Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 128-133
HRG Tokushima: Molecular and Cellular Characterization of
Histidine-Rich Glycoprotein (HRG) Deficiency
By
Toshio Shigekiyo,
Hidemasa Yoshida,
Kazuya Matsumoto,
Hiroyuki Azuma,
Sadao Wakabayashi,
Shiro Saito,
Kazuo Fujikawa, and
Takehiko Koide
From the First Department of Internal Medicine, School of Medicine,
The University of Tokushima, Tokushima; Department of Life Science,
Faculty of Science, Himeji Institute of Technology, Hyogo, Japan; and
Department of Biochemistry, University of Washington, Seattle, WA.
 |
ABSTRACT |
Previously, we found the first congenital deficiency of
histidine-rich glycoprotein (HRG) in a Japanese woman with thrombosis.
To elucidate the genetic basis of this deficiency, we first performed
Southern blot analysis and found no gross deletion or insertion in the
proband's HRG gene. We then examined the nucleotide sequences of all
seven exons of the proband's HRG gene. A single nucleotide
substitution, G to A at nucleotide position 429, which mutates Gly85 to
Glu in the first cystatin-like domain, was found in exon 3 in 13 of 22
amplified clones. This mutation generates a unique Taq I site.
Exon 3 was amplified from the proband, her family members, and 50
unrelated normal Japanese individuals, and Taq I fragmentation
was examined. Fragmentation of exon 3 was observed in one allele of the
genes from the proband and the family members who also have decreased
plasma levels of HRG. Fifty unrelated normal Japanese individuals had a
normal HRG gene, indicating that the G to A mutation is not a common
polymorphism. To elucidate the identified mutation as a
cause for the secretion defect of HRG in the proband's
plasma, we constructed and transiently expressed the recombinant
Tokushima-type HRG mutant (Gly85 to Glu) in baby hamster kidney (BHK)
cells, and examined an intracellular event of the mutant protein. The
results showed that only about 20% of the Tokushima-type HRG was
secreted into the culture medium, and intracellular degradation of the
mutant was observed. Thus, the present study strongly suggests that the
HRG deficiency is caused by intracellular degradation of the Gly85 to
Glu mutant of HRG in the proband.
| |
INTRODUCTION |
HUMAN HISTIDINE-RICH glycoprotein (HRG)
is a single-chain glycoprotein (Mr 67,000) composed
of 507 amino acid residues that circulates in blood at a relatively
high concentration (~100 mg/L).1-3 The HRG gene spans
approximately 11 kb on chromosome 3q28-q29 and consists of seven exons
and six introns.4,5 Platelets store HRG in the 
-granules
and secrete HRG upon thrombin activation.3 HRG
has an unusually high content of proline and histidine residues in the
carboxy-terminal region.2 By structural similarity, it
belongs to the cystatin superfamily, eg, cystatin SN, SA, and C and
high-molecular weight kininogen.4 The histidine-rich
region of HRG is homologous to the corresponding area of high molecular
weight kininogen, a modulator in the contact activation system of blood
coagulation.2
HRG has been shown to interact with plasma proteins involved in blood
coagulation and fibrinolysis such as fibrinogen,6 factor
XIIIa,7 plasminogen,8 and heparin.9
It also interacts with several other blood components:
heme,10 metal ions,10 activated
platelets,11 thrombospondin,12 T
lymphocytes,13 and several complement
factors.14 HRG diminishes heparin activity in plasma;
removal of HRG from the plasma increased the heparin effect on thrombin
inhibition by antithrombin9 and activated protein C
inhibition by protein C inhibitor.15 However, HRG is
reported not to interfere with interactions between antithrombin and
heparan-sulfate on vascular endothelial cells.16 Although
HRG interacts with plasminogen, it probably has no function in the
fibrinolytic system, because a high or low concentration of HRG did not
affect plasminogen activation in the plasma milieu.17 Thus,
the physiologic significance of these molecular interactions of HRG
remains unclear.18
In 1993, we discovered the first case of familial deficiency of HRG in
a 43-year-old Japanese woman with cerebral sinus
thrombosis.19 Laboratory tests for platelet count and
procoagulant activity were normal. The concentrations of various
coagulation factors and fibrinolytic agents were also in the normal
range in the patient's plasma, except for HRG. The patient had only
21% of the normal level of HRG, and the affected family members also
had low levels of HRG. Recently, Souto et al20 have
discovered a second family with this deficiency. The patient developed
pulmonary embolism on two occasions at age 36. Her father had
thrombosis in the central artery of the retina at age 59. These two
family cases suggest that HRG plays an antithrombotic role to prevent
thrombotic disorders.
To elucidate the molecular and cellular mechanism for the first case of
congenital HRG deficiency, which we refer to as "HRG Tokushima,"
we identified a single Gly to Glu mutation in the proband's HRG gene
and examined the secretion of the recombinant Tokushima-type HRG mutant
using transiently transfected baby hamster kidney (BHK) cells and
compared it against the wild-type HRG.
| |
MATERIALS AND METHODS |
Subject.
Clinical and laboratory data relevant to the congenital HRG deficiency
in this report have been previously presented.19 In brief,
the proband was a 43-year-old Japanese woman with right transverse
sinus thrombosis while taking contraceptive medication. Her plasma HRG
level was only 21% of the normal level of 109.5% ± 51.5%
(mean ± 2 SD). The HRG concentration in her plasma determined on
four occasions over 6 months was consistently low. She showed no
clinical signs of liver function abnormality or sepsis. Low levels of
plasma HRG were also found in her aunt, uncle, and two daughters. These
results suggest that congenital HRG deficiency is inherited in this
family.
Enzymes and reagents.
All restriction enzymes and the Wizard Plus Minipreps DNA Purification
System were purchased from Promega (Madison, WI). T7 Taq DNA polymerase
was obtained from Boehringer Mannheim (Indianapolis, IN). The Ultrapure
dNTP set and Sequenase Version 2 Deaza-dCTP kit were purchased from US
Biomedical (Cleveland, OH). X-gal and agarose were obtained from Life
Technologies Inc (Gaithersburg, MD). The GeneClean II kit was obtained
from Bio 101 (La Jolla, CA). The TA cloning kit including the pCR 2.1
vector and INVF
Escherichia coli cells was purchased from
Invitrogen (San Diego, CA). The Multiprime DNA-labeling kit,
deoxycytidine 5-[-32P]triphosphate
([-32P]dCTP), deoxyadenosine
5-[-35S]triphosphate ([-35S]dATP),
and Hybond-N transfer membrane were obtained from Amersham Life Science
(Arlington Heights, IL). ZMB3 expression vector was kindly provided by
Dr Don Foster (ZymoGenetics, Seattle, WA).
DNA isolation and Southern blot analysis.
Blood was drawn from the patient or donors after obtaining informed
consent according to the Declaration of Helsinki. Genomic DNA was
isolated from leukocytes by a standard technique.21 A
plasmid containing the full-length cDNA for human HRG, 
HHRG3,2 was digested with EcoRI. Approximately
2.0-kb EcoRI fragments were isolated using the GeneClean II kit
and radiolabeled with [-32P]dCTP. Ten micrograms of
genomic DNA was digested with either EcoRI, HincII,
Pst I, or Xba I, and the fragments were isolated by
electrophoresis on a 0.7% agarose gel. The DNA fragments were then
transferred to a Hybond-N membrane and hybridized with the
32P-labeled 2.0-kb EcoRI fragments according to a
standard procedure. The membrane was exposed to Kodak X-Omat AR film
(Eastman Kodak, Rochester, NY) at 
80°C with an intensifying
screen.
Polymerase chain reaction and DNA sequence analysis.
Oligonucleotide primers were synthesized based on the intron sequences
approximately 50 bp apart from the intron/exon boundaries of the HRG
gene (Wakabayashi S, Koide T, submitted) using the 380B
DNA Synthesizer (Applied Biosystems, Foster City, CA). Exon 7 was
divided into five segments (7-1 to 7-5) separately amplified using the
primers synthesized similarly as before. The sequences of all primers
used for polymerase chain reaction (PCR) are listed in Table
1. For TaqI fragmentation of exon
3, pair 12 primers were used for amplification. The PCR mixtures
contained 1X PCR buffer (10 mmol/L Tris hydrochloride, pH 8.4, 50
mmol/L KCl, 1.5 mmol/L MgCl2, and 0.01% gelatin), 1 µg
genomic DNA, and 2 U Taq polymerase. Amplification was
performed as follows: denaturation at 94°C for 1 minute, annealing at
55°C for 1 minute, and extension reaction at 72°C for 1.5 minutes
for 35 cycles. The amplified DNA fragments were directly subcloned into
pCR 2.1 vector supplied with the TA Cloning System kit. The DNA
sequence was determined by the dideoxy chain-termination method using
T7 DNA polymerase and [-35S]dATP.
Construction of expression vectors.
A region of nucleotides 118 to 2067 of human HRG cDNA
(HHRG3)2 was used for the expression experiment. Nucleotide
118 is just four bases upstream from the initiation codon ATG, and this
cDNA was subcloned in pUC19 plasmid. A BamHI site was
introduced into this DNA at the 3 end by PCR using a primer,
5-TTGGATCCCTCTTCTCAGGC-3, complementary to nucleotides 1843 to 1862
in which two bases were replaced. The amplified fragment was digested
with EcoRI and BamHI and inserted into ZMB3 expression
vector.22 To avoid incorporation of an unexpected mutation,
the EcoRI/Bal I fragment (nucleotides 118 to 1411) was
replaced by that of the original clone. The 3 region from the
Bal I site to the BamHI site was sequenced to verify
the absence of mutation. The cDNA carrying the mutation found in the
proband was amplified with the above-mentioned primer and the mutated
primer, 5-GTGATCGAACAATGTAAGGT-3. This PCR product was used as one of
the primers for successive second-round PCR. The other was a universal
40 primer. The whole cDNA fragment carrying the mutation thus
amplified was digested with EcoRI and HincII, and
subcloned into pUC19 plasmid to verify incorporation of the mutation
without any other unexpected replacements. The whole cDNA was then
constructed on pUC19 plasmid, released by EcoRI and
BamHI, and ligated into ZMB3 vector.
Cell culture and pulse-chase experiments.
To examine secretion of the Tokushima-type HRG mutant, pulse-chase
experiments were performed using transiently transfected BHK cells as
described previously.23 Briefly, about 5 ×
104 cells in 35-mm dishes were transfected with an
expression plasmid (7.5 µg) by the calcium phosphate method. After
transfection, cells were cultured under a 3% CO2
atmosphere for 16 hours, transferred to 5% CO2, and
further cultured for 24 hours. Then, the cells were incubated in the
medium without Met and Cys for 30 minutes, labeled with 35
µCi EXPRE35S35S (NEN-DuPont, Tokyo, Japan)
per dish for 1 hour, washed with phosphate-buffered saline, and chased
with Dulbecco's modified Eagle's medium/10% fetal calf serum
containing 2 mmol/L Met. At selected time intervals, the
medium was harvested and cells were lysed. The labeled HRG was
immunoprecipitated using affinity-purified rabbit anti-human HRG IgG
and Staphylosorb (Mercian, Tokyo, Japan). After washing,
immunoadsorbed proteins were dissociated by heating at 85°C for 5
minutes, and then electrophoresed on 8% polyacrylamide gel in the
presence of sodium dodecyl sulfate (SDS) and
-mercaptoethanol.24 After fixing the gels, radioactivity
on dried gels was measured quantitatively by the Fujix BAS2000
Bio-Imaging Analyzer system (Fuji Photo Film, Tokyo, Japan).
Autoradiographs were made by exposure to Kodak XAR films at 80°C.
| |
RESULTS |
Southern blot analysis.
To investigate if any gross structural alternation is present in the
HRG gene of the proband, the genomic DNA digested with EcoRI,
HincII, Pst I, or Xba I was subjected to
Southern blot analysis as described earlier. The digestion pattern of
the proband's DNA was indistinguishable from that of normal
individuals (data not shown), indicating that neither gross deletion
nor gross insertion is present in the proband's HRG gene.
DNA sequencing.
The results of Southern blot analysis indicated that a small structural
alternation or a point mutation(s) occurs in the proband's HRG gene.
We then examined the nucleotide sequences of all seven exons of the
proband's DNA. Each exon was amplified by PCR, subcloned into pCR2.1
vector, and transfected into INVINVF cells. At least 10
independent clones were isolated for each exon and sequenced. One
single-base substitution was found in exon 3, and no substitution was
found in the other exons. The amino acid residues of five polymorphic
sites shown by Hennis et al25,26 were identified as Ile162,
Pro186, His322, Arg430, and Asn475, all of which agree with those
originally reported by Koide et al,2 and no polymorphisms
were found in the proband's HRG gene. A single G to A substitution at
nucleotide position 429, which mutates Gly85 to Glu in the first
cystatin-like domain, was found in 13 of 22 different clones analyzed,
and nine clones contained the normal sequence (Fig
1). These results indicate that the proband
carries a heterozygous deficiency.
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| Fig 1.
Nucleotide sequence showing the G to A substitution in
exon 3 of the HRG gene. G to A substitution at nucleotide position 429
mutates Gly85 to Glu in the first cystatin-like domain.
|
|
Taq I digestion of exon 3.
The G429 to A mutation creates a unique Taq I restriction site
(TCGA) in exon 3. Taq I digestion facilitates detection of the
G to A mutation in exon 3. We studied Taq I cleavage of exon 3
of the proband (Fig 2A, IV-3), her family
members, and 50 normal Japanese individuals. The normal exon 3 is
composed of 283 bp, whereas the G to A-mutated exon 3 yields two
fragments, 162 and 121 bp, after digestion with Taq I (Fig 2B).
As predicted, the proband's gene exhibited a composite digestion
pattern indicating that the mutation occurs in one allele of the HRG
gene (Fig 2C). To confirm the genetic basis for the phenotype observed
in this family, we studied Taq I digestion of exon 3 amplified
from other available family members. The aunt (III-21), uncle (III-24),
and daughter (V-1) have been found to have approximately half the
normal plasma level of HRG. These individuals had the same Taq
I digestion pattern of exon 3 as observed for the proband (Fig 2C). A
younger sister (IV-4) and another aunt (III-22) had a normal plasma
level of HRG, and Taq I did not cleave exon 3 amplified from
these individuals. A third uncle (III-6), a parent's cousin (III-13),
and two of the proband's cousins (IV-5 and IV-7) with normal levels of
HRG also had normal exon 3 (data not shown). These results clearly show
that the G429 to A substitution found in exon 3 of the proband's gene
is inherited in this family. We then studied the Taq I
digestion with 50 unrelated healthy Japanese individuals and did not
find any mutations in exon 3 in the population. This study eliminates
the possibility that this mutation is a common polymorphism.
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| Fig 2.
(A) Pedigree of the family with congenital HRG
deficiency.19 Arrow, the proband; £ {, affected
subjects with reduced HRG levels;  , subjects with normal HRG
levels;   , deceased family members;  ,
unexplored subjects. (B) PCR-Taq I digestion analysis of normal
and mutated exon 3. Normal exon 3 is composed of 283 bp and is not
cleaved by Taq I. Mutated exon 3 with 1 Taq I
restriction site (TCGA) is cleaved by the enzyme to form 2 fragments,
162 bp and 121 bp. (C) For each family member, exon 3 amplified by PCR
was digested with Taq I and subsequently analyzed by
electrophoresis on a 2% agarose gel. Lane 1, molecular weight
standard; lanes 2 to 7, Taq I digests of exon 3 derived from
the proband and 5 of her family members.
{/CAPT;;;left;stack}
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|
Secretion of wild-type and Tokushima-type HRG in transiently
transfected BHK cells.
To elucidate if the mutation in HRG Tokushima is responsible for the
secretion defect of the mutant protein in the proband, we performed
pulse-chase experiments of Tokushima-type HRG using transiently
transfected BHK cells, and compared the results with those obtained for
wild-type HRG. Both wild-type and Tokushima-type HRG were detected as
a band of 62 kD (Fig 3, left arrowhead) and
72 kD (right arrowhead) in the cell extracts and culture medium,
respectively, and they were synthesized at nearly equal levels in BHK
cells as detected in cell extracts at time 0 of the chase. During the
chase period, wild-type HRG was secreted into the culture medium, and
essentially no HRG band was detected in the 2-hour chased cell extracts
(Fig 3A). However, the total amount of radioactivity was maintained
during the chase period. On the other hand, only a small amount (20%)
of Tokushima-type HRG was secreted into the culture medium even after
an 8-hour chase, and a decrease in total radioactivity was observed,
suggesting intracellular degradation of the mutant HRG (Fig 3B). The
time to 50% disappearance of total radioactivity from the cell
extracts was estimated to be 1 hour.
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| Fig 3.
Pulse-chase analysis of recombinant wild-type (A) and
Tokushima-type (B) HRG expressed in BHK cells. Transiently transfected
BHK cells were pulse-labeled for 1 hour and chased for 0, 0.5, 1, 2, 4,
and 8 hours. Labeled HRG in cell extracts and culture medium was
immunoprecipitated and analyzed on SDS-PAGE. Left arrowheads indicate
HRG bands within cells, and right arrowheads indicate HRG bands
secreted into culture medium.
|
|
| |
DISCUSSION |
In this study, we showed that a single missense mutation (nucleotide
G429 to A in exon 3) leading to a mutation of Gly85 to Glu in the first
cystatin-like domain on the HRG molecule is a genetic cause for the
first case of congenital HRG deficiency. This is the first report on
the molecular and cellular basis of congenital HRG deficiency.
Hennis et al25,26 discovered five amino acid polymorphisms
in HRG in Dutch families. These mutations occur in both alleles: Ile162
to Thr, Pro186 to Ser, His322 to Arg, Arg430 to Cys, and Asn475 to Ile.
The Pro to Ser mutation changes both the mature HRG mass and the plasma
HRG concentration. The mutated Ser generates one additional
carbohydrate chain in HRG at a newly created carbohydrate-attaching
motif, resulting in a higher-molecular mass HRG (77 kD) than the
normal protein (75 kD on SDS-polyacrylamide gel electrophoresis
[PAGE]). The mean plasma HRG level was highest (156%) in individuals
homozygous for Ser and lowest (93%) in homozygotes for Pro, whereas it
was intermediate (121%) in heterozygotes. Hennis et al also reported
that a dinucleotide polymorphism is located in the intron between the
last two exons of the HRG gene5 and is associated with a
higher level of plasma HRG in a Dutch family.27
To elucidate if the Gly85 to Glu mutation identified in HRG Tokushima
is a cause of congenital I HRG deficiency, we expressed the mutant HRG
in transiently transfected BHK cells and examined the secretion rate
and intracellular events. The results showed that only about 20% of
total Tokushima-type HRG was secreted into the culture medium and most
of the molecules (80%) were degraded within the cells, while
essentially all of the wild-type HRG was secreted within a 2-hour
chase. Thus, the low plasma levels of HRG observed in the proband and
the affected family members are most likely the result of intracellular
degradation of the mutant HRG.
Gly85 and the surrounding sequence in HRG are strictly conserved among
species, including human,2 bovine,28
rabbit,29 and rat 1 and 2 (Wakabayashi S, et al, manuscript
in preparation; Fig 4). This may suggest that Gly85 is indispensable
for correct folding of HRG. In general, glycine is known to be one of
the conservative amino acids and is
important to support the correct conformation of
proteins.30 The glycine located in the turning points of
the backbone of polypeptide chains is irreplaceable by any other amino
acids. Therefore, it is conceivable that the Gly85 to Glu mutation
causes a misfolding of HRG, leading to the degradation. Intracellular
degradation of genetic mutants of plasma proteins causing a type I
deficiency has been demonstrated for 1-antitrypsin
Z-variant31,32 and null-variants33 and the
antithrombin 
Glu (deletion of Glu313) mutant.34 The
sites for intracellular degradation of such misfolded proteins have
been shown to be the endoplasmic reticulum to cis-Golgi
compartment35-37 or the cytosol through the
ubiquitin-proteasome pathway.38-40 To confirm this,
cellular experiments are under way.
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| Fig 4.
Amino acid sequences surrounding Gly85 of human, bovine,
rat, and rabbit HRG. Residues that are identical in all 5 proteins are
blocked. Numbering starts at the N-terminus of human
HRG.2
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| |
FOOTNOTES |
Submitted February 24, 1997;
accepted August 18, 1997.
Supported by a Grant-in-Aid for Scientific Research (06671097) and a
Grant-in-Aid for Scientific Research on Priority Areas (Intracellular
Proteolysis) from the Ministry of Education, Science,
Sports, and Culture of Japan, and Research Grant No. HL16919 from the
US National Institutes of Health.
Address reprint requests to Toshio Shigekiyo, MD, First Department of
Internal Medicine, School of Medicine, The University of Tokushima,
3-18-15 Kuramoto, Tokushima 770, Japan.
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.
| |
ACKNOWLEDGMENT |
The authors are grateful to Dr E.W. Davie for support throughout this
study, Dr D.W. Chung for valuable advice, K. Takeshima for technical
help, J. Harris for synthesis of oligonucleotides, and Dr Don Foster
for supplying ZMB3 expression vector.
| |
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Abstract
Introduction
Abstract