OC 07.2 - Investigating the dominant negative effect of heterozygous F13B gene mutations detected from mild FXIII deficiency patients

Background: Heterozygous mutations in the F13B gene that expresses coagulation FXIII-B subunit are complicit in inducing mild FXIII deficiency states, which although primarily asymptomatic, can cause bleeding when exposed to vascular trauma. The underlying conditional dominant-negative pathomolecular mechanism of these F13B heterozygous mutations is poorly understood

Aims: To investigate if the dominant-negative effect of the F13B gene heterozygous missense mutations is exerted intracellularly or extracellularly

Methods: Secretory behavior of FXIII-B heterozygous variant species was investigated by confocal microscopy using a dual promoter bi-cistronic heterozygous co-expression vector strategy with FXIII-B wild-type (WT) cDNA in the first ORF, fused with an N-terminal ALFA tag and respective FXIII-B variants (Cys5Arg, Cys316Phe, Cys430Phe) in the second ORF fused with a C-terminal HA tag. HepG2 cells were transiently transfected on confocal microscopy slides, fixed and stained with antibodies against the respective tags, ER and Golgi. Microscopic measurements were performed on ZEISS Apotome. Quantitative co-localization analysis was performed with EZColocalization plugin embedded in ImageJ (Figure1). The extracellular effect of the three mutations on the binding energy/formation of FXIIIA2B2 heterotetramer and FXIII-B2 dimer was evaluated on a recently deposited cryo-EM structure of FXIIIA2B2 heterotetramer and another structure extracted from the cryo-EM density maps of free FXIII-B2 subunit generated from rFXIII-B on the Strubitem cryo-EM platform

Results: Selective dominant-negative intracellular accumulation in the ER was observed for the heterozygous species only in the case of Cys5Arg mutant. Additionally, the homozygous mutant species for Cys5Arg mutant also accumulated in ER. All three mutations were observed to be proximal or directly on either the heterotetrameric interface (Cys5Arg) or the free FXIII-B subunit dimeric interface (Cys430Phe and Cys316Phe)

Conclusion(s): An intracellular dominant-negative effect was observed only for Cys5Arg mutant. The dominant-negative effect of all cysteine mutations (including Cys5Arg) is imparted primarily extracellularly by destabilizing the formation of either the FXIII-A2B2 heterotetramer or the FXIII-B2 dimer itself