4 NOVEL MUTATIONS IN DEFICIENCY OF COAGULATION-FACTOR-XIII - CONSEQUENCES TO EXPRESSION AND STRUCTURE OF THE A-SUBUNIT

The characterization of naturally occurring mutations is one way to ap proach functionally significant domains of polypeptides. About 10 muta tions have been reported in factor XIII (FXIII) A-subunit deficiency, but very little is known about the effects of the mutations on the exp ression or the structure of this enzyme. In this study, the recent cry stallization of FXIII A-subunit and determination of the three-dimensi onal model were used for the first time to pursue the structural conse quences of mutations in the A-subunit. The molecular analysis of four families from Sweden, Germany, and Denmark revealed four previously un reported point mutations. Three of the mutations were missense mutatio ns, Arg326 --> Gln, Arg252 --> lie, and Leu498 --> Pro, and one was a nonsense mutation, a deletion of thymidine in codon for Phe8 resulting in early frameshift and premature termination of the polypeptide chai n. In the case of the nonsense mutation, deIT Phe8, the steady-state m RNA level of FXIII A-subunit was reduced, as quantitated by reverse tr anscriptase-polymerase chain reaction and solid-phase minisequencing. In contrast, none of the missense mutations affected mRNA levels, indi cating the possible translation of the mutant polypeptides. However, b y enzyme-linked immunosorbent analysis and immunofluorescence, all the patients demonstrated a complete lack of detectable factor XIIIA anti gen in their platelets. In the structural analysis, we included the mu tations described in this work and the Met242 --> Thr mutation reporte d earlier by us. Interestingly, in the three-dimensional model, all fo ur missense mutations are localized in the evolutionarily conserved ca talytic core domain. The substitutions are at least 15 A away from the catalytic cleft and do not affect any of the residues known to be dir ectly involved in the enzymatic reaction. The structural analyses sugg est that the mutations are most likely interfering with proper folding and stability of the protein, which is in agreement with the observed absence of detectable FXIIIA antigen. Arg326, Arg252, and Met242 are all buried within the molecule. The Arg326 --> Gln and Arg252 --> IIe mutations are substitutions of smaller, neutral amino acids for large, charged residues. They disrupt the electrostatic balance and hydrogen -bonding interactions in structurally significant areas. The Met242 -- > Thr mutation is located in the same region of the core domain as the Arg252 --> IIe site and is expected to have a destabilizing effect du e to an introduction of a smaller, polar residue in a tightly packed h ydrophobic pocket. The substitution of proline for Leu498 is predicted to cause unfavorable interatomic contacts and a disruption of the alp ha-helix mainchain hydrogen-bonding pattern; it is likely to form a ki nk in the helix next to the dimer interface and is expected to impair proper dimerization of the A-subunits. In the case of all four missens e mutations studied, the knowledge achieved from the three-dimensional model of crystallized FXIII A-subunit provides essential information about the structural significance of the specific residues and aids in understanding the biologic consequences of the mutations observed at the cellular level. (C) 1996 by The American Society of Hematology.