1. No category

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1996 87: 141-151
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
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Four Novel Mutations in Deficiency of Coagulation Factor XIII:
Consequences to Expression and Structure of the A-Subunit
By Hanna Mikkola, Vivien C. Yee, Martti Syrjall, Rainer Seitz, Rudolf Egbring, Pia Petrini, Rolf Ljung,
Jorgen Ingerslev, David C. Teller, Leena Peltonen, and Aarno Palotie
The characterization of naturally occurring mutations isone
way t o approach functionally significant domains of polypeptides. About 10 mutations have been reported in factor
Xlll (FXIII) A-subunit deficiency, but very little is known
about the effects of the mutations on the
expression or the
structure of thisenzyme. In this study, the recent crystallizaof the three-dimention ofMlllA-subunit and determination
sional model wereused for the first time
pursue
to the structural consequences of mutations in the A-subunit. The
molecular analysis of fourfamilies from Sweden, Germany,
and Denmark revealed four previously unreportedpoint mutations. Three of the mutations were missense mutations,
Arg326 Oln, Arg252 + Ile, and Leu498 Pro, and one was
a nonsense mutation, a deletion of thymidine in codon for
termination
PheE resulting in early frameshift and premature
of thepolypeptide chain. In the case of thenonsense mutation, delT Phe8, the steady-state mRNA level of FXlll A-subunit was reduced, as quantitated by reverse transcriptasepolymerase chain reactionand solid-phase minisequencing.
In contrast, none of the missense mutations affected mRNA
levels, indicating thepossible translation of the mutant
polypeptides. However, by enzyme-linked immunosorbent analysis and immunofluorescence, all thepatients demonstrated
a complete lack of detectable factor XlllA antigen in their
platelets. In the structural analysis, we included the mutations described in this workand the Met242 Thr mutation
reported earlier by us. Interestingly, in the three-dimensional
model, all fourmissense mutations are localized in theevo-
lutionarily conserved*catalytic core domain. The substitutions are at least 15 A away from the catalytic cleft and do
not affect any of theresidues known t o 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 M l l t A antigen.
Arg326, Arg252, and Met242 are all buried within the molecule. TheArg326 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 islocated in the same region of
the core domain as the Arg252 lle siteand is expected t o
have a destabilizing effect due t o an introduction of a
smaller, polar residue in a tightly packed hydrophobic
pocket. The substitution of proline for Leu498 is predicted
t o 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 t o impair proper dimerizationof theA-subunits. In the case of all fourmissense mutations studied, the
knowledge achieved from the three-dimensional model of
crystallized FXlll A-subunit provides essential information
about the structural significance of the specific residues and
aids in understanding the biologic consequences of themutations observed at the cellular level.
0 1996 by The American Society of Hematology.
C
catalytic A-dimer until it is needed
in
The
A-subunit of FXIII is expressed mainly in some bone marrow-derived cells such as megakaryocytes/platelets and
monocytes/macrophages.’’”5 The intracellular protein is a
homodimer of two A-subunits that are identical to plasma
-+
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ONGENITAL factor XI11 (FXIII) deficiency is a rare
bleeding disorder that is inherited inan autosomal
recessive manner. Deficiency in FXIII leads to a bleeding
diathesis that is characterized by delayed hemorrhages in
soft tissues after initially successful primary clot formation.
In addition to subcutaneous and intramuscular hematomas,
severe bleeding complications such as intracranial hemorrhages occur. The most characteristic feature is neonatal umbilical cord bleeding, which is a symptom that is not associated with other clotting factor deficiencies. Other typical
findings are spontaneous miscarriages in pregnant females
and defective wound healing.’,’
The characteristic symptoms can be understood by the
function of FXIII. FXIII belongs to the family of transglutaminases that are thiol enzymes that catalyze covalent crosslinking of proteins in a variety of tissues and are involved
in processes such as blood coagulation, apoptosis, and keratini~ation.~
Of. ~the transglutaminase superfamily, inherited
disorders have been reported only in the case of FXIII and
keratinocyte transgl~taminase.~.~
FXIII is a transglutaminase
that acts as the final enzyme in the blood coagulation cascade. It catalyzes crosslinking of fibrin molecules converting
the primary blood clot into a stable form. Additionally, FXIII
crosslinks other substrates such as alpha-2-antiplasmin, thus
controlling the rate of fibrinolysis,’ and some extracellular
matrix proteins such as fibronectin and collagen, thus anchoring the clot into the site of i n j ~ r y . ~ , ~
F3111 circulates in plasma as a heterotetramer composed
of two A-subunits that possess the catalytic activity and
two B-subunits that probably serve to carry and protect the
Blood, Vol 87,No 1 (January l), 1996:pp 141-151
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From the Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland; the Laboratory Departmentof Helsinki University Central Hospital, Helsinki, Finland; the Departments of Biochemistry and Biological Structure, University of Washington,
Seattle, WA; the Department of Haematology/Oncology, PhilippsUniversity Hospitals, Marburg, Germany;
the Department of Pediatrics, Karolinska Hospital, Stockholm, Sweden; the Department of
Pediatrics, University of Lund, Malmo, Sweden; th,e Department of
Clinical Immunology, Skejby University Hospital, Arhus, Denmark;
and The Department of Human Molecular Genetics,National Public
Health Institute, Helsinki, Finland.
Submitted February 16, 1995; accepted August 22, 1995.
Supported by the Finnish Cultural Foundation, the Oskar Ojund
Foundation, the Hjelt Research Fund, the American Hearr Association (910080070), the National lnstitutes of Health (HL-50355), and
the Medical Faculty of the University of Lund.
Address reprint requests to Aarno Palotie, MD, PhD, Department
of ClinicalChemistry,University of Helsinki, Meilahti Hospital,
Haartmaninkatu 4, SF-00290 Helsinki, Finland.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8701-0004$3.00/0
141
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142
MIKKOLA ET AL
A-subunits. Intracellularly, the dimer is located in the cytosol. The secretion mechanism of FXIII A-subunit has not
been elucidated, but it does not follow the conventional secretory pathway through endoplasmic reticulum (ER) and
Golgi-complex.
The catalytic 730-amino acid A-subunit of FXIII is a proenzyme that is activated by thrombin cleavage at Arg37, in
the presence of calcium and fibrin(ogen). After activation,
the B-subunits dissociate to yield the dimeric A2 enzyme.I6
The three-dimensional structure determination of the A-subunit zymogen dimer by X-ray crystallography has been reAs shown in Fig 3, each A-subunit monomer is
folded into four sequential domains: from the amino terminus, they are the beta sandwich, the catalytic core, and beta
barrels 1 and 2. The core domain contains most of the residues that are conserved among members of the transglutaminase family, indicating the importance of a conserved core
domain structure for transglutaminase activity. It is in this
core domain that the catalytic triad residues (Cys314,
His373, and Asp396) are located. Each amino terminus activation peptide crosses the dimer interface and occludes the
opening to the active site cavity in the other subunit in the
dimer. To expose the catalytic residues and render them
accessible to substrate, movement of the activation peptide
as well as of the beta barrel 1 domain must occur. In efforts
to obtain a structure of activated FXIII, crystallographic studies of the thrombin-cleaved and high calcium forms of the
protein have been performed. Both the thrombin-cleaved and
high calcium structures are very similar to that of the zymogen; in the latter structure, only local protein conformational
changes in the calcium-binding site could be observed.”
Complementary to the crystallographic structure, the characterization of disease-causing mutations provides biologic
evidence for critical residues and regions of the polypeptide
chain that are essential for maintaining the structure and/or
catalytic properties of the enzyme. In this study, we report
four novel point mutations in the A-subunit gene causing
FXIII deficiency. Our aim has been to clarify the effects of
the different mutations by combining the data from steadystate mRNA and protein expression studies in vivowith
the structural knowledge achieved by the three-dimensional
model of crystallized FXIII A-subunit. The elucidation of
the three-dimensional structure has brought new insights to
understanding the relationship between structure and function of the A-subunit. It is a valuable tool to evaluate the
mechanism by which each mutation exerts its effects by
altering the structure and/or function of FXIII.
MATERIALS AND METHODS
Patients
One German family, one Danish family, and two Swedish families
were studied (Fig 1). The patients’ disorders have been previously
diagnosed by clinical bleeding tendency and immunologic and func(Table 1).
tional determinations of FXIII A-~ubunit~’.~~
Family A from Germany has two affected individuals. The propositus, A-11-10, was born in 1966. His two brothers died neonatally
from intracranial hemorrhages. Patient A-11-10 had umbilical cord
bleeding and an abnormal bleeding tendency with spontaneous subcutaneous hemorrhages.z6Since 1971, he has been treated prophylactically with placenta-derived FXILI concentrates. The second patient
in family A is a girl born in 1986. Her mother is a cousin of the
propositus. Patient A-111-4 demonstrated umbilical cord bleeding
shortly after delivery, and since the first year of life, she has been
in regular prophylactic treatment.
Family B from Sweden has two affected girls born in 1989 and
1992. Patient B-11-3demonstrated neonatal umbilical cord bleeding,
whereafter FXIII deficiency was diagnosed. Since 1 year of age, she
has been treated prophylactically with her father’s plasma. After the
regular prophylactic treatment, she has once had a more severe
bleeding in the muscles associated with a fracture of the tibia. The
bleeding was treated with plasma infusions, and thehealing of wound
and fracture was normal. Disease in the younger girl of the same
family, B-11-4,was diagnosed neonatally because ofher sister’s
disease. She also experienced umbilical cord bleeding, but has since
been treated prophylactically as is her sister without any severe
bleeding symptoms.
Family C from Sweden has one affected girl, C-11-3, born in 1979.
She demonstrated umbilical cord bleeding and, thereafter, a tendency
for ecchymoses and hematomas after mild trauma. FXIII deficiency
was diagnosed at the age of 3 years. She had intracranial bleeding
after a mild trauma on her head at the age of 5 years and a rupture
of the spleen with serious abdominal bleeding at the age of 8 years.
Both emergencies necessitated FXIII substitution therapy, but the
treatment was complicated by inhibitor development against FXIII
concentrates. Induction of immune tolerance by using the Malmo
treatment protocol was attempted, but the treatment had to be interrupted because she had a serious anaphylactoid reaction against
FXIII concentrates.”
Family D from Denmark has one affected male, D-1-2, born in
1945. He demonstrated umbilical cord bleeding at birth. He has been
treated prophylactically with FXIII concentrate.
Analysis of the Gene Encoding FXIII A-Subunit
The analysis of the gene coding for the A-subunit was performed
mainly as described earlier in detail.28The genomic DNA was extracted from peripheral blood leukocytes. Each exon including the
introdexon boundaries of the A-subunit gene was amplifiedby polymerase chain reaction (PCR) with specific primers. (The primer
sequences were mostly the same as described earlier,28thus, only
the primers used to study the mutations reported in this work are
shown in Table 2.) Radioactively labeled PCR fragments were analyzed by single-strand conformation polymorphismz9(SSCP) and the
regions demonstrating an abnormal migration in the renaturing gel
were sequenced directly from the PCR product using solid-phase
purification3’ and the Sanger dideoxy chain termination m e t h ~ d . ~ ’
Each identified mutation was screened among family members
and 20 to 45 control individuals from the specific country. The
screening was performed by solid-phase minisequencing as described earlier?R In principle, the biotinylated PCR product of the
mutated area is captured onto solid support. The incorporation of
radioactive H’-test nucleotides corresponding to wild-type and mutant alleles is tested by guiding the test nucleotides nexttothe
mutation site by a specific primer (Table 2). The incorporation of
the two test nucleotides is analyzed separately, and the radioactivity
is measured by liquid scintillation counter. The genotype is defined
by the ratio of the counts obtained from the mutated andor wildtype test nucleotides.”
Quantitation of FXIII A-Subunit mRNA
The steady-state mRNA levels associated with each mutation were
studied by reverse transcriptase PCRand consequent solid-phase
minisequencing, which facilitates allele-specific quantitation of the
product. RNA
was
extracted by the RNAzol kit (Tel-Tec,
Friendwood, TX). The reverse transcriptase reaction was performed
mainly as described earlier.28The cDNA PCR was performed using
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FACTOR Xlll MUTATIONS
143
A.
5
6
10
Arg-326-Gln
2
3
1
11
12
Arg-252+11e
4
Q)
B.
,,
,,
,,
,,
,
m a
Fig 1. Pedigrees of the families demonstratingthecoseg
regation of the identified mutations. The propositi are marked
by arrows. The mutation genotype in individuals D-11-1 and D11-2 was not studied.
1
primers listed in Table 2. The! relative amount of each mutated
mRNA was quantitated in heterozygotes, where the wild-type
mRNA serves as a standard. The ratio of mutated versus wild-type
mRNA was determined by the ratio of the counts from the two test
nucleotides corrected by the difference of the specific activities (r
value). The ratio was compared to the corresponding ratio in genomic
DNA in heterozygotes, where the two alleles are present in equal
amounts (r = 1).
Immunologic Determination of FXIII A-Subunit by
Enzyme-Linked Immunosorbent Assay (ELISA) and
ImmunoJ7uorescence
The presence of intracellular A-subunit antigen was studied by
immunofluorescenceand ELISA. For immunofluorescence, peripheral
blood smears were prepared from fresh EDTA blood samples and
2
1
air-dried. The preparations were fixedwith 4% paraformaldehyde
immediately before analysis. The slides were washed afterwards with
phosphate-buffered saline (PBS). The preparations were permeabilized, and aspecific-binding was blocked by incubating for 15 minutes
in blocking buffer, which contains 2% saponin and 5% bovine serum
albumin inPBS. The primary antibody, a 1:200 dilution of rabbit
antiserum for FXIII A-subunit (Clotimmun; Behringwerke, Marburg,
Germany) was incubated for 1 hour at room temperature. After several
washes with blocking buffer, a 1:50 dilution of the second antibody,
fluorescein isothiocyanate (FlTC)-conjugated goat anti-rabbit IgG
(Supertechs, Bethesda, MD), was incubated for 30 minutes at m m
temperature. The slides were washed with PBS and covered by Vectashield mounting medium (Vector Laboratories, Burlingame, CA).
The slides were analyzed by a Zeiss Axiophot fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
In the patients where the amount of FXIII A-subunit antigen had
Table 1. Summary of Mutations and Their Expression and Activity in FXIII-Deficient Patients
Family and Origin
A. Germany
B. Sweden
C. Sweden
Patient
Age (yr)
11-10
Arg326 28
111-4
7
11-3
5
11-4
2
11-3
15
-
Mutation
-
Gln
Arg326 Gln
Arg252 -,Ile
Leu498 Pro
Arg661 -t Stop
Leu498 Pro
Arg661 Stop
delT Phe8
--
Allelic mRNA
Normal
Normal
Normal
Normal
c 10%
4 %
A-Subunit Antigen
FXlll Activity
IEPELSA c l %
IFELISA < l %
ELSA c l %
IFIF-
"C-putr inc ~ 2 %
Berichrom <2%
"C-putr inc <2%
Berichrom <5%
"C-putr inc ~ 2 %
Agar act st <3%
Agar act st <3%
IEP-
Agar act st <3%
IFD. Denmark
1-2
49
E. Finland
11-2'
55
delT Phe8
Unknown
Met242 Thr
Arg661 Stop
--
IEP-
Stago ~ 3 %
Normal
ELSA c l %
"C-putr inc < l %
3%
IF-
Abbreviations: IEP, Laurell's immunoelectrophoresisfrom plasma; IF, immunofluorescence from platelets; "C-putr inc, "C-putrescine incorporation into casein; Berichrom, ammonia release assay (Behringwerke);agar act st, agarose gel-embeddedactivity staining; Stago, clot solubility
assay on monochloracetic acid.
The mutations of patient €4-2 have been reported earlier?'
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144
MIKKOLA ET AL
-
to be scrutinized, a more sensitive method, ELISA, was also applied.
The materials for ELISA were supplied by Dr Metzner, Behringwerke AG. The cytoplasmic fraction of three times freezethawed platelets were incubated in microtiter wells that were coated
with rabbit anti-FXIII A-subunit antibody. The detection of the
amount of A-subunit antigen was performed using the peroxidase
reaction.
m:
2- v3
;!e
-
u
2.2 u
P
-
z*
5
0
0;
+z
q z :
V
V
u
e
a
Functional Determination of FXIII A-Subunit
e
$ 3 g l uj
$ 2 is $f 8$
In the patients with missense mutations, the transglutaminase activity was further studied by ''C-putrescine incorporation assay. The
transglutaminase activity was analyzed in three times freeze-thawed
platelets as described earlier."
.q2
Modeling of the Protein Defects
.E 8
0
.-
f i = ?
8I- g
E
V
z u
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t
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.C
Analysis of the Gene Encoding FXIII A-Subunit
E;;EEg$E
c
Using SSCP as an initial screening method and for subsequent direct sequencing of the shifted fragments, four novel
mutations were identified in the gene coding for the A-subunit (Fig 2). Three of the mutations were missense mutations:
a G-to-A transversion in exon 8, causing the substitution of
glutamine for arginine at amino acid position 326; a G-toT transversion in exon 8, resulting in the substitution of
isoleucine for arginine 252; and a T-to-C point mutation in
exon 12, changing the amino acid leucine 498 to proline.
The fourth mutation was a deletion of thymidine in codon
TTT for phenylalanine 8, resulting in a frameshift and a
premature termination codon. No other mutations were detected by sequencing the coding regions.
In family A, patient A-11-10 demonstrated the Arg326
Gln mutation in a homozygous form. Patient A-111-4, whose
mother is a cousin of patient A-11-10, demonstrated the
Arg326 --t Gln mutation in the heterozygous form. In her
paternal allele, patient A-111-4 demonstrated the k g 2 5 2
Ile mutation, thus being a compound heterozygote for the
two mutations.
In family B, the two sisters demonstrated the Leu498
Pro mutation in a heterozygous form. In the other allele,
they had a C-to-T point mutation in exon 14 resulting in
Arg661 4 Stop alteration, which we have previously detected in six Finnish FXIII-deficient families."
In family C , the patient demonstrated the deletion of thy-
z
n
v ) "
r: z
g
gs
4 ;e, m
-
N
N
8
;,
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m
N
0
f
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&J3%2".
;.
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i=?28?$#
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ap
'6 E % g j ; E E g
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i
E 2 8 2 3 P ~ u
:
o+-uauI-_cu
=
zs??z?g 5
0 0
c
;a
a u u c a
V O V O . ? V
o u
U O
5
C
;.5
u : a u : a n c - u ~z
JCI L C% iC: ; C
2
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C
eeeeeegg
c
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RESULTS
O 1
t 1
t o t o T 1 , m
N . - w ~ e c Q r . -
w u J w u l w w w w
1
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.@{@q
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5
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The three-dimensional structure of the A-subunit zymogen dimer,
as determined by single-crystal X-ray diffraction," was used as a
template for modeling the four single-residue missense mutants (see
Fig 3). Coordinates for the wild-type structure were pbtained by
refinement against diffraction data from 10.0 to 2.65 A resolution
using the program X-PLOR.33The geometry for the moslel is good
(root mean square deviation from ideality is 0.013 A for bond
lengths, 2.2" for bond angles, 25.7" for torsion angles, and 1.6"for
improper torsion angles). Individual atomic temperature factors have
been refined (average B = 25.8 A'), and the crystallographic R
factor is 21.6%. Models for the mutants were generated using the
As there are two crystallographically indepengraphics program 0.34
dent subunits in the asymmetric unit of the crystal, the average of
the two values is given for interatomic distances cited in the text.
The coordinates for the experimentally determined wild-type structure have been deposited with the Protein Data Bank (identifying
code: 1 ggt). Figures are drawn with the program MOLSCRIPT.35
c c c c c c
E
= = = _ c = = _ c =
:.0
m
0 ) g ;
2)
m
-T
$E",%
t
t
" 0$ a$ a$ - ; 1
'
2
*
+
+
+
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
FACTOR Xlll MUTATIONS
145
Family A
control
homozygous compound
patient
control
heterozygous
patient
C A T G C A T G
G T A C G T A C
4-
4-
G -> A
Arg326 -> Gln
G -> T
Arg252 -> Ile
Family B
Family C
0
compound
heterozygous
patient
control
control
C A T GC A T G
homozygous
patient
C A T GC A T G
del T
Phe8 -> frameshift
T -> C
Leu498 -> Pro
Fig 2. Sequencing gels demonstrating four novel point mutations are shown. (a) The mutations in family A are a G-to-A transversion in
exon 8 that changes the codon CGA into CAA, resulting in Arg326 -, Gln substitution(homozygous patient A-11-10), and a G-to-T transversion
in exon 6 that changes the codon AGA into ATA, resulting in Arg252
Ile substitution (compound heterozygous patient A-111-41. (b) The
CTG into CCG, resulting in Leu498 Pro substitution (cornpound
mutations in family B are a T-to-C transversion in exon 12 that alters the codon
heterozygous patient 8-11-3). and the Arg661 -, Stop mutation (reported earlier; not shown in the figure). (c) The mutation in family C is a
deletion of thymidine in codonT7T for phenylalanine 8 in exon 2 altering the reading frame after alanine 7 (homozygous patient C-11-31. The
same mutation was detectedin heterozygous form in family D.
+
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MIKKOLA ETAL
146
Table 3. Relative Quantitation of AllelicmRNA
Transcripts in Heterozygotes
Mutated Allele
Test
Nucleotides
(mutanthild
type)
Arg326 Gln
Arg252 Ile
Leu498 Pro
delT Phe 8
T/C*
T/G
CTT
G/T
-+
r
Appr Mutant
Level
mRNA
DNA
cDNA
Gen
0.88 2 0.21
0.90 z 0.22
1.07 f 0.24
0.04 z 0.03
0.87 2 0.16
0.91 t 0.16
1.17 2 0.13
1.02 z 0.12
Normal
Normal
Normal
<5%
The r value has been obtained from the amount of radioactivity
measured from the H3 test nucleotides incorporated into the RT-PCR
product (mutant cpm/wild typecpm, corrected by the specific activities of thenucleotides).
Abbreviations: Gen DNA, genomic DNA; appr mutant mRNA level,
approximate mutant mRNA level.
* The Arg326 Gln mutation was analyzed from the antisense direction.
antigen in platelets was analyzed using ELISA and/or immunofluorescence (Fig 3). In addition to the patients described
in
thisreport, a Finnishpatientwhowaspreviouslyshownto
represent a compound
heterozygote
for
the
Met242
-P
Thr and the Arg661 + stop mutations was also included in
the analysis. Using polyclonal monospecific antiserum for
the A-subunit, the antigen level in all the patients’ platelets
was below the detection limit (Table l ) .
Analysis of Transglutaminase Activih
In the case of the patients with missense mutations, isolated platelets were also assayed for transglutaminase activity using the “C-putrescine incorporation method. Transglutaminase activity could notbe demonstrated in any of the
cases (Table l ) .
+
midine in a homozygous form. Additionally, the same mutation was detected in the Danish patient D-1-2 in the heterozygous form, whereas in his other allele, the mutation has not
been detected.
To discriminate between a disease mutation and a common polymorphism, the prevalence of each mutation in the
normal population was analyzed by solid-phase minisequencing. From each country, 25 to 45 control individuals
were screened for the specific mutations, but none of the
mutations were found in the 50 to 90 chromosomes studied.
Analwis of the Steady-state mRNA Level of FXIII ASubunit
The steady-state transcript levels ofthemutated alleles
were analyzed in both the affected individuals and heterozygous carriers. The method for quantitation was solid-phase
minisequencing, which facilitates specific detection of different allelic products. Relative quantitation of the allelic transcripts was performed in heterozygotes, where the wild-type
transcript serves as a quantitative standard (Table 3).
All three missense mutations (Arg326 + Gln, Arg252 -*
Ile,and Leu498 -* Pro) resulted in a steady-state mRNA
level equal to the wild-type allele. This is demonstrated by
the ratio of the incorporated mutant versus wild type test
nucleotides in minisequencing. The ratio was close to I in
the cDNA PCR products of the heterozygotes carrying the
missense mutations, and equal to the corresponding ratio in
genomic DNA. In contrast, the steady-state mRNA levels of
the two nonsense mutations (delT Phe8 and Arg661 + Stop)
were reduced drastically. The allelic transcript with the delT
Phe8 mutation in the heterozygous carriers was hardly detectable, which is approximated to be less than 5% of the level
of thewild-typeallele
(Table 3). The levelofArg66I
-*
Stop mRNA was reduced to less than IO%, which is a finding
that has been consistent in the Finnish patients with the same
mutation.2x
Analysis of the Intracellular Antigen bv ELBA and
Immunoplrorescence
To study further the expression of the mutated alleles at
the cellular level, the presence of intracellular FXIlI A-subunit
Modeling of the Protein Defects Caused by Individual
Mutations
Met242 -* Thr. Residue Met242 is nearthe carboxyl
terminus of a helix in the core domain. near the beta sandwich. The sidechain of this residue is surrounded by a predominantly hydrophobic environment (Fig 4A). Replacing
this methionine with a threonine residue isprobably the
least extreme of the four single-residue missense mutations
presented here, as it is possible for the sidechain hydroxyl
group of the threonine to form a hydrogen-bond with a
nearby residue (Thr242 OH to 0 Leu239 = 2.6 A). However,
substitution with the smaller threonine residue would create
a vacuum in the structure, which in turn could lead to a
misfolded or destabilized folded molecule (Fig 4B).
Arg 252 + Ile. Residue Arg252 is located in a loop
between two core domain helices that are near the beta sand-
Fig 3. Stereoview of the domain structure of the FXlll
A subunit,
as determined by x-ray crystallography. The protein is drawn as a
coil with the central catalytic core domain in bold and the three
surrounding beta domains. An asterisk marks the location of the
active site ofthe molecule. From the amino terminus activation
peptide, the four sequential domainsare beta sandwich, catalytic core,
beta barrel 1, and beta barrel 2. Alpha carbon atoms belonging t o
Thr, 2. Arg-252 Ile, 3.
the four missense mutations (1. Met242
Arg326 Gln, 4. Leu498 Pro, and 5. Arg661- Stop) are represented
as white spheres.
-
-
-
-
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
147
FACTOR Xlll MUTATIONS
Fig 4. Structural consequencesofthe Ma242+Thr mutaion. The
protein backbone is shown as a ball-and-stick structure. Sidechain
fragments of Met242 (A), Arg252, and the modeled Thr242 mutant
(B) are also shown. TheMet242 is a buried residue whose sidechain
packs against that of Arg252 in a roughly antiparallel menner. Replacement of Met242 with a smallerthreonine residue creates a destabilizing void in thestructure. Although the chemical environment
at this site can accommodatethe Thr242 mutant to some extent (a
possible hydrogen bond to the mainchain of Leu239 is shown as a
dashed line), the substitution of a small polar residue for a buried
hydrophobicamino acidis expected to yield a destabilized structure.
the other two catalytic triad residues (His373 and Asp396)
are located. Asn207 is at the carboxyl terminus of another
core domain helix (residues 198 to 207) that
is distant in
terms of amino acid sequence and is partially exposed to
solvent. The hydrogen bonds involving the Arg326 sidechain
atoms serve to tie the active site helix to other parts of the
core domain. Replacing Arg326 with a glutamine residue
would at best preserve only one of the hydrogen bonds to
the carbonyl oxygen atom
of Ala332 andeliminate the interaction with Asn207 (Fig 6B). The charge on the guanido
group of Arg326 has no obvious compensation, and it seems
probable that the helix dipole provides the c~mpensation.~~
Replacing Arg by a neutral residue in this location almost
certainly upsets the electrostatic balance
in this region of the
protein. A third consequence of substitution with the smaller
glutamine sidechainis the creationof a void within the molecule. As a result, itis likely that the Arg326 Gln mutation
would either prevent the foldingof the coredomain or lead
to a folded but destabilized structure.
Leu498 Pro. TheresidueLeu498islocatednearthe
carboxyl terminus of a helix that is on the surface of the catalytic core domain Fig 7).Atthecarboxylterminusof
this
helixis a turn containingresiduesatthedimer
inmface
(Ty1-500
Gly501);
,
the turn leads to a short segment of beta
structure and thento a flexible extended stretch of peptide that
links the catalytic core and beta barrel1 domains. The Leu498
sidechain is fairly accessible to solvent and not tightly packed
in a hydrophobic envhnment. The mainchain phi torsion angle
of -68" is close to the ideal value -60"
of for a proline residue;
however, mutating Leu498to a proline would generatea number of short interatomic contacts involving proline ring carbon
atoms and render the residue's mainchain nitrogen atom
unavailable to participate in the hydrogen-bond scaffold in the
+
+
wich. The sidechainof this residue is completely buried and
participates in a number of hydrogen-bonding interactions
with two residues that are slightly amino terminal to it (Fig
SA; Arg252 NE to 0 Met247 = 3.1 A, Arg252 NH1 to OD1
Asp243 = 2.8 A, Arg252 NH2 to OD1 Arg243
= 2.7 A,
Arg252 NH2 to 0 Met247 = 2.9 A). These hydrogen bonds
are critical in securing the conformation of the loop. The
arginyl charge in this case is compensated by the carboxyl
ofAsp243.ReplacingArg252withanisoleucineresidue
wouldhaveseveraldeleteriouseffects.First,thesmaller
isoleucine sidechain would resultin a vacuum in the molecule, because Arg252 is a buried residue. Second, all
of the
hydrogen-bonding interactions involving the Arg252 sidechain would be eliminated; as the isoleucine sidechain is not
able to make any hydrogen bonds to stabilize the structure
(Fig 5B). Third, the lack of electrostatic compensation would
likely cause Asp243 to rotate into the solvent and disrupt
the mainchain conformation for a substantial section of the
sequence. The Arg252-B Ile mutant, which is a substitution
of a small hydrophobic residue for a large, buried, charged
amino acid, would likely interfere with protein folding
or
result in a misfolded or destabilized structure.
Arg326 -B Gln. ThecatalyticCys314residue is atthe
amino terminus of the longest helix in the subunit; Arg326
Fig 5. Structural consequencesof the Arg252 Ile mutation. Here
is located near the carboxyl end of this helix. The Arg326
protein loop containing Arg29 isrendered in ball-and-stick form
residue is completely buried in the core domain.
Its sidechain the
with only mainchain atoms drawn. Sidechain fragments for Arg252
nitrogen atoms are involved in hydrogen bonds to mainchain
(A), Asp243, and the modeled lle252 mutant (B) are also shown. In
carbonyl oxygen atoms belonging to two other core domain the wild-type structure, Arg252forms a salt bridge with Asp243 and
also hydrogen bonds to the mainchain carbonyl of Met247; these
residues (Fig 4A; Arg326
NE to 0 Ala332 = 2.9 A, Arg326
interactions are represented by dashed lines. The Arg252
Ile mutaNH1 to 0 Asn207 = 2.6 A, Arg326 NH2 to 0 Ala332 =
tion involves the removal of a buried,positively charged residue,the
3.0 A). Ala332 is in the beta strand immediately following
elimination of structural electrostatic interactions, and the introductheCys314-containing, or activesitehelix; this strand is
tion of a smaller sidechain fragment. This mutation is expected to
prevent proper folding or yield an unstable, misfolded structure.
located in the central beta sheet in the core domain, where
+
-
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MIKKOIA ET AL
148
DISCUSSION
Fig 6. Structural consequencesofthe Arg326 -.Gln mutation. The
protein backbone near Arg326 is shown as solid lines connecting
alpha carbon atoms. The sidechain
fragments of Arg326 (A) and the
modeled Gln326 mutant (B) are drawn as ball-and-stick structures,
as arethe mainchain atomsof the two residues (As11207 and Ala332).
which are hydrogen-bonded to the Arg326 sidechain. Thehydrogen
bond interactions are represented by dashed lines. Also shown are
the sidechain fragments of the catalytic triad residues (Cys314,
His373, and Asp3961, which are a little more than 15 A away. The
Gln mutation, which involves the loss ofa positively
Arg326
charged sidechain,the removal of two structural hydrogen bond interactions, andthe introduction of a smaller sidechain fragment, are
expected to prevent proper protein folding or yield a destabilized
structure.
-
Compared with other congenital bleeding disorders, significantlyless is knownaboutthegeneticbackgroundof
FXIII deficiency.Untilnow,about
10 different mutations
have been reported by us and
others in the gene of FXIII
A-subunit. So far, no large deletions or other genomic rearrangements have been detectedin this gene. The mutations
reported thus far consist of point mutations that result
in
premature terminationof t r a n s l a t i ~ n , ~possible
~ * " ~ ~ ~defective
~plicing'~
or amino acid alterati0n,2~.~~.'"'
or minor deletions
that result in frameshift (Fig 9):'In
the case of the amino
acid alterations, the actual consequences of the mutations
have been unclear due to the lack of both structural knowledge of the protein and expression data of the mutations.
In this study,we have analyzed the molecular basis
of the
disease in fourFXIII-deficientfamilies.UsingSSCPand
direct sequencing, we have identified four novel mutations
in the geneof FXIII A-subunit. Three of the mutations were
point mutations resulting in a single-amino acid alteration
(Arg326 + Gln,Arg252 + ne. andLeu498 + Pro), and
one of them was a deletion of thymidine resulting in early
frameshift and termination of translation (del T Phe8). The
Arg326 + Glnmutation in theGermanfamilyhas
been
detectedindependentlyalso by Dr U. Grundmann,Behringwerke, (personal communication, November 1994).
The mutations reported earlier
in the geneof the A-subunit
have been mostly uniquefor each family. The only exception
has been the Arg66
1 + stop mutation that we have previously
detected in six Finnish families, reflecting the enrichment of
one mutation due to founder effect in the genetic isolate of
helii (Fig 7). These effects would forcea kink in the helix that
may in itself interfere with the folding of the subunit
or destabilize the structure. Kinking of the helix would at the very least
result in altering the position and conformation of the tum at
its carboxyl terminus, which would disturb some of the subunitto-subunit interactions that occur at the dimer interface. The
influence of the helix kink may extend to altering the conformationandflexibilityofthenearbylinkingpeptide,whichwe
postulate is importantin the activation of the enzyme." Substitution of Leu498 with a proline residue, then, is predicted to
interfere with the correct folding of the subunit. The misfolding
of the structure is expected to result in impaired dimerization.
Arg661- Srop. The A r g 6 6 1 residue is located at the beginning of the third beta strand in the carboxyl terminus barrel 2
domain. A premature stop of translation at residue 661 would
yield a shortened polypeptide chain that is missing70 residues
second subunit
second subunit
at the carboxyl terminus. As a result, more than two thirds of
in dimer
in dimer
the barrel 2 domain, containing five of the seven beta strands,
is deleted from the molecule (Fig 8). The missing portion of
thesubunitincludesthefour-strandedbetasheetthatpacks
Fig 7. Structural consequences of the Leu498 Pro mutation.
against the catalytic core domain and one of the exposed strands
Stereoview of the protein near bu498. The chain of the subunit
containing the Leu498 residue is shown as crosshatched ball-andin the second, exposed beta sheet in the barrel 2 domain. It is
stick model. The open ball-and-stick
model is thesecond subunit in
unlikely that the barrel 2 residues that are translated will fold
the A2 dimer. Sidechain fragments of the wild-type Leu498 and of
intothetwobetastrandsobservedinthewild-typecrystal
the modeled Pro498 mutant are labeled. Substitution of the leucine
structure,becausetheprematurestopmutationleadstothe
by a proline residue at thisposition creates a numberof unfavorable
short contacts and disrupts the mainchainhydrogen
bonding
exposureofthehydrophobicfaceofthetwo-strandedbeta
(dashed line) that is characteristic of alpha helix structure. The exsheet.Inthe
Arg661
Stopmutant,thebarrel
2 residues
pected result is a distortion of the helix causing it to kink, which in
that are translated are expected to fold into a smaller, altered
turn would change the conformation of the subunit at the dimer
conformation that is very different from the structure of the
interface. The Leu498
mutation is expected to yield a misfoldedstrucbarrel 2 domain in the wild-type structure.
ture that is
destabilized or incapable ofdimerizing properly.
-
+
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FACTOR Xlll MUTATIONS
149
In the caseof each of the six patients included in our study,
a complete lack of FXIII A-subunit antigen and activity in
plasma and/or platelets was demonstrated. This suggests that
the mutations would result in either the absence of synthesis
of the A-subunit or strongly altered properties
of the mutant
protein.
In the case of the two nonsense mutations, the Arg661
Stop mutation and the frameshift mutation caused by the
deletion of thymidine, the steady-state transcript levels were
verylow.Thereductionofsteady-statemRNAlevelhas
been associated with stop-mutations and frameshift mutations also in other genes?* probably because of low efficiency of mRNA processing and nucleocytoplasmic trans+
port:'"
-
Fig 8. Structural consequences of the Arg661 Stop mutation.
This residueis located in the carboxylterminus beta banel 2 domain;
its alpha carbonatom is shown as a large, labeled sphere. The
polypeptide chain aminoterminal to this residue is drawn as a coil structure, while the portion
of the molecule carboxyl terminal t o Arg661
is shown as smaller, alpha carbon spheres connected by thin lines.
A premature end of translation at position 661 would lead to a loss
of most of the barrel2 domain, andas a result, the protein is expected
to misfold into a structure whose carboxyl terminus is very different
from that observed in the wild
type.
Finland.= Now the Arg661 -+ Stop mutation has also been
found in a Swedish family in the heterozygous form. The
deletion of thymidine in phenylalanine8 is also anew example of a more widely spread mutation in FXIII A-subunit. It
was detected in a Swedish patient in the homozygous form
and in a Danish patient in the heterozygous form.
activation R-sandwich
peptide
B
II
+
R-barrel
0-barrel1
domain
core
2
Cys-314
H19373 l 1Asp396
184
I
51
731
5
, ,.........,.,.,...,.,. . . . . . . . . ., ,...... . ........,.,. . . . . . .
..
,_....._._._...:,:,:,:
.. .. ..,..........
,......__
_ . . .::::.:.:.:
. . . .: .:_........
, , , . ......................................
. . . . . . .,
, , . , .(, _
. (.,.,.,.(.,.,.,.(,,.
..........................
37 43
I
In the case of both nonsense mutations, the absence of
detectable A-subunit antigen would be explained by the deficiency in mF2NA template for protein synthesis. Even if
some of the Arg661
Stop mutant polypeptide would be
translated, its beta-2 barrel is expected to fold into a very
different form due to the lack of more than two thirds of its
amino acid sequence. In the case of the deletion of thymidine,onlysevenaminoacidsoftheA-subunitsequence
would be in proper reading frame, thus obviously leading to
complete lack of the polypeptide. Interestingly, the patient
C-II-3, who is homozygous for the frameshift mutation, has
developed inhibitors against FXIII substitution therapy. It
could be speculated whether the mutation type would have
a predisposing effect to the formation
of an antibody. Inhibitors against FXIII substitution therapy in congenital FXIII
deficiency are very rare.In addition to this patient, only two
other cases have been reported, but in neither of them is
anything
known
about
mutation
the Furthermore,
about 20 cases of acquired inhibitors against endogenous
FXIII have been reported:'
628
._....._
.............................................
........................
.. .. .. .. .. . . ......
,.,.,.,.............. . . . . . . . . . . , . , . . . . .
m
IV
v
VI
vn
VI11
IX
x
XI
XI1
-
m1
XIV
xv
I l77
Am-260'
C~S
Arg-326 +Qln
Fig 9. Sequential arrangement of the Fxlll A-tubunit polypeptide and the corresponding cDNA showing the lmation of the mutations
reported thus far. The position of the exons areindicated by roman numbers and the sizes of the exons are marked. Theactivation peptide
and the four sequential domains as well as the three catalytic triad residues are markedon the polypeptide. Seven of eight clear missense
mutations are located in the core domain, further indicating the signFflcance of the conserved structure of the core domain. (Forfurther details
of the mutations, see for references: delAGGlu43," Arg681 His/splicing,= Argl7l stop," Am60 Lys," Tyr441 -. Stop," Gly501 + Arg."
Arg260 Qs," Ala394 + Val.")
-
-
-
-
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
150
MIKKOLA ET AL
In contrast with the nonsense mutations, the
mRNAs of all
three different missense mutations (Arg252 -+ ne, Arg326 -+
Gln, and Leu498 Pro) were expressed in amounts compa-+
rable with normal, based on quantitation of the levels of
the two different allelic transcripts in heterozygotes. These
results, as well as the previous mRNA results from the
Met242 Thr mutation, are in agreement with the general
observations that missense mutations do not alter the quantity of mRNA. Nevertheless, in none of the cases included
in this study could any A-subunit antigen be demonstrated
in the platelets by immunofluorescence.
In an effort to understand the possible effects of the three
missense mutations identified in this work and the Met242
Thr and Arg661 Stop mutations reported earlier by us,28
each mutation site was mapped onto the three-dimensional
structure of the wild-type A2 zymogen that had been determined by X-ray crystallographic method^.'^ The four missense mutations are located in the core domain, but none
of them affects any of the catalytic triad (Cys314, His373,
Asp396) or other residues likely to be directly involved in
cataly~is.'~.'~
Furthermore, all four mutation sites are located
at least 15 A from the active site of the molecule, and thus,
the mutations are not expected to interfere with the enzymatic mechanism. Our structural analysis of the mutations
indicates that the consequences are more likely to be disruption of proper protein folding or destabilization of the ensuing structure.
The Arg326 "* Gln mutation is a clear example of an
amino acid substitution likely to prevent protein folding or
to result in a misfolded or unstable structure. This arginine
is absolutely conserved among 16 members of the calciumdependent transglutaminase family, including even the nonenzymatic human protein 4.2, which is an indication of its
structural importance. Substitution of the buried, positively
charged Arg326 with asmaller, uncharged glutamine residue
is predicted to create a destabilizing void in the molecule,
eliminate two of the three hydrogen bonds observed in the
crystal structure, and destroy the electrostatic balance of the
molecule.
Arg252 is conserved among 13 of the 16 transglutaminase
sequences. Two of the transglutaminase proteins that do not
have an arginine at this position (P 4.2 and annulin) lack the
aspartic acid at position 243 as well. The third, rat prostate
transglutaminase, has a shortened loop in this region. The
Arg252 "* Ile mutation is another example of substitution of
a buried, charged arginine residue with a smaller, uncharged
(in this case, hydrophobic) amino acid that is incapable of
participating in the same hydrogen bonding and electrostatic
interactions. As a result, this mutation is also predicted to
interfere with protein folding or to lead to a misfolded or
destabilized structure.
Met242 is another core domain residue with a sidechain
buried in the molecule. A methionine in this position is
peculiar to FXIII; 13 of the other transglutaminases have a
slightly shorter hydrophobic leucine residue at this position.
The Met242 Thr substitution is expected to have a less
drastic effect than the other three missense mutations, although the mutant is also predicted to lead to a misfolded
or destabilized folded molecule. It is interesting to note that
the side-chain moieties of Met242 and Arg 252 pack against
-+
-+
-+
-+
each other in an antiparallel manner. Both the Met242
Thr and Arg252 + Ile mutations yield smaller buried amino
acid sidechains at the mutation site and a vacuum in the
same region of the core domain. Although these mutation
sites are located far from the active site of the molecule
(about 25 A), the alteration of amino acid size and chemical
nature brought about by the residue substitutions maybe
enough to destabilize the molecule or even interfere with
proper folding.
The Leu498 residue is conserved among only four of the
transglutaminase sequences. This is not surprising, as this
residue, located in a core domain alpha helix, is partially
exposed to solvent, and this site is, thus, expected to more
easily accommodate some variation in amino acid type. The
substitution of leucine 498 witha proline is a mutation,
however, that has dramatic structural consequences. The
constraints of the proline ring introduce unfavorable close
interatomic contacts and disrupt the mainchain hydrogenbonding pattern that is characteristic of helix structure. As
a result, the proline mutant is predicted to fold with a kinked
helix, which in turn is expected to destabilize the molecule
or even interfere with dimer formation.
Our analyses of these four missense mutations predict that
each would interfere with folding or lead to misfolded and
unstable structures, which, in turn, is assumed to result in
premature intracellular degradation of the mutant polypeptides. These results are consistent with the immunologic
finding in each case of no detectable FXIII A-subunit antigen.
-+
ACKNOWLEDGMENT
WeaddressspecialthankstoDrLaszloMuszbekforhelpful
discussions andDrRiittaKekoIll;ikiforhelpandencouragement
with this work. We also thank Eija Hhiiliiinen, Sinikka Laine, and
Tuula Salmivaara for excellent technical assistance.
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