Functional evaluation of complement factor I variants by immunoassays and SDS-PAGE

Frontiers in Immunology

Immunology

Marit Pullen¹  Nicole C. A. J. van de Kar²  Siem J. van de Peppel²  Laura M. Baas²  Lambertus P. van den Heuvel³  Per H. Nilsson⁴  Dick J. Sjöström⁴  Alexandra Gerogianni⁴ 

[1] Department of Genetics, Radboud University Medical Center, Nijmegen, Netherlands;Department of Pediatric Nephrology, Radboud University Medical Center, Amalia Children’s Hospital, Nijmegen, Netherlands;Department of Pediatric Nephrology, Radboud University Medical Center, Amalia Children’s Hospital, Nijmegen, Netherlands;Department of Genetics, Radboud University Medical Center, Nijmegen, Netherlands;Department of Pediatrics/Pediatric Nephrology, University Hospitals Leuven, Leuven, Belgium;Department of Development and Regeneration, University Hospitals Leuven, Leuven, Belgium;Linnaeus Centre for Biomaterials Chemistry, Linnaeus University, Kalmar, Sweden;Department of Chemistry and Biomedicine, Linnaeus University, Kalmar, Sweden;

关键词: factor I;    co-factor activity;    functional assay;    complement regulation;    factor H;    complement receptor I;   

DOI  :  10.3389/fimmu.2023.1279612

 received in 2023-08-18, accepted in 2023-10-16,  发布年份 2023

来源: Frontiers

【 摘 要 】

Factor I (FI) is an essential regulator of the complement system. Together with co-factors, FI degrades C3b, which inhibits further complement activation. Genetic mutations in FI are associated with pathological conditions like age-related macular degeneration and atypical hemolytic uremic syndome. Here, we evaluated eight recombinant FI genetic variants found in patients. We assessed FI’s co-factor activity in the presence of two co-factors; Factor H and soluble CR1. Different analytical assays were employed; SDS-PAGE to evaluate the degradation of C3b, ELISA to measure the generation of fluid phase iC3b and the degradation of surface-bound C3b using a novel Luminex bead-based assay. We demonstrate that mutations in the FIMAC and SP domains of FI led to significantly reduced protease activity, whereas the two analyzed mutations in the LDLRA2 domain did not result in any profound changes in FI’s function. The different assays employed displayed a strong positive correlation, but differences in the activity of the genetic variants Ile55Phe and Gly261Asp could only be observed by combining different methods and co-factors for evaluating FI activity. In conclusion, our results provide a new perspective regarding available diagnostic tools for assessing the impact of mutations in FI.

【 授权许可】

Unknown   
Copyright © 2023 Gerogianni, Baas, Sjöström, van de Kar, Pullen, van de Peppel, Nilsson and van den Heuvel