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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2830-2838
Molecular Mechanisms of Type II Factor XIII Deficiency: Novel
Gly562-Arg Mutation and C-Terminal Truncation of the A
Subunit Cause Factor XIII Deficiency as Characterized in a Mammalian
Expression System
By
Nobumasa Takahashi,
Hiroaki Tsukamoto,
Hideaki Umeyama,
Giancarlo Castaman,
Francesco Rodeghiero, and
Akitada Ichinose
From the Department of Molecular Patho-Biochemistry, Yamagata
University School of Medicine, Yamagata, Japan; the Department of
Physical Chemistry, Kitasato University School of Pharmaceutical
Sciences, Tokyo, Japan; the Department of Haematology, San Bortolo
Hospital, Vicenza, Italy.
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ABSTRACT |
To explore the biological and clinical implications of the
structure/function relationships in factor XIII, mutations in two patients with type II deficiency were identified and characterized in a
mammalian expression system. Nucleotide sequence analysis of the A
subunit gene showed that case no. 1 had a deletion of 4 bp (AATT) in
exon XI and that, in case no. 2, Gly562 (GGG) had been replaced by
Arg(AGG). The deletion in case no. 1 leads to a premature
termination at codon 464. Restriction digestion of amplified DNAs
confirmed that both cases were homozygous for their respective
mutations. Reverse transcription-polymerase chain reaction analysis demonstrated that the level of mRNA was greatly reduced in
case no. 1, whereas the level of mutant mRNA expressed in case no. 2 was normal. Molecular modeling calculated that Arg562 changed the
conformation of the A subunit, suggesting misfolding and/or destabilization of the molecule. To determine how these mutations impaired synthesis of the A subunit, recombinant A subunits bearing the
mutations were expressed in mammalian cells. Pulse-chase experiments showed that the mutants were synthesized normally but disappeared rapidly, whereas the wild-type remained. These results indicate that
both mutant proteins with an altered conformation become prone to rapid
degradation, resulting in factor XIII deficiency in these patients.
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INTRODUCTION |
COAGULATION FACTOR XIII is a plasma
transglutaminase consisting of two catalytic A and two noncatalytic
B subunits.1-3 A combination of cDNA cloning
and amino acid sequence analysis established the primary structures of
both subunits.4,5 The A subunit contains an active site
Cys314, and the B subunit is composed of 10 tandem repeats termed Sushi
domains.6 We previously characterized the genes for both
the A and B subunits of human factor XIII.7,8 The gene for
the A subunit spans more than 160 kb and consists of 15 exons
interrupted by 14 introns. The gene for the B subunit is approximately
28 kb in length and is composed of 12 exons interrupted by 11 introns.
Determination of the genomic organization and sequence for both
subunits has made it possible to characterize factor XIII deficiency at
the DNA level.9
Factor XIII deficiency has been classified into two
categories10: type I deficiency, characterized by the lack
of both the A and B subunits; and type II deficiency, characterized by
the lack of the A subunit alone. To clarify the genetic bases of these diseases, two cases of type I deficiency were examined at the DNA
level,11 and a patient with complete B subunit deficiency was also studied.12 The insertion of a triplet created a
stop codon in the gene for the B subunit in the former cases, and a single base deletion caused the splicing defect in its mRNA in the
latter case. An additional substitution of T for G in the latter case
replaced Cys430 with Phe in the seventh Sushi domain and resulted in
impaired intracellular transport of the B subunit.13 Accordingly, it was shown that type I deficiency can be caused by
defects in the gene for the B subunit.14
Mutations in the gene for the A subunit have also been identified in
patients with type II factor XIII deficiency (ie, the A subunit
deficiency).9,15-26 These mutations are highly
heterogeneous and include a variety of nonsense mutations, small
deletions, and insertions with or without out-of-frame shift/premature
termination, splicing abnormalities, etc. Various missense mutations in
the A subunit will also result in type II deficiency. The mechanisms of
defective A subunit biosynthesis caused by the missense mutations have
not been clarified, mainly because in vitro expression systems were
unavailable using mammalian cells. Recently, we succeeded in
synthesizing both the A and B subunits in baby hamster kidney (BHK)
cells and an abnormal B subunit of type I deficiency in COS
cells.13,27 However, no abnormal A subunit of type II
deficiency has been characterized in a mammalian expression system to
date.
It is important to closely examine amino acid substitutions and
deletion of peptide regions to understand the structure/function relationships of the factor XIII molecule as well as its clinical implications in factor XIII deficiency. In this study, we identified a
4-bp deletion and a Gly562-Arg mutation in the gene for the A subunit
of factor XIII in two patients (cases no. 1 and 2) with A subunit
deficiency28 and predicted altered structures of the mutant
A subunits by molecular modeling. In addition, we present for the first
time the successful expression of novel mutant A subunits in BHK cells
and provide evidence indicating that the mutants are degraded rapidly
in the synthesizing cells.
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MATERIALS AND METHODS |
Venous blood was drawn from normal individuals and from two patients
with A subunit deficiency28 and their family members after
informed consent had been obtained. Both cases had essentially no
enzymatic activity for factor XIII and the A subunit antigen (<5% of
normal by Laurell's method). The A subunit was also absent in the
patients' platelets.11 Genomic DNA and RNA samples were prepared from peripheral blood cells by standard techniques, as described previously.11,12
Southern blotting.
Ten micrograms of genomic DNA was digested with 20 U of EcoRI
restriction enzyme at 37°C for 2 hours and applied to a 0.4% Agarose gel. The DNA fragments were transferred to a nylon membrane. The membrane was hybridized overnight at 65°C with a
32P-labeled cDNA encoding the A subunit5 and
exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY)
at  80°C.
Polymerase chain reaction (PCR).
One microgram of genomic DNA was amplified using 5.0 U of Thermus
aquaticus DNA polymerase (Promega, Madison, WI) in a
100 µL reaction mixture. A total of 17 pairs of gene-specific primers were designed from the nucleotide sequence of the normal A subunit gene7 (Fig 1, top). After 30 to
35 cycles of amplification, 9 µL of each reaction mixture was applied
to a 1.5% Agarose gel.
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| Fig 1.
(Top) Gene for the A subunit and mutations identified.
Exons are indicated by wide vertical bars and Roman numerals, and
introns are indicated by capital letters. Each exon and its boundaries and the 5 -flanking regions were amplified one by one using 17 pairs of primers (arrows under exons). Solid and open circles with
sequences represent homozygous causative mutations and changes known as
common polymorphisms, respectively. Normal sequences are followed by
those corresponding to substitutions found in the probands' DNAs. For
analysis of the two mutations identified, two amplified fragments were
digested with appropriate endonucleases as indicated by their names and
arrows. (Bottom) mRNAs for the A subunit in the probands. Three regions
of exons II-IV, X-XIII, and XI-XIII were amplified by RT-PCR. A solid
line represents the expressed mRNA, whereas a broken line indicates a
possible mRNA that was not detected.
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Nucleotide sequence analysis.
Amplified DNA samples were digested with restriction enzymes to
generate proper ends for ligation into sequencing vectors. The DNA
sequence of an insert was obtained using the dideoxynucleotide method29 with ABI sequence analyzer 373A (Perkin-Elmer,
Norwalk, CT). To minimize the possibility of obtaining the
DNA sequences with misincorporation by the DNA polymerase, 10 or more
samples of each amplified region of the A subunit were examined.
Detection of mutations by restriction digestion.
For restriction enzyme analysis to detect the presence of the 4-bp
deletion in exon XI and the replacement of G by A in exon XII (Fig 1,
top), 15 µL of the amplified DNA samples was incubated with
Tru9I and Hpa II endonucleases (Boehringer Mannheim
GmbH, Mannheim, Germany), respectively. After digestion
for 2 hours, half of each sample was applied to a 2% Agarose gel.
Reverse transcription-PCR (RT-PCR) assay of mRNA for the A subunit.
RT of the total RNA (1.0 µg) was performed using an oligo dT
(dT18) primer and Superscript II RNase H
(GIBCO-BRL, Grand Island, NY), as described
previously.11 One fiftieth of the synthesized first-strand
cDNA was used for PCR in a reaction mixture of 50 µL by employing two
pairs of primers separately; for exons II-IV of the A subunit (Fig 1,
bottom), 5-ATGTCAGAAACTTCCAGGACC-3 (sense) and
5-CGTGTCTGTTTCTGGGTTTCG-3 (antisense); for human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal
control, 5-CATCACCATCTTCCAGGAGC-3 (sense) and
5-TAAGCAGTTGGTGGTGCAGG-3 (antisense). After 25 cycles,
the reaction mixtures were electrophoresed in a 2% Agarose gel. The gel was subjected to quantitation of the amplified products by Densitograph 3.01 Imaging Analyzer (Atto Inc, Tokyo, Japan). The fluorescence intensity of the PCR product for the A subunit was normalized to that for GAPDH.
One fiftieth of the synthesized first-strand cDNA was also used for PCR
in a reaction mixture of 50 µL to amplify two regions (Fig 1, bottom)
by employing two pairs of primers: for exons X-XIII, 5-CATCAAGCACGGCCATGTCTGC-3 (sense) and
5-CTTGATGATGATCTCAGG-3 (antisense); for exons XI-XIII,
5-TCCGGAACAACAGCCACAACCG-3 (sense) and
5-CTTGATGATGATCTCAGG-3 (antisense). To detect extremely small amounts of cDNAs, prolonged amplification was performed using 35 to 40 cycles. Amplified fragments were subjected to digestion by either
Tru9I or Hpa II to detect expressed mutations.
Molecular modeling.
To predict the tertiary structure of the mutant molecule, molecular
modeling was performed by using the coordinates of the A subunit
defined by X-ray crystallography.30 To discuss the structural change caused by the Gly562-Arg mutation, we used a partial
molecule from residues 190 to 628 consisting of domains II (residues
190-319), III (320-513), and IV (514-628) according to the domain
structure defined by Kurochkin et al.31 Hydrogen atoms,
except for those belonging to hydrocarbons in the native XIIIA
(190-628), were energetically optimized, as were the mutated Arg562
residue and hydrogens in the mutant XIIIA (190-628). The initial
conformation of the Arg residue was determined by using the chimera
system for homologous protein modeling32 and was inserted
into the model for the mutant. Energy optimization was performed using
a program called APRICOT33 with a united atom force field
of AMBER.34 All the coordinates, except the
mutated Arg562 residue and hydrogen atoms that bound to nitrogens and oxygens, were fixed (constrained) to those experimentally determined by
Yee et al30 (identifier 1ggt in the Protein
Data Bank). Therefore, the omission of the C-terminal domain (domain V)
does not affect the calculated results for the wild-type and the
Gly562-Arg mutant. In addition, domain V exists a distance from domains
II, III, and IV where the mutation is located.
Construction of expression vectors.
A Pst I cDNA fragment of 2.3 kb coding for the A subunit of
human factor XIII5 was subcloned into pBluescriptII SK(+)
(Stratagene, La Jolla, CA) for mutagenesis. In vitro
mutagenesis was performed by recombinant PCR technique35
using oligonucleotide primers: for 4-bp deletion,
5-AGTTTCTTAATGTCACGAGCGTTCACCTGTTCAA-3 (sense), from the
middle 5-GCCACCCACATTGGGAAATTGTGACCAAACAAATTG-3 (sense), and 5-CTTGATGATGATCTCAGG-3 (antisense); for Gly562-Arg
mutation, 5-(CATCAAGCACGGCCATGTCTGC)-3 (sense), from the
middle 5-GCCTTCAGGACCCTGGTGTAGAAGGTG-3 (antisense) and
5-CACCTTCTACACCAGGGTCCTGAAGGC-3 (sense), and 5-GGAAACAGCTATGACCATG-3 (antisense). The mutations in the
cDNAs were confirmed by dideoxy sequencing. The cDNAs for the wild-type and the mutants were released by HindIII/XbaI from the
pBluescriptII vector and inserted into expression vector pcDNA3
(Invitrogen, San Diego, CA).
Cell culture and transfection.
BHK cells were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin, streptomycin, and neomycin, GIBCO BRL). Approximately 3 × 106 BHK cells were transfected with 10 µg of an
expression plasmid by the calcium phosphate method. Two days after
transfection, cells were grown in DMEM containing 0.5 mg/mL G418
(GIBCO-BRL) as a selective agent. All cell cultures were maintained in
an incubator humidified with 5% CO2 at 37°C.
Pulse-chase experiments.
For metabolic radiolabeling, 2 × 106 BHK cells in
6-well (37-mm) plates were incubated in Met-free DMEM for 1 hour at
37°C. The cells were then labeled with 50 µCi/well of
[35S]Met in Met-free DMEM with 10% dialyzed FBS for 30 minutes (pulse) and further incubated in DMEM supplemented with
unlabeled Met (chase) and 10% FBS. The chase was terminated at various
time intervals; culture media and cell lysates were harvested and
stored at 80°C.
Immunoprecipitation of the A subunit was performed by incubating the
supernatant of the cell lysates and culture medium with the anti-A
subunit antiserum, as described previously.13 Bound antigen
was applied to electrophoresis on a 10% sodium dodecyl sulfate
(SDS)-polyacrylamide gel. The gel was dried and the radioactive band
for the A subunit was quantified using a FLA-2000 Fluoroimage Analyzer
(Fuji Photo Film, Tokyo, Japan).
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RESULTS |
Genomic Southern blotting analysis and in vitro amplification of the A
subunit gene.
To test for the presence of large deletion(s), insertion(s), and
recombination(s), genomic Southern blotting analysis was performed
first. EcoRI-digested genomic DNAs of two Italian cases and
normal individuals showed an identical pattern (data not shown). To
search for possible smaller mutation(s) in and around the exons by PCR,
17 pairs of amplification primers were used, and these produced 17 different DNA fragments, respectively (data not shown). The sizes of
these fragments were the same as predicted from the normal genomic
sequence.7 These results confirmed that there was no gross
change(s) in the probands' genes.
Identification of the 4-bp deletion and Gly562-Arg mutation.
Nucleotide sequences of the 17 amplified fragments were then examined
to search for differences from the normal sequence.7 When
exon XI of case no. 1 was sequenced, all of 15 ssDNA samples showed
AAATTGTGA (Fig 2, top), whereas the normal
sequence is AAATTAATTGTGA. One obvious result of this 4-bp
deletion is the creation of a new termination codon at amino acid
position 464 (Fig 2, top).
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| Fig 2.
(Top) Nucleotide sequence of a part of exon XI for normal
and case no. 1. The 4-bp deletion is boxed. Codon 464 becomes a termination signal because of a reading frame-shift. (Bottom) Nucleotide sequence of a part of exon XII for normal and case no. 2. Mutated nucleotide A is indicated in a box, and a normal Gly562 residue
is substituted with an abnormal Arg sequence.
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Three additional changes in the nucleotide sequence were found in case
no. 1 (Fig 1, top), but all of these were known genetic polymorphisms
in the coding regions: Val34-Leu(GTG-TTG) in exon II,
Pro331-Pro(CCA-CCC) in exon VIII, and
Pro564-Leu(CCG-CTG) in exon XII.7,18 No other
mutations were detected in and around the 15 exons or in a section of
the 5-flanking region. This region of about 300 bp, 1.3 kb
upstream from the 5-end of exon I, contains several TATA and
CAAT boxes (A. Ichinose, unpublished data). Taken together, these results suggest that the 4-bp deletion is very likely
causative for the A subunit deficiency in case no. 1.
When exon XII of case no. 2 was examined, all of 12 ssDNA samples
demonstrated AGG for Arg in place of the normal sequence of
GGG coding for Gly562 (Fig 2, bottom). Another nucleotide substitution, Val34-Leu (GTG-TTG), was identified in exon II; this is the
same known polymorphism as found in case no. 1. No other mutation was detected in and around all exons or in a section of the
5-flanking region. Accordingly, it was concluded that the
Gly562-Arg mutation was responsible for the A subunit deficiency in
case no. 2.
Detection of mutations by restriction enzyme digestion.
Because the deletion of 4-bp (AATT) in the DNA of case no. 1 destroys
an intrinsic Tru9I site (TTAA), a fragment spanning exon XI was
amplified and digested with Tru9I endonuclease. Digestion of
the 291-bp fragment from a normal individual yielded two bands of 165 bp and 126 bp (Fig 3, left), whereas the
287-bp fragment of the proband remained unchanged. The proband's
mother showed both cleaved and uncleaved bands. These results indicated
that case no. 1 was a homozygote and that the mother was a heterozygote of this mutant allele. This same mutant allele was not found in 54 normal Italian individuals (data not shown).
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| Fig 3.
Restriction digestion with Tru9I
endonuclease of the amplified products of the exon XI for the 4-bp
deletion (left) and with Hpa II endonuclease of the amplified
products of exon XII for the Gly562-Arg mutation (right).
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Because the G-A substitution in case no. 2 destroyed an intrinsic
Hpa II site (CCGG), a fragment spanning exon XII was amplified and digested with Hpa II endonuclease. Digestion of the 389-bp fragments from a normal individual and the proband's brother (Fig 3,
right) yielded three fragments of 208, 116, and 65 bp. In contrast, the
digest of the 389-bp fragment from the proband yielded two fragments of
208 and 181 bp. Thus, the brother was normal, and case no. 1 was
homozygous for the Gly562-Arg mutation. This mutant allele also was not
found in 53 normal Italian individuals (data not shown).
Semiquantitation of mRNA for the A subunit.
Because neither factor XIII activity in plasma28 nor the A
subunit antigen in platelets11 was detected in either
patient, the mRNA for the A subunit was examined. An RT-PCR assay was
performed to estimate the amount of mRNA for the A subunit in
peripheral blood cells, where the A subunit may be aberrantly
expressed, although the site of biosynthesis for the plasma A subunit
is still unknown. A 543-bp amplified fragment for the A subunit and a
253-bp fragment for GAPDH were obtained by RT-PCR from a normal individual (Fig 4). Under experimental
conditions, a linear relationship was observed between the amounts of
total RNA used and the fluorescence intensity of these bands (data
not shown). A very faint band for the A subunit was detected in
case no. 1, whereas a band was clearly observed in case no. 2 (Fig 4).
The ratio of the A subunit mRNA/GAPDH mRNA was 1.0, 0.04, and 1.0 for a
normal subject, case no. 1, and case no. 2, respectively. Thus, the
amount of the mRNA in case no. 1 was markedly reduced, strongly
suggesting that the mutant mRNA is not synthesized as stably as normal
mRNA. In contrast, the amount of the mRNA of case no. 2 was comparable
to that of the normal individual.
Detection of the mutant transcript for the A subunit.
A band of normal size was obtained in the amplified sample from case
no. 2 by using primers for exons XI-XIII. Digestion with Hpa II
of the 290-bp fragment from case no. 2 yielded three bands of 155, 133, and 2 bp (the last fragment was unresolved), whereas a normal fragment
produced four bands of 133, 90, 65, and 2 bp (Fig 5, right), indicating that the mRNA
from case no. 2 contained the Gly562-Arg mutation. These results
confirmed that the mRNA with the Gly562-Arg mutation was the sole
transcript expressed in case no. 2.
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| Fig 5.
RT-PCR and RFLP of a fragment containing exons XI-XIII
for the Gly562-Arg mutation in case no. 2. An RT-PCR product was
digested with Hpa II. For the results shown here, amplification
was performed for 35 cycles.
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No discrete band was obtained when the mRNA sample from case no. 1 was
amplified, even by prolonged RT-PCR for 35 to 40 cycles using a pair of
primers for exons X-XIII. Accordingly, PCR-restriction fragment length
polymorphism (RFLP) analysis could not be performed for
case no. 1.
Molecular modeling.
To calculate the effects of the Gly562-Arg mutation on the structure of
the mutant A subunit, molecular modeling was performed. The solvent
accessibility of the Arg562 side chain was 47% for domain IV alone and
6% for domains II, III, and IV, indicating that the Arg562 residue of
the mutant was present on the surface of domain IV and on the
interdomain surface between domains IV, II, and III of the molecule
(refer to the coordinates for the mutant molecule presented at
http://prtds.pharm.kitasato-u.ac). Domain IV interacts with each of
domains II and III, and Arg562 in domain IV interacts with several
amino acid residues on the surface of domains II and III. Although the
Gly562 residue is completely conserved among
transglutaminases,13 the structure of the A subunit was
partly compatible with that of the elongated side chain of the mutated
Arg residue. However, there were atomic pairs that had distances
shorter than van der Waals contacts between the Arg and other residues
(Fig 6, top right); these included Phe559,
Tyr560, Thr561, Val563, His605, and Gln622 in domain III, which were
about 2.0 Å distant from the Arg residue; for domain IV, Ser368 and
Val369 were at about 2.6 Å.
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| Fig 6.
(Top) Close-up model view of the native (left, Gly562) or
mutant (right, Arg562) A subunit. The tertiary structure of the normal
A subunit is based on x-ray diffraction
analysis.30 The main chain of the protein is
drawn as an A carbon trace. Side chain groups of labeled residues are
shown as ball-and-stick structures. Although the mutant Arg562 residue
can be accommodated by the chemical structure, it generates unfavorable
short contacts with neighboring residues. Accordingly, the substitution
of a small residue by a large charged amino acid is expected to yield
an unstable, misfolded structure. (Bottom left) Model view for the A
subunit dimer including the normal domain II, the first half of domain
III, the second half of domain III in a monomer, and the same domains
II and III in the counterpart monomer. Domains IV and V are not shown.
A total of 268 residues consisting of the second half of domain III and
domains IV and V are removed from the molecule in the deletion mutant
of case no. 1. The C-terminal Leu463 residue of the truncated molecule
is shown. Pro383 and Asp384 residues contact with the second part of
domain III, which is absent in the mutant. (Bottom right) Closer view
of the environment around domains II and III. Because premature
termination at position 464 would lead to the loss of a C-terminal part
of the core domain and the entire domains IV and V (barrel 1 and 2),
the protein is expected to misfold and/or be incapable of dimer
formation.
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The molecular structure of the 464Stop mutant is truncated to 463 residues by the 4-bp deletion. In the native form, the first half of
domain III (residues 320-463) makes contact with domain II and the
second half of domain III (residues 464-513) over a wide area (Fig 6,
bottom left). When the second half of domain III is absent, as in the
deletion mutant of case no. 1, the surfaces of domain II and the first
half of domain III become exposed extensively to solvents. Thus, it may
be said that the surfaces of the mutant molecule will be susceptible to
proteolytic degradation. The dimer formation of the mutant A subunit
must also be impaired by the truncation of residues 464-731, because,
in the native A subunit, this second part of domain III in one monomer
contacts residues Pro383 and Asp384 directly in the counterpart monomer
(Fig 6, bottom right).
Pulse-chase experiments in mammalian expression system.
To evaluate the intracellular stability of the newly synthesized A
subunit, the wild-type and two mutants were examined by pulse-chase
studies. RT-PCR analysis, using mRNA samples obtained from cells
transfected with the expression vectors, showed that there were
comparable amounts of the transcripts for both the wild-type and two
mutants (data not shown).
Nascent wild-type A subunit seen at 0 hours gradually decreased in the
cells but did not appear in the culture medium at all (Fig 7, top right), as described
previously.27 At 24 hours, the band of the wild-type A
subunit was still intense (64% of 0 hours). In contrast, the mutant A
subunits in the cell lysates rapidly decreased with time but, again,
did not appear in the culture medium (Fig 7, bottom). The disappearance
of a truncated 48-kD band in the 4-bp deletion mutant was much faster
than that for the Gly562-Arg mutant (0% within 2 hours v 8 hours, Figs 7 and 8). The complete loss of
both mutants in BHK cells indicated that these molecules degraded
rapidly in an intracellular compartment(s).
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| Fig 7.
Pulse-chase study of wild-type and mutant A subunits in
BHK cells. Pulse-chase experiments were performed in a mammalian
expression system as described in Materials and Methods. Radiolabeled
cell lysates and culture media of cells were taken at various time intervals and immunoprecipitated with an antiserum against the A
subunit, followed by electrophoresis on 10% SDS-polyacrylamide gels.
Top left, mock; top right, wild-type; bottom left, the 4-bp deletion
(4-bp del); bottom right, the Gly562-Arg mutant.
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| Fig 8.
Time course of the radiolabeled bands for wild-type and
mutant A subunits in pulse-chase study. The radioactivity of the band for the recombinant A subunit was measured by a fluoroimage analyzer as
described in Materials and Methods. ( ) Wild-type, ( ) the 4-bp
deletion (ie, C-terminal truncation), and ( ) the Gly562-Arg mutation.
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These results strongly suggest that the mutant A subunits are unstable.
This is in good agreement with the results obtained by molecular
modeling as described above.
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DISCUSSION |
Factor XIII deficiency is caused by defects in either the A or B
subunit of the gene.14 These can include a variety of
missense and nonsense mutations, small deletions and insertions with or without out-of-frame shift/premature termination, splicing
abnormalities, etc.11-26 Therefore, mutations causing
factor XIII deficiency are highly heterogeneous. In the present study,
nucleotide sequence analysis of amplified DNAs showed that cases no. 1 and 2 with severe factor XIII deficiency28 had a deletion
of 4 bp in exon XI and a mutation of Gly562-Arg in exon XII of the gene
for the A subunit, respectively.
The premature termination newly created by the 4-bp deletion leads to a
marked reduction in the mRNA level of the A subunit in case no. 1. However, the mechanism by which a new in-frame termination codon
results in a decrease in the concentration of steady-state cytoplasmic
mRNA has not been understood to date. As discussed by
Cooper,36 nonsense mutations would halt the pulling process
of the pre-mRNA through the splicing apparatus and the nuclear pores,
leaving the RNA molecule vulnerable to RNase digestion. Another model
is proposed whereby detection of an in-frame termination codon by
putative nuclear scanning machinery would result in a slowing down of
mRNA splicing/translocation. Despite the fact that the steady-state
mRNA of the mutant allele was not detectable in peripheral blood cells
from case no. 1, the level of mutant mRNA was comparable to that of the
wild-type in the transfected mammalian cells, strongly suggesting that
this mutation impairs some steps involved in pre-mRNA processing before the completion of the mature mRNA. The marked reduction in the level of
A subunit mRNA in case no. 1 would also result in a marked decrease in
production of the A subunit protein.
The premature termination at codon 464, in turn, results in the
deletion of the C-terminal third of the A subunit molecule (268 amino
acids, residues 464-731) and in the creation of a C-terminal truncated
protein. Because domains I and II (residues 1-319) are structurally
independent of domains III, IV, and V,30 domains I and II
of the mutant will fold like that of the native A subunit. It is
generally accepted that a hydrophobic core in a domain plays essential
roles in protein folding. Because residues 320-463 nearly complete the
hydrophobic core in domain III, it is likely that the 464Stop mutant
folds in a manner similar to the native A subunit. Because the missing
C-terminal portion is essential for interdomain interactions and dimer
formation of the A subunit, the truncated protein of 463 residues is
predicted to be unstable. This hypothesis has been confirmed by
pulse-chase experiments in a mammalian expression system in which the
truncated protein of 48 kD was degraded rapidly inside BHK cells. The
disappearance of the truncated mutant was even faster than that of the
Gly562-Arg mutant. Accordingly, it may be said that the A subunit
deficiency in case no. 1 was caused by two steps: first, by a severe
reduction in its mRNA level; and, second, by the instability of its
truncated molecule.
Alternatively, without residues 464-513, domain III may not be able to
fold like the native domain III, in which case the result would also be
the premature degradation of the entire mutant protein. However, this
hypothesis cannot be substantiated at present.
Although this 4-bp deletion was previously identified at the DNA level
in a compound heterozygote of French origin,23 it was not
characterized further. A founder effect for this mutation is not
likely, because none of the three polymorphisms found in our Italian
homozygote was detected in the heterozygote mentioned above. This
deletion mutation may be a recurring one, because the direct repeat of
the AATT sequence7 at the deletion site in exon XI seems to
be a hotspot for slippage of DNA polymerases.
The mRNA level of the Gly562-Arg mutation was not reduced at all in
case no. 2. Gly562 is highly conserved throughout all 15 members of the
transglutaminase family37,38; therefore, it may play an
important role(s) in the enzymatic function and/or structural
integration of transglutaminases. This amino acid is located on the
interface of domains IV, II, and III, based on the three-dimensional
structure of the molecule.30 It may be said that the
Gly562-Arg mutant protein cannot fold into exactly the same structure
as the wild-type, especially in the formation of the domain complex of
II, III, and IV. Even if the gross structure consisting of domains II,
III, and IV for the mutant were close to the native x-ray structure,
molecular modelling showed a space somewhat smaller than required for
the elongated side chain of Arg562. Furthermore, molecular mechanics predicted that the Gly562-Arg mutation increased the total potential energy of the A subunit molecule by +73.4 kcal/mole (Tsukamoto et al,
unpublished data), suggesting that the mutation from Gly to Arg makes the molecule less stable than the native form. Therefore, the Gly562-Arg mutation is not adaptable for the A subunit molecule. This hypothesis is consistent with the results obtained by pulse-chase experiments that showed that the Gly562-Arg mutant was synthesized normally, but degraded rapidly in cells. Accordingly, the Gly562-Arg mutation leads to deficiency of the A subunit not only in
plasma28 but in platelets as well.11
It may be that transport and secretion of the mutant A subunit are
impaired in addition to its folding capabilities; however, these
questions must remain unanswered until the mechanisms of its normal
biosynthesis are better understood.
Eight additional missense mutations have been reported, including
Asn60-Lys,19 Met242-Thr,18
Arg252-Ile,22 Arg326-Gln,22 Arg408-Gln,20 Leu498-Pro,22
Gly501-Arg,24 and Leu667-Pro.19 To our
knowledge, two of these mutants, Asn60-Lys and Gly501-Arg, have been
expressed in yeast24 to date, but not in mammalian cells.
It was concluded by Coggan et al24 that the Asn60-Lys mutant was very unstable and/or the subject of increased
proteolytic degradation. However, this conclusion may not always be
valid in vivo, because the quality control and proteolytic degradation mechanisms for abnormal molecules in mammalian cells may differ from
those in other systems. For example, C-terminal truncated mutants
(including one composed of 462 residues) were expressed in
Escherichia coli at levels comparable to those of the
wild-type,39 whereas our mutant of 463 residues was lost
within 2 hours in mammalian cells, indicating that the effects of
mutations on protein biosynthesis depend on the type of expression
systems used. We have recently developed mammalian expression systems
both for the A and B subunits9,13 and have confirmed that
both recombinant A and B subunits were indistinguishable from the
native A and B subunits of plasma factor XIII in terms of their
physical and functional properties.27 Thus, in this study,
we present for the first time the successful expression of novel mutant
A subunits in mammalian cells and provide evidence indicating that
rapid degradation of the A subunit mutants in the synthesizing cells leads to type II factor XIII deficiency.
Finally, this study has shown that molecular modeling is, at least to
some extent, useful in predicting possible effects of missense
mutations on the structure and stability of the A subunit, because an
expression analysis of each mutant individually would require a great
outlay of time and resources.
| |
FOOTNOTES |
Submitted July 24, 1997;
accepted December 2, 1997.
Supported in part by research grants from the Ministry of Education,
Science and Culture, Japan (08457271), by the Ichiro Kanehara
Foundation (Japan), and by the Japan Research Foundation for Clinical
Pharmacology.
Address reprint requests to Akitada Ichinose, MD, PhD, Department of
Molecular Pathological Biology, Yamagata University School of Medicine,
Iida-Nishi 2-2-2, Yamagata, 990-9585 Japan.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
After completion of this manuscript, Mikkola et al40
reported an expression of A subunit mutants in COS cells.
The authors thank Drs V.C. Yee and D.C. Teller for providing
the coordinates for the A subunit; Dr P. Bishop for the A subunit
antibody; J. Harris for synthesis of the oligonucleotides; E. Espling
for his excellent assistance; Drs T. Yamazaki, T. Izumi, and T. Saito for their helpful discussion; and L. Boba for her help in the preparation of the manuscript.
| |
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Abstract
Introduction
Abstract
