Identification and characterization of two missense mutations causing factor XIIIA deficiency

In this study, two amino acid substitutions, Arg260His and Val414Phe, have been identified in the factor XIIIA subunits of factor XIII deficient patie nts of Syrian and Indian descent, respectively. To confirm the deleterious effects of these substitutions, both variant sequences have been engineered into cDNA clones and the mutant enzymes expressed in yeast. Determination of the transglutaminase activity and immune detection of the mutant enzymes together with mRNA hybridization revealed that the mutations dramatically reduce both the catalytic activity and the level of enzyme expressed in yea st. The mutations Arg260His and Val414Phe occur within the 'core' domain of the enzyme. Computer modelling of the mutant enzymes reveals that the subs titution of the Arg260 by His results in the loss of a conserved electrosta tic interaction whereas the effect of the Val414Phe substitution is a conse quence of the large increase in side-chain volume. Although both mutations do not effect the active site directly, they are predicted to reduce the st ability of the enzyme. The effects of these two amino acid substitutions on enzyme expression and three-dimensional structure strongly confirm that residues which are locate d outside of the active site can have a significant effect on protein stabi lity and function.