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: Markus J Harder, Markus Anliker, Britta Höchsmann, Thomas Simmet, Markus Huber-Lang, Hubert Schrezenmeier, Daniel Ricklin, John D Lambris, Paul N Barlow, Christoph Q Schmidt
• Journal: Journal of immunology (Baltimore, Md. : 1950) (J Immunol) Vol. 196 Issue 2 Pg. 866-76 (Jan 15 2016) ISSN: 1550-6606 [Electronic] United States
• PMID: 26643478 (Publication Type: Comparative Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2016 by The American Association of Immunologists, Inc.

Chemical References

• CFHR1 protein, human
• Complement C3b Inactivator Proteins
• Complement Inactivating Agents
• Recombinant Proteins
• Complement Factor H

Topics

• Amino Acid Sequence
• Complement C3b Inactivator Proteins (immunology)
• Complement Factor H (chemistry, immunology)
• Complement Inactivating Agents (chemical synthesis, immunology, pharmacology)
• Complement Pathway, Alternative (immunology)
• Electrophoresis, Polyacrylamide Gel
• Enzyme-Linked Immunosorbent Assay
• Flow Cytometry
• Humans
• Molecular Sequence Data
• Polymerase Chain Reaction
• Protein Binding
• Recombinant Proteins (chemical synthesis, immunology, pharmacology)