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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4300-4308
Factor XII Tenri, a Novel Cross-Reacting Material Negative Factor XII
Deficiency, Occurs Through a Proteasome-Mediated Degradation
By
Shinichi Kondo,
Fuminori Tokunaga,
Seiji Kawano,
Yoichi Oono,
Shunichi Kumagai, and
Takehiko Koide
From the Department of Life Science, Faculty of Science, Himeji
Institute of Technology, Harima Science Garden City, Hyogo; the
Department of Clinical Medicine, Kobe University, Kobe; and the
Department of Hematology, Tenri Yorozu Hospital, Tenri, Japan.
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ABSTRACT |
A homozygous cross-reacting material negative factor XII-deficient
patient with 3% antigen and activity levels of factor XII was screened
for the identification of a mutation at the genomic level. Low-ionic
strength single-stranded conformation polymorphism (SSCP) analysis and
sequence analysis showed that the proband's gene for factor XII had an
A G substitution at nucleotide position 7832 in exon 3, resulting in a Tyr34 to Cys substitution in the NH2-terminal type II domain of factor XII. We designated
this mutation as factor XII Tenri. Mutagenic polymerase chain reaction (PCR), followed by KpnI digestion, showed a homozygous mutation in the proband's gene and heterozygous mutations in his parents and
sister. Immunoprecipitation and Western blot analyses of plasma samples
from the factor XII Tenri family indicated that the proband had a trace
amount of variant factor XII with an apparent molecular mass of 115 kD,
which was converted to the normal 80-kD form after reduction,
suggesting that factor XII Tenri was secreted as a disulfide-linked
heterodimer with a  35-kD protein, which we identified as
 1-microglobulin by immunoblotting. Pulse-chase
experiments using baby hamster kidney (BHK) cells showed that
Tenri-type factor XII was extensively degraded intracellularly, but the
addition of cystine resulted in increased secretion of the mutant.
Using membrane-permeable inhibitors, we observed that the degradation occurred in the pre-Golgi, nonlysosomal compartment and a proteasome appeared to play a major role in this process. On the basis of these in
vitro results, we speculate that the majority of the factor XII Tenri
is degraded intracellularly through a quality control mechanism in the
endoplasmic reticulum (ER), and a small amount of factor XII Tenri that
formed a disulfide-linked heterodimer with
1-microglobulin is secreted into the blood stream.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN COAGULATION factor XII (Hageman
factor) is a single chain glycoprotein (Mr 80,000) that
circulates in blood as an inactive zymogen at concentrations of 29 to
40 µg/mL.1,2 Human factor XII is composed of 596 amino
acid residues with N-linked and O-linked carbohydrate
chains.3-5 The domain organization of factor XII is
analogous to those of several fibrinolytic proteins, including
tissue-type plasminogen activator and urokinase. Factor XII binds to
negatively-charged molecules such as kaolin, ellagic acid, dextran
sulfate, sulfatide, and endotoxin.1,2 The binding is
required not only for the expression of factor XIIa proteolytic activity, but also for the cleavage of factor XII by kallikrein. The
human factor XII gene is located on the chromosomal band 5q33-qter, and
12 kb of the entire genomic sequence with 14 exons and 13 introns have
been analyzed.6 Factor XII is converted to a two-chain serine protease ( -factor XIIa) with an NH2-terminal
heavy chain (Mr 50,000) and a COOH-terminal light chain
(Mr 28,000) that is capable of initiating the intrinsic pathway
of blood coagulation, kinin production, and
fibrinolysis.1,2 The heavy chain of factor XIIa contains
five different domains including a type I and a type II domain of
fibronectin, two epidermal growth factor (EGF)-like
domains, a kringle domain, and a proline-rich domain, whereas the light
chain consists of a typical trypsin-like serine protease
domain.3-5 On activation, further cleavages take place in
the heavy chain, resulting in the production of  -factor XIIa consisting of the nonapeptide (Asn335-Arg343) and disulfide-linked 28-kD protease domain.7
Hereditary factor XII deficiency is clinically asymptomatic, but can be
detected by an in vitro test due to a prolonged activated partial
thromboplastin time (APTT). Typically, homozygous or compound heterozygous carriers exhibit almost no (<1%) factor XII activity as
compared with normal subjects, whereas heterozygotes display intermediate activity. Most of these subjects also lack immunologically detectable factor XII and are referred to as cross-reacting material (CRM) negative.8,9 The majority of CRM negative factor XII deficiency cases are known to be caused by mutations such as a change
in the splice sites or a deletion of portions of the coding sequence
resulting in a frameshift.10 Hageman trait, the best known
factor XII deficiency, is associated with an aberrant TaqI restriction site in intron B, a putative regulatory
site.11,12 In contrast, factor XII Washington D.C., a CRM
positive factor XII deficiency, contains a Cys571 to Ser replacement,
resulting in the disruption of a disulfide-linkage between
Cys540-Cys571 near the active site Ser544.13 Factor XII
Locarno, another CRM positive factor XII deficiency, is induced by an
amino acid substitution of Arg353 by Pro, resulting in a loss of the
kallikrein cleavage site at Arg353-Val354.14 Recently,
Schloesser et al15 investigated 31 unrelated factor
XII-deficient patients and identified two missense
mutations, Arg398Gln and Leu395Met, as well as two CRM positive factor XII deficiencies caused by amino acid substitutions of Asp442Asn and Gly570Arg. Kanaji et
al16 reported an abnormal factor XII in which Arg126 has
been replaced by Pro, and a common HgaI polymorphism (46 C/T)
in the 5'-untranslated region is associated with low
translational efficiency resulting in a decreased antigen level of
factor XII.17
We report here a Japanese family with CRM negative factor XII
deficiency, which we designated as factor XII Tenri. We demonstrated that the proband, who showed 3% antigen and activity levels of factor
XII, had an amino acid substitution of Tyr34Cys in the type
II domain, and a small amount of the patient's factor XII found in
plasma was a heterodimeric complex with 1-microglobulin. Moreover, pulse-chase analyses, using stably-transfected wild-type and
Tenri-type factor XII cDNAs in baby hamster kidney (BHK) cells, showed
that most of the synthesized mutant factor XII was degraded by a
proteasome through the quality control mechanism in the endoplasmic reticulum (ER).
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MATERIALS AND METHODS |
Subject.
A 29-year-old Japanese man was found to have a prolonged APTT (58.2 seconds) before a reconstruction operation of his nasal septum,
although he had no history of abnormal bleeding. Both his antigen and
activity levels of factor XII were determined as 3% of normal.
Hemostatic laboratory findings were as follows (% values represent
relative activity unless otherwise stated): prothrombin time, 11.3 seconds; fibrinogen, 2.2 mg/mL; factors XI, 105%; X, 100%; IX, 98%;
VIII, 82%; VII, 98%; and V, 72%; prothrombin, 85%; antithrombin,
97%; and 2-plasmin inhibitor, 98%. From the laboratory
investigation and the patient's pedigree (Fig 1), we diagnosed the
proband as CRM negative homozygous factor XII deficiency, which we
named factor XII Tenri.
Enzymes, antibodies, and reagents.
Restriction endonucleases and DNA modifying enzymes were purchased from
Takara Shuzo (Kyoto, Japan), Toyobo (Osaka, Japan), and New England
Biolabs (Beverly, MA). Thermo Sequenase cycle sequencing kit and
Sculptor in vitro mutagenesis system were the products of Amersham Life
Science (Tokyo, Japan). GeneAmp PCR reagent kit, AmpliWax PCR Gem 100, and AmpliTaq DNA polymerase were obtained from Takara Shuzo.
EXPRE35S35S (mixture of 72%
L-[35S]Met and 18%
L-[35S]Cys) was purchased from NEN-DuPont Japan
(Tokyo, Japan). Chloroquine and brefeldin A were obtained from Sigma
Chemicals (St Louis, MO). Leupeptin,
[L-3-trans-ethoxycarbonyloxiran-2-carbonyl]-L-leucine(3-methylbutyl)amide (E64d), carbobenzoxy-Leu-Leu-lucinal (LLL), and
carbobenzoxy-Leu-leucinal (LL) were the products of Peptide Institute
(Osaka, Japan). Lactacystin was a generous gift from Dr Satoshi Omura
(The Kitasato Institute, Tokyo, Japan). Rabbit and goat
antihuman factor XII antisera were purchased from Calbiochem (San
Diego, CA) and Nordic Immunology (Tilburg, The Netherlands),
respectively. Rabbit antihuman 1-microglobulin antibody
was obtained from Cosmobio (Tokyo, Japan). Oligonucleotides were
synthesized by an Applied Biosystems 394 DNA/RNA synthesizer (Applied
Biosystems, Foster City, CA). All other reagents were of
the highest quality commercially available.
Polymerase chain reaction (PCR).
Leukocytes from peripheral blood samples were prepared using Lymphoprep
(Daiichi Chemicals, Tokyo, Japan), and the leukocyte DNA was obtained
by proteinase K digestion and phenol extraction, followed by ethanol
precipitation. All of the exons and flanking regions for the factor XII
gene were amplified by a DNA Thermal Cycler (Perkin-Elmer Cetus
Instruments, Norwalk, CT). Primers used were designed as listed in
Table 1. To prevent a nonspecific annealing of primers to genomic DNA,
a solution containing 200 µmol/L deoxyribonucleoside 5'-triphosphates
(dNTPs) mixture and 100 pmol each of 5'- and
3'-primers was sealed by AmpliWax. Then, 10 µL of 10x PCR
buffer, 1 µg of leukocyte DNA, and 1 µL of AmpliTaq DNA
polymerase were overlaid. The hot-start PCR reaction was performed for
30 cycles as: denaturation at 94°C for 1 minute, annealing at
63°C for 1 minute, and polymerization at 72°C for 2 minutes.
Screening of mutation site and sequence analysis.
To detect the mutation site in the factor XII Tenri gene, low-ionic
strength single-stranded conformation polymorphism (LIS-SSCP) analysis
was performed.18 Briefly, 1 µL of PCR product was mixed with 20 µL of LIS solution (10% sucrose/0.01% Bromophenol
Blue/0.01% xylene cyanol). After heat denaturation at 97°C for 2 minutes, 4 to 10 µL of sample was applied to a 10% polyacrylamide
gel and electrophoresed in 45 mmol/L Tris/45 mmol/L boric
acid/1 mmol/L EDTA, pH 8.0 at 22°C. Single-strand DNA fragments
were detected by silver staining.
For sequence analyses, PCR fragments were purified by Centricon 100 (Amicon, Beverly, MA) and digested by restriction enzymes at the sites designated in 5'- and 3'-primers, and
subsequently ligated to pUC118. At least six independent plasmids were
sequenced using Thermo Sequenase cycle sequencing kit and a DSQ-1
automated DNA sequencer (Shimadzu, Kyoto, Japan).
Mutagenic PCR and KpnI digestion.
To confirm the assigned mutation site in factor XII Tenri gene, a
mutagenic primer, exon III-33M was designed as
5'-AGCCCTGCCACTTCCCCTTCCGGT-3' (mutagenic G is
underlined) to create a KpnI recognition site in the PCR
product from the normal factor XII gene. PCR amplification by primers
exon III-33M and exon III-32A (Table 1) was performed as described
above. The PCR products were purified by Centricon 100, digested with 6 U of KpnI at 37°C for 2 hours, and then electrophoresed in
a 3% agarose gel.
Western blotting.
To examine the secretion of Tenri-type factor XII in the proband's
plasma, immunoprecipitation followed by Western blotting was performed.
Briefly, 450 µL of plasma from either the Tenri family or normal
individuals was reacted with rabbit antihuman factor XII antiserum and
then with protein A-agarose (Sigma). The immunoprecipitates were
electrophoresed in an 8% sodium dodecyl sulfate (SDS)
gel,19 and transferred to a polyvinylidene difluoride membrane (Millipore, Japan, Tokyo). The membrane was blocked overnight with 1% bovine serum albumin in 25 mmol/L Tris-HCl, pH 7.4/0.15 mol/L
NaCl. Factor XII antigen was detected by incubating the membrane with
biotinylated anti-factor XII antibody followed by the avidin-peroxidase
detection system (Vector Laboratories, Burlingame, CA) as
described.20 To examine the heterodimer complex of factor XII with 1-microglobulin, the membrane was reacted with
biotinylated rabbit anti-1-microglobulin antibody and
detected by the same method as described above.
Site-directed mutagenesis and construction of the expression
vectors.
A full-length (1.6 kb) cDNA for human factor XII,4
generously provided by Dr Ross T.A. MacGillivray (University of British Columbia, Vancouver, British Columbia, Cananda), was ligated to the
XbaI site of pUC118. Single-strand DNA was prepared by the addition of helper phage M13K07 and kanamycin. To prepare the Tenri-type factor XII cDNA, site-directed mutagenesis was performed using a Sculptor in vitro mutagenesis kit and a mutagenic
oligonucleotide of 5'-TTCCAGTGCCACCGGCAGCT-3'
(mutagenic G is underlined) according to the manufacturer's
instructions. The mutation was confirmed by sequence analysis.
Wild-type and Tenri-type factor XII cDNAs in pUC118, thus prepared,
were transferred to XbaI site of the pcD2-SR expression vector21 and purified by CsCl gradient ultracentrifugation. BHK cells, supplied from Japan Cancer Research Resources Bank (Tokyo,
Japan), were maintained in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (Filtron, Brooklyn, Australia) and antibiotic-antimycotic liquid (GIBCO BRL Life
Technology, Grand Island, NY). A total of 7 µg of expression vector
was transfected into 2 × 105 cells by the calcium
phosphate coprecipitation method.21 To obtain stable
transfectants, cells were selected by the addition of 400 µg/mL G418
to the medium. A pool of stably-transfected BHK cells was used in
subsequent experiments.
Pulse-chase, immunoprecipitation, and gel electrophoresis.
To examine the secretion rate of Tenri-type factor XII, pulse-chase
experiments were performed as described previously.22,23 Briefly, stably-transfected BHK cells (5 × 105 cells)
were starved for 30 minutes with DMEM lacking Met and Cys (Sigma)/10%
dialyzed fetal bovine serum, and subsequently labeled with 100 µCi/mL
of EXPRE35S35S for 30 minutes. Cells were
chased with DMEM/10% fetal bovine serum/ 2 mmol/L Met, and cystine
(0.5 mmol/L) was added to the chase media in selected experiments.
After incubation for the indicated times, conditioned media were
harvested and cells were lysed by 1% NP-40/0.1% SDS
solution.22,23 The 35S-labeled factor XII was
immunoprecipitated using goat antihuman factor XII antiserum and
Staphylosorb (Mercian, Tokyo, Japan). After washing, immunoadsorbed
proteins were dissociated by heating at 85°C for 5 minutes, and
then electrophoresed. The radioactivities in dried SDS-gels were
measured quantitatively by a Fujix BAS2000 Bio-Imaging Analyzer system
(Fuji Photo Film, Tokyo, Japan).
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RESULTS |
Identification of the mutation site in factor XII Tenri gene.
Figure 1A shows a pedigree of the factor
XII Tenri family. Both factor XII antigen and activity levels in the
proband were quantitated as 3% of normal, suggesting homozygous CRM
negative factor XII deficiency as described in Materials and Methods.
The proband's parents, who are married consanguineously, in addition to his sister, also showed reduced antigen and activity levels (35%) of factor XII, suggesting heterozygous deficiency. The proband's brother showed 67% antigen and 68% activity levels, suggesting normality. None of the family members had a bleeding tendency or a history of thromboembolism. To elucidate the molecular basis of factor XII Tenri, we first amplified all 14 exons and flanking
regions of factor XII gene from the proband by PCR using the primers
listed in Table 1, then analyzed them
by the LIS-SSCP method.18 As shown in Fig 1B, a PCR
fragment derived from exon 3 of the proband exhibited a slower
migration than that of a normal subject. No aberrant migrations on
LIS-SSCP analysis were observed for the other PCR fragments (data not
shown). Sequence analysis of exon 3 from the proband showed a
substitution of A to G at nucleotide position 7832 (Fig 1C). All eight
sequenced clones had the identical mutation, suggesting homozygous
mutation at this site. This mutation induced an amino acid substitution
of Tyr34 (TAC) to Cys (TGC) in the type II domain
of factor XII. Sequence analysis of all other exons and flanking
regions indicated no mutations. An aberrant TaqI restriction
site in intron B, which is well-known as a cause for Hageman trait, was
not detected in factor XII Tenri gene (data not shown).
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| Fig 1.
Identification of the mutation site in factor XII Tenri.
(A) Pedigree of the factor XII Tenri family. Affected subjects with
reduced factor XII levels are shadowed and the proband is indicated by
an arrow. Deceased family members and unexplored subjects are indicated
by  / or  / and n.t., respectively.
Antigen/activity levels of factor XII are shown. (B) LIS-SSCP analysis
of exon 3 of factor XII gene. PCR fragments of exon 3 derived from a
normal subject (left) and the proband (right) were analyzed by LIS-SSCP
as described in Materials and Methods. (C) Nucleotide sequence showing
the A to G substitution in exon 3 of the factor XII Tenri gene. This
substitution mutates Tyr34 to Cys in the type II domain of factor XII.
(D) PCR-Kpn I digestion analysis of the factor XII Tenri
family. For each member, exon 3 amplified by PCR as described in
Materials and Methods was digested with KpnI and subsequently
analyzed by agarose gel electrophoresis. PCR product from normal allele
showed a cleaved fragment of 141 bp, whereas that from Tenri-type
allele showed an uncleaved band of 163 bp.
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To confirm the mutation site in the genes from the members of the
factor XII Tenri family, we performed mutagenic PCR followed by
KpnI digestion analyses (Fig 1D). The mutagenic primer was designed
to change the codon (CAG) for Gln33 to CGG to
create a KpnI site (GGTACC) in the normal allele, but
not in the Tenri-type allele. After a 2-hour digestion by KpnI,
PCR products derived from the younger brother and normal individuals
showed a cleaved fragment of 141 bp, while the PCR fragment from the
proband was uncleaved and remained as a 163-bp fragment. PCR products
from the parents and sister of the proband showed two fragments of 163 bp and 141 bp, suggesting heterozygosity. These results were consistent
with the antigen and activity levels of factor XII in the Tenri family.
A portion of the secreted Tenri-type factor XII is complexed with
1-microglobulin.
The proband of factor XII Tenri showed low, but detectable (3%),
antigen and activity levels of factor XII in plasma. This suggests that
a small amount of variant factor XII having Cys34 is secreted into the
blood stream. To demonstrate Tenri-type factor XII in plasma, we
performed immunoprecipitation followed by Western blotting using
anti-factor XII antibody. Under nonreducing conditions, immunoblots of
factor XII derived from the proband's plasma showed a 115-kD band, in
addition to a faint 80-kD band (Fig 2A,
lane 1). In contrast, plasma samples from his parents, sister, and brother exhibited a major 80-kD band that was the same size as normal
factor XII (Fig 2A, lanes 2 through 5). To elucidate the presence of a
115-kD variant factor XII among family members, overexposure of the
membrane was performed (Fig 2B), which showed that immunoblots of
parents and sister, ie, heterozygous deficients, also had a very faint
115-kD band. This suggested that the 115-kD band was derived from the
factor XII Tenri allele. After reduction of the SDS sample with
2-mercaptoethanol, all family members showed only a single band of
80-kD (Fig 2C), indicating that the variant factor XII was complexed
with a 35-kD protein through a mixed disulfide linkage. We suspected
the protein to be 1-microglobulin because
1-microglobulin has a molecular mass of 33 kD and is known to form a disulfide bond to some abnormal coagulation factors with a free Cys residue introduced into the molecule.24,25 To confirm this possibility, the same preparation in lane 1 of Fig 2A
was blotted using anti-1-microglobulin antibody. Under these conditions, the 115-kD band was detected by this antibody (Fig 2A, lane 6). Furthermore, we could not detect a 115-kD
band by anti-1-microglobulin antibody in normal plasma.
Factor XII dimer (160 kD) or albumin-bound form (145 kD) of the
factor XII Tenri was not observed in the proband's plasma (data not
shown).
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| Fig 2.
Western blot analysis of the factor XII Tenri family.
Factor XII in plasmas of the proband (lane 1), his father (lane 2),
mother (lane 3), sister (lane 4), and brother (lane 5) were
immunoprecipitated by anti-factor XII antibody. After heat
dissociation, immunoprecipitates were electrophoresed on 8%
SDS-polyacrylamide gels under nonreducing (A and B) or reducing (C)
conditions. Electrophoresed proteins were transferred to polyvinylidene
difluoride membranes and then blotted by the biotinylated anti-factor
XII antibody followed by avidin-peroxidase. (B) is the overexposure of
the high molecular mass region of (A). The sample in lane 6 in (A) was
the same as in lane 1, but was blotted by the biotinylated
anti-1-microglobulin antibody.
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Cellular basis for the secretion defect of factor XII Tenri.
To elucidate the molecular basis whereby substitution of
Tyr34Cys causes a reduction of factor XII antigen level in
plasma, we performed pulse-chase experiments of the Tenri-type factor XII using stably-transfected BHK cells and compared the results with
that of wild-type factor XII. Nascent wild-type and Tenri-type factor
XIIs were detected as a single band of 76 kD in the cell extracts
(Fig 3A). The intracellular Tenri-type
factor XII was sensitive to endoglycosidase H, suggesting the presence
of high mannose-type oligosaccharides attached to the polypeptide in
the pre-Golgi compartment (data not shown). By kinetic analysis of the
wild-type factor XII, almost all of the pulse-labeled radioactivity was
recovered from the media after a 4-hour chase, and the total amount of
radioactivity was maintained during the chase period (Fig 3B, left
panel). In contrast, Tenri-type mutant showed impaired secretion and a
significant reduction (50%) in the total amount of radioactivity
was observed during an 8-hour chase, suggesting intracellular
degradation of Tenri-type factor XII (Fig 3B, right panel).
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| Fig 3.
Pulse-chase analyses of wild-type and Tenri-type
factor XIIs in stably-transfected BHK cells. (A) Stably-transfected BHK
cells were pulse-labeled for 30 minutes with 100 µCi/mL
EXPRE35S35S and chased for 0, 1, 2, 4, 6, and 8 hours. Labeled factor XIIs from cell extracts and from culture media
were immunoprecipitated and analyzed on 8% SDS-polyacrylamide gel
electrophoresis. (B) In kinetic analyses, the amount of radioactivity
in the pulse-labeled cell extracts was taken as 100%, and relative
radioactivities of intracellular and secreted fractions are shown by
and  , respectively. The sum of the radioactivities of both
fractions at each time is shown by  . (C) BHK cells expressing
Tenri-type factor XII were pulse-labeled for 30 minutes with 100 µCi/mL EXPRE35S35S (lane 1) and chased
in DMEM for 8 hours in the absence (lane 2) or presence (lane 3) of
excess amounts of cystine. Factor XII was immunoprecipitated from the
pulse-labeled cell extracts (lane 1) and chased media (lanes 2 and 3),
and then electrophoresed on an 8% SDS-polyacrylamide gel.
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As shown in Fig 2, we found that an
1-microglobulin-bound form of factor XII Tenri was
secreted in the proband's plasma. Although BHK cells do not synthesize
1-microglobulin constitutively, we speculated that a
mixed-disulfide formation through the free sulfhydryl group of Cys34 in
Tenri-type factor XII might enhance the secretion. To test this
hypothesis, pulse-chase experiments were performed in the absence or
presence of excess amounts of cystine (Fig 3C). Compared with its
secretion in the absence of cystine (lane 2), a larger amount of the
mutant was secreted in the presence of 0.5 mmol/L cystine (lane 3).
Thus, we conclude that the addition of excess amounts of cystine,
presumably resulting in the mixed-disulfide formation, resulted in the
increased secretion of Tenri-type factor XII from BHK cells.
Proteasome-mediated degradation of factor XII Tenri.
To localize the site and identify the protease(s) responsible for the
intracellular degradation of factor XII Tenri, we next examined the
effects of various inhibitory reagents on the degradation of Tenri-type
factor XII expressed in stably-transfected BHK cells (Fig 4). Taking the amount of radioactivity
of factor XII in the cell extracts immediately after pulse-labeling as
100%, 97% of wild-type factor XII was detected in the
16-hour-chased media (Fig 4, left panel, lane 2 v 1). In
contrast, >85% of the Tenri-type factor XII disappeared during the
16-hour chase (Fig 4, right panel, lane 2 v 1). In
the presence of 5 µg/mL brefeldin A, which blocks intracellular
transport by redistribution of Golgi proteins into the
ER,26 wild-type factor XII accumulated in the cells and
60% of the radioactivity was recovered from the cell extracts after
a 16-hour chase (Fig 4, left panel, lane 3). However, in the case of
Tenri-type factor XII, 76% of the total radioactivity disappeared
during a 16-hour chase in the presence of brefeldin A, suggesting that
the Tenri-type factor XII was selectively degraded in a pre-Golgi
compartment (Fig 4, right panel, lane 3). In the presence of brefeldin
A, the intracellular Tenri-type factor XII exhibited a slower
migration, which may be due to the processing of carbohydrate chains by
the incorporated Golgi enzymes as reported.27 Among the
lysosomotropic agents, 100 µmol/L chloroquine (Fig 4, lane 4), 30 mmol/L NH4Cl (data not shown), and 100 µmol/L leupeptin (lane 5), a lysosomal protease inhibitor of microbial
origin,28 had no effect on the degradation of Tenri-type
factor XII. These results suggest that the degradation of Tenri-type
factor XII occurs in a nonlysosomal compartment. In the presence of
chloroquine, secreted wild-type factor XII showed faster migration than
others (Fig 4, left panel, lane 4). This is probably due to prevention of terminal saialic acid transfer as described
previously.29 Among the membrane-permeable protease
inhibitors we examined, 100 µmol/L E64d (Fig 4, lane 6) and 20 µmol/L LL (Fig 4, lane 7), which are inhibitors for intracellular
cysteine protease calpain,30 showed no inhibitory effects
on the degradation of Tenri-type factor XII or secretion of wild-type
factor XII. However, 20 µmol/L LLL (Fig 4, lane 8) and 20 µmol/L
lactacystin (Fig 4, lane 9), two chemically different potent proteasome
inhibitors,31,32 strongly inhibited the degradation of
Tenri-type factor XII. As a result, Tenri-type factor XII was retained
in the cells, while these proteasome inhibitors essentially showed no
effect on the secretion of wild-type factor XII (Fig 4, left panel,
lanes 8 and 9). Other peptidyl-aldehyde inhibitors of proteasome such as
carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal
and
carbobenzoxy-L-isoleucyl- -t-butyl-L-glutamyl-L-alanyl-L-leucinal showed similar inhibitory effects as LLL and lactacystin on the intracellular degradation of Tenri-type factor XII (data not shown). These results suggest that a proteasome plays a role in the
intracellular degradation of the Tenri-type factor XII.
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| Fig 4.
Effects of various inhibitors on the secretion and
degradation of wild-type and Tenri-type factor XIIs. Stably transfected
BHK cells expressing wild-type (left panel) or Tenri-type (right panel)
factor XII were pulse-labeled for 30 minutes with 100 µCi/mL
EXPRE35S35S and then chased for 16 hours in the
presence of various inhibitors. Factor XII in the cell extracts and
culture media were subjected to immunoprecipitation followed by
SDS-PAGE analysis. Lane 1, sample from pulse-labeled cells; lane 2, sample from 16-hour chased cells without inhibitors; lanes 3 through 9, samples from 16-hour chased cells in the presence of inhibitors; lane
3, 5 µg/mL brefeldin A; lane 4, 100 µmol/L chloroquine; lane 5, 100 µg/mL leupeptin; lane 6, 100 µmol/L E64d; lane 7, 20 µmol/L LL;
lane 8, 20 µmol/L LLL; and lane 9, 20 µmol/L lactacystin. Positions
of factor XII are indicated by arrows.
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DISCUSSION |
In this study, we show that a novel type of missense mutation (exon 3, 7832AG), leading to the substitution of Tyr34 to Cys in the
NH2-terminal type II domain of the factor XII molecule, is
the genetic basis for a CRM negative factor XII deficiency (Fig 1).
Hereditary factor XII deficients in the factor XII Tenri family are
clinically asymptomatic, but were found by chance due to the
prolongation of the APTT by laboratory tests before
surgery.8,9 CRM negative factor XII deficiency is mainly
associated with an additional TaqI restriction site in intron
B, which is known as the Hageman trait.11,12 Recently,
Schloesser et al10 identified a mutation that was induced
by the presence of a TC transition 224 bp upstream of exon 3 and shown to be associated with a mutation in the 5'-flanking
region of the factor XII gene (exon 1, -8GC). Two missense
mutations resulting in CRM negative factor XII deficiency were also
reported by Schloesser et al15; ie, Leu395Met and Arg398Gln in exon 10, but the mechanism whereby the mutations lead to factor XII deficiency is not known. Thus, to our knowledge, our
data is the first report on the cellular analysis of a CRM negative
factor XII deficiency induced by an amino acid substitution.
A small amount (3%) of mutant factor XII was secreted into the
proband's plasma. We showed by immunobloting that the majority of the
secreted Tenri-type factor XII was complexed with
1-microglobulin (Fig 2). 1-Microglobulin,
a lipocalin superfamily protein, is known to be synthesized in the
liver as an NH2-terminal portion of the light chain
precursor of inter--trypsin inhibitor.33,34 A
furin-like protease is responsible for the cleavage of the dibasic amino acids in the central portion, resulting in the production of two
functionally different proteins.34 Human
1-microglobulin is a glycoprotein with an apparent
molecular mass of 33 kD and is detected in various body fluids as a
monomer or as a complexed form with IgA. The protein has three Cys
residues, two of which are involved in the formation of an
intramolecular disulfide bridge, while the other is covalently bound to
IgA.33 Previously, Wojcik et al24 reported that
a mutant factor IX, factor IX Zutphen, had a Cys18Arg
mutation resulting in the disruption of Cys18-Cys23 linkage. This
mutant factor IX, which exhibited 65% antigen and <4% activity
levels, was identified as a heterodimer with
1-microglobulin in patient's plasma. In a subsequent
report,25 these investigators extensively examined the
intermolecular complex between 1-microglobulin and
mutants of protein C and prothrombin. The protein C mutants with
Arg-1Cys, Arg9Cys, or Ser12Cys were shown
to be present in plasma as a complex with
1-microglobulin, whereas a prothrombin mutant with
Tyr44Cys was not. They concluded that unpaired Cys residues
in the propeptide or in the NH2-terminal half of the Gla
domain of vitamin K-dependent proteins formed a complex with 1-microglobulin.25 In the present report, we
show that not only vitamin K-dependent proteins, but the
Tyr34Cys mutation in type II domain of factor XII, also
caused complex formation with 1-microglobulin. In
addition to 1-microglobulin binding, a variety of
modifications to the newly-introduced Cys are reported in clotting
factors. For example, albumin was bound to fibrinogen Milano VII ( chain Ser358Cys)35 and fibrinogen Fukuoka II (B chain Gly15Cys)36, and free Cys was
attached in cases of fibrinogen Osaka II ( chain
Arg275Cys)37 and antithrombin Toyama
(Arg47Cys)38,39. It is interesting to note that a
CRM positive factor XII deficiency, factor XII Washington D.C., has an
amino acid substitution of Cys571Ser, which resulted in a generation of free Cys540.13 Unlike Tenri-type factor XII,
however, factor XII Washington D.C. was efficiently secreted into the
blood stream.
In pulse-chase experiments, Tenri-type factor XII was poorly secreted
from stably-transfected BHK cells and the total amounts of
radioactivity decreased to 50% during an 8-hour chase (Figs 3A and
B) and to 15% during a 16-hour chase (Fig 4). Thus, an extensive intracellular degradation of Tenri-type factor XII was suggested by these experiments. Although endogenous
1-microglobulin expression was negligible in BHK cells,
the addition of cystine, which presumably enhanced a mixed
disulfide-linkage formation of 1-microglobulin with
Tenri-type factor XII, improved secretion of the mutant, suggesting
resistance to intracellular degradation as a result of blocking the
free sulfhydryl group (Fig 3C). The degradation occurred in pre-Golgi,
nonlysosomal compartments, and a proteasome appeared to play a critical
role in this degradation (Fig 4). This type of degradation is referred
to as "ER-associated degradation" or "quality control in the
ER" (see reviews in Kopito40 and Kim and
Arvan41). Currently, various nascent proteins that fail to
fold or to oligomerize correctly are known to be degraded through this
mechanism. We have shown that protein C synthesized in the presence of
warfarin22 and type I (secretion defect) antithrombin
deficiency mutants42 were degraded by the quality control
in the ER. Tenri-type factor XII presumably folded incorrectly due to
the introduction of Cys34 and was retained in the ER associating with
molecular chaperones, eg, calnexin, calreticulin, and Ig binding
protein (BiP).43 Finally, misfolded
Tenri-type factor XII was degraded by a proteasome presumably after the
retrograde transport from the ER to the cytosol through a
translocon.40,41 It should be noted that a trace amount of
1-microglobulin-bound form of factor XII Tenri escaped
the quality control mechanism and was secreted into the blood stream.
The difference in the association of ER chaperones with Tenri-type
factor XII, with or without binding to 1-microglobulin,
remains to be investigated.
| |
ACKNOWLEDGMENT |
We thank Drs Satoshi Omura and Ross T.A. MacGillivray for supplying us
with lactacystin and the factor XII cDNA, respectively. We also thank
Dr Sadao Wakabayashi and Kazuya Hara of our department for helpful
discussion and technical assistance, respectively.
| |
FOOTNOTES |
Submitted November 4, 1998; accepted February 18, 1999.
Supported by a Grant-in-Aid for Scientific Research on Priority Areas
(Intracellular Proteolysis) from the Ministry of Education, Science,
and Culture of Japan (to T. K.).
S.K. and F.T. contributed equally to this study.
Address reprint request to Takehiko Koide, DSc, Department of Life
Science, Faculty of Science, Himeji Institute of Technology, Harima
Science Garden City, Hyogo 678-1297, Japan; e-mail: koide{at}sci.himeji-tech.ac.jp.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
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ABSTRACT
INTRODUCTION