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A novel method for real-time analysis of the complement C3b:FH:FI complex reveals dominant negative CFI variants in Age-related Macular Degeneration

Lookup NU author(s): Dr Thomas Hallam, Tom Cox, Dr Kate Smith-Jackson, Dr Vicky Brocklebank, April Baral, Nik Tzoumas, Professor David SteelORCiD, Dr Edwin Wong, Victoria Shuttleworth, Professor Claire Harris, Professor Kevin MarchbankORCiD, Professor David KavanaghORCiD

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Abstract

Age-related macular degeneration (AMD) is linked to 2 main disparate genetic Q8pathways: a chromosome 10 risk locus and the alternative pathway (AP) ofcomplement. Rare genetic variants in complement factor H (CFH; FH) andfactor I (CFI; FI) are associated with AMD. FH acts as a soluble cofactor tofacilitate FI’s cleavage and inactivation of the central molecule of the AP, C3b.For personalised treatment, sensitive assays are required to define thefunctional significance of individual AP genetic variants. Generation ofrecombinant FI for functional analysis has thus far been constrained byincomplete processing resulting in a preparation of active and inactiveprotein. Using an internal ribosomal entry site (IRES)-Furin-CFI expressionvector, fully processed FI was generated with activity equivalent to serumpurified FI. By generating FI with an inactivated serine protease domain (S525AFI), a real-time surface plasmon resonance assay of C3b:FH : FI complexformation for characterising variants in CFH and CFI was developed andcorrelated well with standard assays. Using these methods, we furtherdemonstrate that patient-associated rare genetic variants lacking enzymaticactivity (e.g. CFI I340T) may competitively inhibit the wild-type FI protein. Thedominant negative effect identified in inactive factor I variants could impact onthe pharmacological replacement of FI currently being investigated for thetreatment of dry AMD.

Publication metadata

Author(s): Hallam TM, Cox TE, Smith-Jackson K, Brocklebank V, Baral AJ, Tzoumas N, Steel DH, Wong EKS, Shuttleworth VG, Lotery AJ, Harris CL, Marchbank KJ, Kavanagh D

Publication type: Article

Publication status: Published

Journal: Frontiers in Immunology

Year: 2022

Volume: 13

Online publication date: 28/12/2022

Acceptance date: 14/11/2022

Date deposited: 21/12/2022

ISSN (electronic): 1664-3224

Publisher: Frontiers Research Foundation

URL: https://doi.org/10.3389/fimmu.2022.1028760

DOI: 10.3389/fimmu.2022.1028760

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