title: Molecular characterization of p.Asp77Gly and the novel p.Ala163Val and p.Ala163Glu mutations causing protein C deficiency.
abstract: BACKGROUND
  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.
  
  
  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.
  
  
  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.
authors.affiliation: Division of Clinical Laboratory Science, University of Debrecen, Debrecen, Hungary.
authors.forename: Kitti B
authors.initials: KB
authors.lastname: Kovács
authors.affiliation: Department of Pediatrics, University of Debrecen, Debrecen, Hungary.
authors.forename: István
authors.initials: I
authors.lastname: Pataki
authors.affiliation: Department of Preventive Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: Helga
authors.initials: H
authors.lastname: Bárdos
authors.affiliation: Division of Clinical Laboratory Science, University of Debrecen, Debrecen, Hungary.
authors.forename: Attila
authors.initials: A
authors.lastname: Fekete
authors.affiliation: Division of Rare Diseases, University of Debrecen, Debrecen, Hungary.
authors.forename: György
authors.initials: G
authors.lastname: Pfliegler
authors.affiliation: Division of Clinical Laboratory Science, University of Debrecen, Debrecen, Hungary.
authors.forename: Gizella
authors.initials: G
authors.lastname: Haramura
authors.affiliation: Division of Clinical Laboratory Science, University of Debrecen, Debrecen, Hungary.
authors.forename: Réka
authors.initials: R
authors.lastname: Gindele
authors.affiliation: Haemostasis, Thrombosis and Vascular Biology Research Group of the Hungarian Academy of Sciences.
authors.forename: István
authors.initials: I
authors.lastname: Komáromi
authors.affiliation: Department of Pediatrics, University of Debrecen, Debrecen, Hungary; Haemostasis, Thrombosis and Vascular Biology Research Group of the Hungarian Academy of Sciences.
authors.forename: György
authors.initials: G
authors.lastname: Balla
authors.affiliation: Department of Preventive Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: Róza
authors.initials: R
authors.lastname: Ádány
authors.affiliation: Division of Clinical Laboratory Science, University of Debrecen, Debrecen, Hungary; Haemostasis, Thrombosis and Vascular Biology Research Group of the Hungarian Academy of Sciences.
authors.forename: László
authors.initials: L
authors.lastname: Muszbek
authors.affiliation: Division of Clinical Laboratory Science, University of Debrecen, Debrecen, Hungary. Electronic address: zsbereczky@med.unideb.hu.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Bereczky
mesh_terms: D005260:Female; D006801:Humans; D008875:Middle Aged; D008958:Models, Molecular; D009154:Mutation; D020125:Mutation, Missense; D011486:Protein C / Q000378:metabolism*; D020151:Protein C Deficiency / Q000235:genetics*
chemical_list: D011486:Protein C
keywords: Expression study; Missense mutation; Molecular modeling; Protein C deficiency; Secretion defect