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Four novel mutations in deficiency of coagulation factor XIII: consequences
to expression and structure of the A-subunit
H Mikkola, VC Yee, M Syrjala, R Seitz, R Egbring, P Petrini, R Ljung, J Ingerslev, DC Teller, L Peltonen and A Palotie
Department of Clinical Chemistry, University of Helsinki, Finland.
The characterization of naturally occurring mutations is one way to
approach functionally significant domains of polypeptides. About 10
mutations have been reported in factor XIII (FXIII) A-subunit deficiency,
but very little is known about the effects of the mutations on the
expression or the structure of this enzyme. In this study, the recent
crystallization of FXIII A-subunit and determination of the
three-dimensional model were used for the first time to pursue the
structural consequences of mutations in the A-subunit. The molecular
analysis of four families from Sweden, Germany, and Denmark revealed four
previously unreported point mutations. Three of the mutations were missense
mutations, Arg326-->Gln, Arg252-->Ile, 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
chain. In the case of the nonsense mutation, delT Phe8, the steady-state
mRNA level of FXIII A-subunit was reduced, as quantitated by reverse
transcriptase-polymerase chain reaction and solid-phase minisequencing. In
contrast, none of the missense mutations affected mRNA levels, indicating
the possible translation of the mutant polypeptides. However, by
enzyme-linked immunosorbent analysis and immunofluorescence, all the
patients demonstrated a complete lack of detectable factor XIIIA antigen in
their platelets. In the structural analysis, we included the mutations
described in this work and the Met242-->Thr mutation reported earlier by
us. Interestingly, in the three-dimensional model, all four missense
mutations are localized in the evolutionarily conserved catalytic 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 directly involved in the
enzymatic reaction. The structural analyses suggest 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-->Ile 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-->Ile site and is expected to
have a destabilizing effect due to an introduction of a smaller, polar
residue in a tightly packed hydrophobic pocket. The substitution of proline
for Leu498 is predicted to cause unfavorable interatomic contacts and a
disruption of the alpha-helix mainchain hydrogen-bonding pattern; it is
likely to form a kink 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 missense 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.
Volume 87,
Issue 1,
pp. 141-151,
01/01/1996
Copyright © 1996 by The American Society of Hematology
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