ORIGINAL ARTICLESEffect of four missense mutations in the factor XIII A-subunit gene on protein stability: studies with recombinant proteinsVysokovsky, Alex*; Rosenberg, Nurit*; Dardik, Rima; Seligsohn, Uri; Inbal, AidaAuthor Information Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, and Sackler Faculty of Medicine, Tel Aviv University, Israel *Contributed equally as first authors. Received 23 October, 2005 Revised 1 December, 2005 Accepted 6 December, 2005 Correspondence and requests for reprints to Aida Inbal, MD, Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, Israel 52621 Tel: +972 3 530 2104; fax: +972 3 535 1568; e-mail: [email protected] Blood Coagulation & Fibrinolysis: March 2006 - Volume 17 - Issue 2 - p 125-130 doi: 10.1097/01.mbc.0000214707.65750.f4 Buy Metrics 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. © 2006 Lippincott Williams & Wilkins, Inc.