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Molecular characterization of p.Asp77Gly and the novel p.Ala163Val and p.Ala163Glu mutations causing protein C
Kitti B Kovács1, István Pataki2, Helga Bárdos3
1Division of Clinical Laboratory Science, University of Debrecen, Debrecen, Hungary.
Insights
Protein C deficiency mutations can cause severe thrombosis. While the 77Gly mutation allows protein secretion, 163Val and 163Glu mutations lead to misfolding, intracellular retention, and polyubiquitination, impacting anticoagulant function.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Protein C (PC) is a critical anticoagulant, and its deficiency, caused by gene mutations, elevates thrombosis risk.
- Severe PC deficiency in neonates, particularly homozygous or compound heterozygous forms, can be life-threatening.
Observation:
- Investigated three missense mutations (p.Asp77Gly, p.Ala163Glu, p.Ala163Val) in two patients with distinct thrombotic events.
- Assessed mutant PC protein fate, including secretion, polyubiquitination, intracellular localization, and function using cell-based assays and microscopy.
- Employed molecular modeling and dynamics simulations to elucidate structural consequences of mutations.
Findings:
- The p.Ala163Val and p.Ala163Glu mutants exhibited undetectable secretion, intracellular co-localization with the 26S proteasome, and polyubiquitination, indicating degradation.
- The p.Asp77Gly mutant was secreted with wild-type-like activity, suggesting intact function but potential impacts on protein complex stability or clearance.
- Mutations p.Ala163Val and p.Ala163Glu induced misfolding, altering EGF2 domain positioning and causing secretion defects.
Implications:
- Identifies distinct molecular mechanisms for PC deficiency, differentiating between secretion defects and potential stability/clearance issues.
- Highlights the role of protein misfolding and proteasomal degradation in severe thrombophilia associated with specific PC mutations.
- Provides insights for potential therapeutic strategies targeting protein folding or degradation pathways in Protein C deficiency.
Introduction:
Protein C (PC) is a major anticoagulant and numerous distinct mutations in its coding gene result in quantitative or qualitative PC deficiency with high thrombosis risk. Homozygous or compound heterozygous PC deficiency usually leads to life-threatening thrombosis in neonates.
Patients And Methods:
The molecular consequences of 3 different missense mutations of two patients have been investigated. The first patient suffered from neonatal purpura fulminans and was a compound heterozygote for p.Asp77Gly and p.Ala163Glu mutations. The second patient had severe deep venous thrombosis in young adulthood and carried the p.Ala163Val mutation. The fate of mutant proteins expressed in HEK cells was monitored by ELISA, by Western blotting, by investigation of polyubiquitination and by functional assays. Their intracellular localization was examined by immunostaining and confocal laser scanning microscopy. Molecular modeling and dynamics simulations were also carried out.
Results And Conclusions:
The 163Val and 163Glu mutants had undetectable levels in the culture media, showed intracellular co-localization with the 26S proteasome and were polyubiquitinated. The 77Gly mutant was secreted to the media showing similar activity as the wild type. There was no difference among intracellular PC levels of wild type and mutant proteins. The 163Val and 163Glu mutations caused significant changes in the relative positions of the EGF2 domains suggesting misfolding with the consequence of secretion defect. No major structural alteration was observed in case of 77Gly mutant; it might influence the stability of protein complexes in which PC participates and may have an impact on the clearance of PC requiring further research.
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