title: Comparative Analysis of Novel Complement-Targeted Inhibitors, MiniFH, and the Natural Regulators Factor H and Factor H-like Protein 1 Reveal Functional Determinants of Complement Regulation.
abstract: The serum proteins factor H (FH), consisting of 20 complement control protein modules (CCPs), and its splice product FH-like protein 1 (FHL-1; consisting of CCPs 1-7) are major regulators of the alternative pathway (AP) of complement activation. The engineered version of FH, miniFH, contains only the N- and C-terminal portions of FH linked by an optimized peptide and shows ∼ 10-fold higher ex vivo potency. We explored the hypothesis that regulatory potency is enhanced by unmasking of a ligand-binding site in the C-terminal CCPs 19-20 that is cryptic in full-length native FH. Therefore, we produced an FH variant lacking the central domains 10-15 (FHΔ10-15). To explore how avidity affects regulatory strength, we generated a duplicated version of miniFH, termed midiFH. We compared activities of FHΔ10-15 and midiFH to miniFH, FH, and FHL-1. Relative to FH, FHΔ10-15 exhibited an altered binding profile toward C3 activation products and a 5-fold-enhanced complement regulation on a paroxysmal nocturnal hemoglobinuria patient's erythrocytes. Contrary to dogma, FHL-1 and FH exhibited equal regulatory activity, suggesting that the role of FHL-1 in AP regulation has been underestimated. Unexpectedly, a substantially increased avidity for complement opsonins, as seen in midiFH, did not potentiate the inhibitory potential on host cells. In conclusion, comparisons of engineered and native FH-based regulators have identified features that determine high AP regulatory activity on host cells. Unrestricted availability of FH CCPs 19-20 and an optimal spatial orientation between the N- and C-terminal FH regions are key.
authors.affiliation: Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, 89081 Ulm, Germany;
authors.forename: Markus J
authors.initials: MJ
authors.lastname: Harder
authors.affiliation: Institute of Transfusion Medicine, University of Ulm, 89081 Ulm, Germany; Institute of Clinical Transfusion Medicine and Immunogenetics, German Red Cross Blood Service Baden-Württemberg - Hessen and University Hospital Ulm, 89081 Ulm, Germany;
authors.forename: Markus
authors.initials: M
authors.lastname: Anliker
authors.affiliation: Institute of Transfusion Medicine, University of Ulm, 89081 Ulm, Germany; Institute of Clinical Transfusion Medicine and Immunogenetics, German Red Cross Blood Service Baden-Württemberg - Hessen and University Hospital Ulm, 89081 Ulm, Germany;
authors.forename: Britta
authors.initials: B
authors.lastname: Höchsmann
authors.affiliation: Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, 89081 Ulm, Germany;
authors.forename: Thomas
authors.initials: T
authors.lastname: Simmet
authors.affiliation: Department of Traumatology, Center of Surgery, University of Ulm, 89081 Ulm, Germany;
authors.forename: Markus
authors.initials: M
authors.lastname: Huber-Lang
authors.affiliation: Institute of Transfusion Medicine, University of Ulm, 89081 Ulm, Germany; Institute of Clinical Transfusion Medicine and Immunogenetics, German Red Cross Blood Service Baden-Württemberg - Hessen and University Hospital Ulm, 89081 Ulm, Germany;
authors.forename: Hubert
authors.initials: H
authors.lastname: Schrezenmeier
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19102;
authors.forename: Daniel
authors.identifier: http://orcid.org/0000-0001-6140-0233
authors.initials: D
authors.lastname: Ricklin
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19102;
authors.forename: John D
authors.initials: JD
authors.lastname: Lambris
authors.affiliation: School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, United Kingdom; and School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JJ, United Kingdom.
authors.forename: Paul N
authors.initials: PN
authors.lastname: Barlow
authors.affiliation: Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, 89081 Ulm, Germany; christoph.schmidt@uni-ulm.de.
authors.forename: Christoph Q
authors.initials: CQ
authors.lastname: Schmidt
mesh_terms: D000595:Amino Acid Sequence; D003180:Complement C3b Inactivator Proteins / Q000276:immunology*; D017242:Complement Factor H / Q000737:chemistry / Q000276:immunology*; D051056:Complement Inactivating Agents / Q000138:chemical synthesis / Q000276:immunology / Q000494:pharmacology*; D003170:Complement Pathway, Alternative / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000276:immunology / Q000494:pharmacology*
chemical_list: C517621:CFHR1 protein, human; D003180:Complement C3b Inactivator Proteins; D051056:Complement Inactivating Agents; D011994:Recombinant Proteins; D017242:Complement Factor H