Abstract
Congenital FXIII deficiency is a rare bleeding disorder in which mutations are detected in F13A1 and F13B genes that express the two subunits of coagulation FXIII, the catalytic FXIII-A, and protective FXIII-B. Mutations in FXIII-B subunit are considerably rarer compared to FXIII-A. Three mutations in the F13B gene have been reported on its structural disulfide bonds. In the present study, we investigate the structural and functional importance of all 20 structural disulfide bonds in FXIII-B subunit. All disulfide bonds were ablated by individually mutating one of its contributory cysteine’s, and these variants were transiently expressed in HEK293t cell lines. The expression products were studied for stability, secretion, the effect on oligomeric state, and on FXIII-A activation. The structural flexibility of these disulfide bonds was studied using classical MD simulation performed on a FXIII-B subunit monomer model. All 20 FXIII-B were found to be important for the secretion and stability of the protein since ablation of any of these led to a secretion deficit. However, the degree of effect that the disruption of disulfide bond had on the protein differed between individual disulfide bonds reflecting a functional hierarchy/diversity within these disulfide bonds.
Highlights
Coagulation Factor XIII (FXIII) is a pro-transglutaminase that acts at the terminal stage of the blood coagulation cascade and is responsible for covalent cross-linking of pre-formed fibrin polymers making them resistant to premature fibrinolysis [1]
The FXIII-B subunit protein which is built from ten sushi domains, each of which contains two disulfide bonds displays such diversity
Mutations on these disulfide bonds have been reported in the two forms of congenital bleeding disorders associated with mutations in the FXIII-B subunit gene, i.e., the inherited severe FXIII deficiency and the mild FXIII deficiency [13,14]
Summary
Coagulation Factor XIII (FXIII) is a pro-transglutaminase that acts at the terminal stage of the blood coagulation cascade and is responsible for covalent cross-linking of pre-formed fibrin polymers making them resistant to premature fibrinolysis [1]. FXIII exists as a zymogenic heterotetramer with non-covalently associated dimers of its catalytic FXIII-A and non-catalytic/carrier FXIII-B subunits Thrombin activates this zymogenic complex by cleaving an N-terminal 37 amino acid long region on the FXIII-A subunit called the activation peptide (FXIII-AP). The FXIII-B protein is a traditionally secreted protein (bearing an N-terminal 20 amino acid long signal peptide) expressed in hepatocytes [17,18,19] It associates with the FXIII-A subunit in the plasma to form the heterotetrameric complex. Three of the mutations detected in the FXIII-B subunit causing either severe or mild inherited FXIII deficiency, occur on the cysteines forming the structural disulfide bonds [5,13,14]. Even though the disulfide bonds present on each sushi domain of this subunit might be structural in nature, its relative
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