Effect of four missense mutations in the factor XIII A-subunit gene on protein stability: studies with recombinant proteins

• Published in: Blood coagulation & fibrinolysis Vol. 17; no. 2; p. 125
• Main Authors: Vysokovsky, Alex, Rosenberg, Nurit, Dardik, Rima, Seligsohn, Uri, Inbal, Aida
• Format: Journal Article
• Language: English
• Published: England 01.03.2006
• Subjects: Animals; Cercopithecus aethiops; COS Cells; Cytoplasm - genetics; Cytoplasm - metabolism; Endoplasmic Reticulum - genetics; Endoplasmic Reticulum - metabolism; Factor XIII - genetics; Factor XIII - metabolism; Factor XIII Deficiency - genetics; Factor XIII Deficiency - metabolism; Gene Expression; Humans; Mutagenesis; Mutation, Missense; Protein Subunits - genetics; Protein Subunits - metabolism; Protein Transport - genetics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 0957-5235
• DOI: 10.1097/01.mbc.0000214707.65750.f4

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.