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Effect of four missense mutations in the factor XIII A-subunit gene on protein stability: studies with recombinant
Alex Vysokovsky1, Nurit Rosenberg, Rima Dardik
1Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, Israel.
Abstract:
Four missense mutations in the factor XIII A-subunit gene, Arg260Leu, Ala318Val, Thr398Asn and Gly210Arg, were previously reported by us in patients with severe factor XIII deficiency. The objective of our study was to discern the effect of all four mutations on the stability and intracellular localization of the factor XIII A-subunit by their expression in COS cells. In-vitro mutagenesis, transient expression of the mutants in COS cells and subsequent pulse-chase analyses were carried out. Intracellular localization of wild-type and mutant proteins was analyzed by immunohistochemistry using a monoclonal antibody against factor XIII A-subunit. Pulse-chase analyses of metabolically labeled proteins demonstrated rapid intracellular degradation of each mutant protein as compared with wild type. Immunocytochemical and immunofluorescence analyses disclosed that wild-type and all four mutant factor XIII A-subunit proteins were diffusely distributed within the cytoplasm but not in the endoplasmic reticulum of the COS-7 cells. The Arg260Leu, Ala318Val, Thr398Asn and Gly210Arg mutations in FXIII A-subunit cause rapid intracellular degradation of the corresponding mutated protein.
Insights
Four mutations in the factor XIII A-subunit gene lead to rapid intracellular degradation of the mutated protein, impacting factor XIII deficiency. This study investigated the stability and localization of these FXIII A-subunit mutations.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Severe factor XIII deficiency can be caused by mutations in the factor XIII A-subunit gene.
- Four specific missense mutations (Arg260Leu, Ala318Val, Thr398Asn, Gly210Arg) were previously identified in patients.
Purpose of the Study:
- To investigate the impact of four identified factor XIII A-subunit gene mutations on protein stability and intracellular localization.
- To understand the molecular mechanisms underlying severe factor XIII deficiency.
Main Methods:
- In-vitro mutagenesis was used to create the mutant factor XIII A-subunit genes.
- COS cells were used for transient expression of wild-type and mutant proteins.
- Pulse-chase analysis and immunohistochemistry were employed to assess protein stability and localization.
Main Results:
- All four mutant factor XIII A-subunit proteins exhibited rapid intracellular degradation compared to the wild-type protein.
- Immunocytochemical and immunofluorescence analyses showed diffuse cytoplasmic distribution for both wild-type and mutant proteins, excluding the endoplasmic reticulum.
Conclusions:
- The Arg260Leu, Ala318Val, Thr398Asn, and Gly210Arg mutations in the factor XIII A-subunit result in accelerated intracellular degradation of the protein.
- This rapid degradation likely contributes to the pathogenesis of severe factor XIII deficiency.
