASP-1 for plasminogen. When bound to CRASP-1, plasminogen is functionally active and cleaves the chromogenic substrate S2251 and fibrinogen. Thus, FHR-1 and plasminogen, in addition to the host immune regulators Factor H and FHL-1 are ligands for CRASPs, suggesting a role for FHR-1 and plasminogen in immune evasion of B. burgdorferi. doi:10.1016/j.molimm.2007.06.195 P117 Mapping of ligand binding sites gives further insight into FHR-4 function Mario Hebecker a , Michael Mihlan b , Peter F. Zipfel b , Mih´aly J´ozsi a a

Junior Research Group Cellular Immunobiology, Germany of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology—Hans Kn¨oll Institute, Jena, Germany b Department

The human factor H-related protein FHR-4 exists in two isoforms. The short isoform FHR-4B has been characterized and shown to interact with C3b. The long isoform FHR-4A consists of nine short consensus repeat (SCR) domains and seems to be the dominant form in plasma. SCR1-4 of FHR-4A is related to SCR5-8, due to a duplication event. The function of FHR-4 is unknown and therefore we aimed to identify binding domains within the molecule. To map the specific ligand binding sites we generated recombinant fragments of FHR-4A, representing SCR1-3, SCR4-9, SCR5-7 and SCR8-9. The interaction of these fragments with complement C3b, iC3b and C3d was analyzed by ELISA. SCR49 and SCR8-9, but not the other fragments, bound to C3b and its cleavage products, indicating that the binding site is located within the C-terminal two SCRs, which are related to SCR19-20 of factor H (FH). Since the binding was stronger for the SCR49 fragment, SCR4 of FHR-4A, which has a high similarity to SCR19 of FH, may contribute to C3b binding. FHR-4A binds to C-reactive protein (CRP), and ELISA assays with the FHR-4A fragments showed a strong binding of the SCR1-3 fragment and no binding of the related SCR57 fragment. In addition, FHR-4B, which consists of SCR1 and SCR6-9 of FHR-4A, also bound CRP. These results suggest that the CRP binding site is located within SCR1, which is related to SCR6 of FH. In summary, we localize complement C3b and CRP binding domains of FHR-4A. The binding characteristics of the protein fragments give further insight into function of the FHR-4 proteins, as the separation of binding sites allows simultaneous binding of FHR-4A to C3b/iC3b/C3d and CRP. Thus, our data indicate a role of FHR-4 in innate immune response and inflammation. doi:10.1016/j.molimm.2007.06.196