A novel method for real-time analysis of the complement C3b:FH:FI complex reveals dominant negative CFI variants in age-related macular degeneration

Age-related macular degeneration (AMD) is linked to 2 main disparate genetic pathways: a chromosome 10 risk locus and the alternative pathway (AP) of complement. Rare genetic variants in complement factor H ( CFH; FH ) and factor I ( CFI; FI ) are associated with AMD. FH acts as a soluble cofactor t...

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Published in:	Frontiers in immunology Vol. 13; p. 1028760
Main Authors:	Hallam, Thomas M., Cox, Thomas E., Smith-Jackson, Kate, Brocklebank, Vicky, Baral, April J., Tzoumas, Nikolaos, Steel, David H., Wong, Edwin K. S., Shuttleworth, Victoria G., Lotery, Andrew J., Harris, Claire L., Marchbank, Kevin J., Kavanagh, David
Format:	Journal Article
Language:	English
Published:	Switzerland Frontiers Media S.A 28.12.2022
Subjects:	aHUS (atypical haemolytic uraemic syndrome) AMD (age-related macular degeneration) Complement Complement C3b - genetics Complement Factor H - genetics Complement Factor I - genetics Factor H Factor I Humans Immunology Macular Degeneration - genetics Surface plasmon resonance Factor H AMD (age-related macular degeneration) PNH (paroxysmal nocturnal haemoglobinuria) C3G (C3 Glomerulopathy) Complement Surface plasmon resonance aHUS (atypical haemolytic uraemic syndrome) Factor I
ISSN:	1664-3224, 1664-3224
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Summary:	Age-related macular degeneration (AMD) is linked to 2 main disparate genetic pathways: a chromosome 10 risk locus and the alternative pathway (AP) of complement. Rare genetic variants in complement factor H ( CFH; FH ) and factor I ( CFI; FI ) are associated with AMD. FH acts as a soluble cofactor to facilitate FI’s cleavage and inactivation of the central molecule of the AP, C3b. For personalised treatment, sensitive assays are required to define the functional significance of individual AP genetic variants. Generation of recombinant FI for functional analysis has thus far been constrained by incomplete processing resulting in a preparation of active and inactive protein. Using an internal ribosomal entry site (IRES)-Furin- CFI expression vector, fully processed FI was generated with activity equivalent to serum purified FI. By generating FI with an inactivated serine protease domain (S525A FI), a real-time surface plasmon resonance assay of C3b:FH:FI complex formation for characterising variants in CFH and CFI was developed and correlated well with standard assays. Using these methods, we further demonstrate that patient-associated rare genetic variants lacking enzymatic activity (e.g. CFI I340T) may competitively inhibit the wild-type FI protein. The dominant negative effect identified in inactive factor I variants could impact on the pharmacological replacement of FI currently being investigated for the treatment of dry AMD.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Reviewed by: József Dobó, Hungarian Academy of Sciences (MTA), Hungary; Ronald Paul Taylor, University of Virginia, United States This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology Edited by: Brian V. Geisbrecht, Kansas State University, United States These authors share first authorship
ISSN:	1664-3224 1664-3224
DOI:	10.3389/fimmu.2022.1028760