Impaired protein folding, dimer formation, and heterotetramer assembly cause intra- and extracellular instability of a Y283C mutant of the A subunit for coagulation factor XIII.

Factor XIII (XIII) is a heterotetramer consisting of two catalytic A subunits (XIIIA) and two noncatalytic B subunits (XIIIB). We examined the molecular mechanisms of a Y283C mutation which had previously been identified in a patient with XIIIA deficiency. The recombinant Y283C protein was labile when expressed in MEG-01 cells, which can endogenously synthesize XIIIA. We also included two other mutants, G562R and I464stop, previously characterized in a non-XIIIA-producing cell line. All these mutants exhibited decreased thermostability and resistance against proteolytic digestion when compared with the wild-type. Gel-filtration analysis revealed that the mutants were in monomer form, while the wild-type formed a dimer. These results were consistent with the prediction by molecular modeling that the mutant molecules would be misfolded. Although assembly of a heterotetramer with XIIIB was demonstrated for Y283C, its binding ability was 10% that of the wild-type. No complex formation was observed for the G562R or I464stop mutants. The wild-type was stabilized in plasma by complex formation with XIIIB, resulting in an increased resistance against proteolytic digestion. In contrast, the mutants were unstable in plasma even in the presence of XIIIB. Thus, impaired folding, dimer formation, and heterotetramer assembly of the mutant XIIIAs lead to both intra- and extracellular instability, which must be responsible for XIIIA deficiency in the patient.