Asp68His mutation in the A1 domain of human factor V causes impaired secretion and ineffective translocation

Abstract

Congenital factor V (FV) deficiency is a rare inherited disorder. We determined the mechanism of a missense mutation, Asp68His, in the A1 domain of the FV protein, is associated with severe FV deficiency. We characterized the mutant FV-Asp68His protein using in vitro expression studies by using specific secretion and degradation pathway inhibitors and analysed the intracellular translocation of the mutant protein by immunofluorescence staining. The Asp68His mutation caused very low levels of FV protein in the conditioned media, with normal specific FV activity. Similar mRNA degradation rates between FV-wild-type (wt) and FV-Asp68His mRNA showed that the Asp68His mutation does not affect FV expression at the transcriptional level. A specific secretion pathway inhibitor, brefeldin A, was used to demonstrate that the lower efficiency of transport to the outside of the cell for FV-Asp68His mutant protein compared with that of the FV-wt protein. Furthermore, we showed that the Asp68His mutation resulted in increased intracellular degradation through a MG132-mediated proteasomal degradation pathway. In the transfected cell lysates, FV-wt protein had multiple posttranslational modified forms, but the FV-Asp68His protein was not completely glycosylated. We further observed that the FV-Asp68His protein was retrieved in the endoplasmic reticulum only and did not undergo transport to the Golgi apparatus, leading to impaired secretion. These results strongly suggest that the Asp68His mutation may result in intracellular defective trafficking and enhanced degradation, and impaired secretion of FV protein.

High Mutational Heterogeneity, and New Mutations in the Human Coagulation Factor V Gene. Future Perspectives for Factor V Deficiency Using Recombinant and Advanced Therapies

“…This wide range of mutational changes observed in the F5 gene is related to a large number of highly variable effects at a functional and molecular level. Some of the consequences of the different mutations include alterations in the stability of FVa [ 61 ]; alterations in the splicing [ 62 , 63 ] and in the biogenesis of FVa [ 64 , 65 , 66 , 67 ], mainly due to secretion disturbances; and mutations affecting the binding sites of the protein to certain activation or inactivation factors [ 68 ]. The mutations described in this study are related to the appearance of the stop codon, which results in a non-functional truncated protein.…”

A Novel Phenotype of the Factor 5 Gene Mutation (Homozygote Met1736Val and Heterozygote Asp68His) Is Associated With Moderate Factor V Deficiency

“…Another mutation profile of FV deficiency was reported by Paraboschi et al in 2020 ( 16 ). In Taiwan, studies about FV deficiency are limited and focus on the A1 domain mutations, such as Asp68His and His147Arg ( 17 ). In this study, we report the cases of two patients with FV deficiency wherein we sequenced their F5 genes.…”

Genetic Confirmation of Congenital Factor V Deficiency in Korean Patients

“…1 ). A review of the literature and databases showed that the above three F5 mutations had been reported previously [ 1 2 3 4 ]. The mother of proband 1 was heterozygous for Asp96His, and the daughter of proband 2 was heterozygous for Asn2010_Ser2011del ( Table 1 ).…”

“…Asp96His and Arg2202Cys in the present study are located in A1 and C2 domains, respectively. Asp96His (Asp68His in the mature protein) was previously described in four unrelated Chinese patients with FVD and was caused by the disruption of salt bridge and subsequent interference in protein expression [ 1 2 ]. Arg2202Cys was reported in a Chinese patient [ 2 ].…”

“…Asn2010_Ser2011del destabilizes C1-A3 interaction, thus affecting the folding, stability, and secretion of FV [ 3 ]. Thus, detection of these F5 mutations in Asian patients suggests shared genetic backgrounds among Asian ethnicities [ 1 2 3 4 ].…”