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Blood, 1 February 2002, Vol. 99, No. 3, pp. 931-938
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Missense mutations in the  3 subunit have a
different impact on the expression and function between
 IIb3 and
v3
Seiji Tadokoro,
Yoshiaki Tomiyama,
Shigenori Honda,
Hirokazu Kashiwagi,
Satoru Kosugi,
Masamichi Shiraga,
Teruo Kiyoi,
Yoshiyuki Kurata, and
Yuji Matsuzawa
From Department of Internal Medicine and Molecular
Science, Graduate School of Medicine, Osaka University, Japan; and
Department of Blood Transfusion, Osaka University Hospital, Japan.
 |
Abstract |
 IIb 3 and
v3 belong to the 3
integrin subfamily. Although the 3 subunit is a key
regulator for the biosynthesis of 3 integrins, it
remains obscure whether missense mutations in 3 may
induce the same defects in both IIb3 and
v3. In this study, it is revealed
that thrombasthenic platelets with a His280Pro mutation in
3, which is prevalent in Japanese patients with
Glanzmann thrombasthenia, did contain significant amounts of
v3 (about 50% of control) using
sensitive enzyme-linked immunosorbent assay. Expression studies showed
that the His280Pro3 mutation impaired IIb3 expression but not
v3 expression in 293 cells. To extend these findings, the effects of several 3 missense
mutations leading to an impaired IIb3
expression on v3 function as well as
expression was examined: Leu117Trp, Ser162Leu, Arg216Gln, Cys374Tyr,
and a newly created Arg216Gln/Leu292Ser mutation. Leu117Trp and
Cys374Tyr 3 mutations did impair
v3 expression, while Ser162Leu,
Arg216Gln, and Arg216Gln/Leu292Ser mutations did not. With regard to
ligand binding function, Ser162Leu mutation induced especially distinct effects between 2 3 integrins: it markedly impaired
ligand binding to IIb3 but not to
v3 at all. These data clearly demonstrate that the biosynthesis and the ligand binding function of
IIb3 and those of
v3 are regulated in part by different
mechanisms. Present data would be a clue to elucidate the regulatory
mechanism of expression and function of 3 integrins.
(Blood. 2002;99:931-938)
© 2002 by The American Society of Hematology.
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Introduction |
Integrins are a family of cell surface
molecules that mediate cellular attachment to the extracellular matrix
and cell cohesion and are involved in such diverse biologic processes
as thrombus formation, angiogenesis, inflammation, and
embryogenesis.1 Integrins are heterodimers, and
3 is one of 8 known subunits. IIb3 and v3
belong to the 3 integrin subfamily and share the same
subunit (3).2,3
IIb3, whose expression is restricted to
the megakaryocyte/platelet lineage, is a prototypic integrin that
functions as a physiologic receptor for fibrinogen and von Willebrand
factor and plays a crucial role in normal hemostasis and platelet
aggregation.4 On the other hand,
v3 is expressed in a number of tissues,
such as platelets, endothelial cells, smooth muscle cells, and
osteoclasts, and plays a key role in cell proliferation, cell
migration, angiogenesis, and bone resorption.5-7
Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding
disorder characterized by a quantitative or qualitative abnormality of
IIb3 and caused by a defect in either the
IIb or 3 gene.8-11
The quantitative abnormality in GT can be divided into 2 groups: type I
has a severe IIb3 deficiency (< 5% of normal) with no or minimal clot retraction, and type II has a moderate
IIb3 deficiency (10%-20% of normal)
with normal or only moderately diminished clot
retraction.8 The numbers of IIb3 and v3
expressed on the platelet surface are 40 000 to 80 000
molecules per platelet and about 100 molecules per platelet, respectively.12 Previous studies have shown that
IIb3 and v3
are synthesized by a similar mechanism.13 The IIb v
and 3 subunits are synthesized from separate messenger
RNA transcripts, and the 3 subunit becomes associated
with either proIIb or prov, single-chain precursor forms of subunits, in the endoplasmic reticulum. The
proIIb3 and
prov3 complex are then transported to the
Golgi apparatus, where pro subunits undergo sugar modification and
endoproteolytic cleavage into heavy and light chains. After these
processing events within the Golgi apparatus, the mature IIb3 and v3
complex is rapidly transported to the cell surface.13,14 Consistent with these biosynthetic processes, GT patients with mutations in the 3 gene that cause impaired
synthesis of 3 are deficient in both
IIb3 and
v3, while patients with mutations in the
IIb gene are deficient only in
IIb3 and have normal or even increased
v3 on their platelets.12
Thus, the level of v3 expression appears
to be a useful marker to differentiate patients with a genetic defect
located in the 3 gene and those in the
IIb gene.12,15 However, it
remains obscure whether missense mutations in the
3 subunit may induce the same defects in both
3 integrins.
In this study, we examined the effects of several 3
missense mutations, including a His280Pro mutation, on the expression and function of these 3 integrins.
| |
Materials and methods |
Antibodies and antagonists
Rabbit polyclonal antisera specific for
IIb3 and AP2
(IIb3-specific monoclonal antibody
[MoAb]) were generously provided by Dr Thomas J. Kunicki (The Scripps
Research Institute, La Jolla, CA).16 AP3
(3-specific MoAb) was generous gift from Dr Peter Newman
(The Blood Center of Southeastern Wisconsin, Milwaukee, WI).17 PAC-1 (a ligand mimetic MoAb) binds specifically to
activated IIb3 and was kindly provided by
Dr Sanford Shattil (The Scripps Research Institute).18
PT25-2 (IIb3-specific MoAb) activates IIb3 and was a kind gift from Drs Makoto
Handa and Yasuo Ikeda (Keio University, Tokyo, Japan).19
LM609 (v3 complex-specific MoAb) and
LM142 (v-specific MoAb) were generously provided by Dr David Cheresh
(The Scripps Research Institute).20 TP80 (IIb-specific MoAb) and MOPC21 (mouse myeloma immunoglogulin [Ig] G1)
were purchased from Nichirei (Tokyo, Japan) and Sigma Chemical (St
Louis, MO), respectively. RGDW (Arg-Gly-Asp-Trp) peptide and FK633
(peptidomimetic antagonist specific for
IIb3) were generously provided by Dr Jiro
Seki (Fujisawa Pharmaceutical, Osaka, Japan).21
Cyclo(RGDfV) (cyclo(-Arg-D-Gly-D-Asp-D-Phe-L-Val-D-)) peptide specific
for v3 was a generous gift from Merck
(Darmstadt, Germany).22
GT patient Osaka-5
Patient Osaka-5, a product of nonconsanguineous parents, was a
33-year-old Japanese woman who was diagnosed as a typical GT. Clot
retraction by MacFarlane's method was normal (40%; normal values
40%-60%). An immunoblot assay using rabbit polyclonal antisera specific for IIb323 revealed
that the amounts of IIb and 3 in
platelets from patient Osaka-5 were 6% and 8% of control platelets,
respectively (data not shown). Although the amounts of
IIb3 in Osaka-5 did not fulfill the
criteria for type II GT (10%-20% of normal), normal clot retraction
of Osaka-5 platelets strongly suggested that she was classified as type
II rather than type I GT.
Flow cytometry and immunoblot assay
Flow cytometric analysis using various MoAbs and immunoblot
assay using rabbit polyclonal antisera specific for
IIb3 were performed as previously
described.23,24 To examine the expression of
v3 on platelets, Alexa-conjugated goat
F(ab')2 antimouse IgG (Molecular Probes, Eugene, OR) was
used instead of fluorescein isothiocyanate (FITC)-conjugated goat
antimouse IgG because of its higher sensitivity.
Quantitative ELISA
Quantitative enzyme-linked immunosorbent assay (ELISA) was
performed to examine the amounts of v3 in
platelet lysates from control subjects or patient Osaka-5. In brief,
1 × 106/µL washed platelets were solubilized in 0.05 M
Tris-buffered saline, pH 7.4, containing 1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, and 100 µg/mL leupeptin (Sigma). After
centrifugation at 10 000g for 10 minutes, 100 µL lysate
was applied to the wells of a microtiter tray, each containing 0.25 µg fixed LM609. After incubation for 60 minutes the tray was washed 6 times, and biotinylated LM142 was added to each well for 60 minutes.
After washing 6 times, the bound LM142 was detected using an
avidin-biotin-alkaline phosphatase complex (Vector, Burlingame, CA)
and ELISA amplification system (Life Technologies, Gaithersburg, MD).
Standard curve was obtained using purified
v3 purchased from Chemicon International
(Temecula, CA).
Amplification and analysis of platelet RNA
Total cellular RNA of platelets was isolated from 30 mL of whole
blood, and IIb or 3 messenger RNA was specifically
amplified by reverse transcription-polymerase chain reaction (RT-PCR),
as previously described.25 The primers for the
amplification of IIb or 3 messenger RNA and
conditions for RT-PCR were described elsewhere.25,26
Nucleotide sequences of PCR products were determined by using Taq
DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems, Foster
City, CA).
Allele-specific restriction enzyme analysis
Amplification of the region around exon 5 of the
3 gene was performed by using primers IIIaE5,
5'-CTCTACCAGTGACATGGCTG-3' (sense, nucleotide [nt] 17 365-17 384 in
the 3 gene), and IIIaE6, 5'-GCCAGAGATCTCACCATGG-3' (antisense, nt 17 607-17 589) using 250 ng
DNA as a template.27 The first-round PCR products were reamplified using primers IIIaE5 and IIIaE6BspHI,
5'-CATGGTAGTGGAGGCAGAGTCA-3' (antisense, nt
17 593-17 572, mismatched sequence underlined). PCR products were
then digested with restriction enzyme BspHI. The resulting
fragments were electrophoresed in a 6% polyacrylamide gel.
Construction of 3 expression vectors
The wild-type IIb and 3 complementary DNAs
(cDNAs) cloned into a mammalian expression vector pcDNA3 (Invitrogen,
San Diego, CA) were generously provided by Dr Peter Newman (Milwaukee,
WI). The full-length v cDNA was generously provided by Dr David
Cheresh (La Jolla, CA) and shuttled into pcDNA3. To construct the
expression vectors containing the 887C (Pro280) form of
3 cDNA, PCR-based cartridge mutagenesis was performed.
The 1654-base pair (bp) region (nt 350-2003) of platelet
3 cDNA from patient Osaka-5, who was homozygous for
887C, was amplified by RT-PCR using primers IIIa1A, 5'-CCATCCAAGTGCGGCAGGTGG-3' (sense, nt 350-370), and
IIIa8AflII, 5'-GCATCCTTGCCAGTGTCCTTAAG-3' (antisense, nt
2003-1981). The amplified fragments were digested with KpnI
and AflII, and the resulting 1526-bp fragments (nt 456-1981)
were extracted using GeneClean II kit (Bio 101, La Jolla, CA). The
530-bp fragments extending from the beginning of the open reading frame
to nucleotide 455 were obtained by digesting the full-length of
3 cDNA with BamHI and KpnI. These
2 fragments were double-inserted into the pcDNA3 digested with
BamHI and AflII. The fragments inserted were
characterized by sequence analysis to verify the absence of any other
substitutions and the proper insertion of the PCR cartridge into
the vector.
For the introduction of other missense mutations in 3
leading to Leu117 Trp, Ser162Leu, Arg216Gln, or Cys374Tyr,
we carried out the overlapping extension PCR, as previously
described.26 For example, to generate the Leu117Trp
3 (Trp1173) mutant, we synthesized
mismatched sense primer IIIa117Trp-s, 5'-GGACATCTACATCTGGATGG-3' (nt 384-403, mismatched sequence underlined), and antisense primer IIIa117Trp-as, 5'-GACAGGTCCATCCAGTAGTAG-3' (nt 410-390, mismatched sequence underlined). PCR was performed by using
3 cDNA as a template and primers pcDNA3-s,
5'-GGCTAACTAGAGAACCCACTG-3' and IIIa117Trp-as, or primers IIIa117Trp-s
and IIIa1 5'-GCGGGTCACCTGGTCAG-3' (antisense, nt 654-648). The 2 individually amplified PCR products were mixed and used as a template
of PCR using primers pcDNA3-s and IIIa1. The amplified PCR
products were digested with KpnI, and then the fragments
were introduced into pcDNA3 as described above. The fragments inserted
were characterized by sequence analysis.
Ten micrograms of wild-type or mutant 3 construct was
cotransfected into human embryonic kidney 293 cells (106
cells) with 10 µg wild-type IIb or v construct by the calcium phosphate method as previously described.28 The 293 cells
transiently expressing IIb3 or
v3 were obtained and analyzed 2 days
after transfection. In selected experiments, 100 ng green fluorescent protein (GFP) expression vector pEGFP-C1 (Clontech, Palo Alto, CA) was
cotransfected with 3 and either IIb or v construct into 293 cells to monitor transfection efficiency. The cells were cultured in Dulbecco modified medium with 10% fetal calf serum.
Surface labeling of the transfected cells
Surface proteins of the transfected cells were biotinylated 2 days after transfection, and immunoprecipitation using MoAbs was
performed as previously described.28
Metabolic label with [35S]methionine and pulse
chase
Metabolic labeling of transfected cells was performed one
day after transfection as previously described.25 The
cells were incubated with 0.4 mCi/mL (14.8 MBq)
[35S]methionine for 30 minutes, and then the medium was
changed to Dulbecco modified medium/10% fetal calf serum with 50 µg/mL nonradioactive methionine. Cells were equally divided into 3 dishes and chased after 0.5, 2, and 22 hours, respectively, and
immunoprecipitation was performed.25
Fibrinogen binding assay
Soluble fibrinogen binding assay was performed as previously
described.29 For fibrinogen binding to
v3, 50 µL aliquots of
v3-transfected cells
(1.5 × 105) in Ca++-free Tyrode-HEPES buffer
containing 1 mM MgCl2 were incubated with MoAb LM142
specific for v (5 µg/mL) for 30 minutes on ice. After washing, 1 mM MnCl2 was added into the cell suspension to induce a
high-affinity state of v3. Cells were
then incubated with FITC-fibrinogen (150 µg/mL) in the presence or
absence of 1 mM RGDW or 50 µM cyclo(RGDfV) (an
v3 antagonist) and
phycoerythrin-conjugated antimouse IgG (1:5 dilution, Serotec, Oxford,
United Kingdom) for 25 minutes at 22°C and then incubated with
propidium iodine (Sigma) for 5 minutes at 22°C. After washing,
fibrinogen binding (FL1) was analyzed on the gated subset of single,
v3-expressing (FL2) live cells (propidium
iodine-negative, FL3). Specific fibrinogen binding was defined as that
inhibited by 50 µM cyclo(RGDfV). For fibrinogen binding to
IIb3,
IIb3-transfected cells were examined in
the presence or absence of 10 µM FK633 (an
IIb3 antagonist) with 1 mM
CaCl2 and 10 µg/mL PT25-2 (an
IIb3-activating antibody). The following
procedures were the same as described above.
| |
Results |
Expression level of IIb3 and
v3 on platelets from thrombasthenic
patient Osaka-5
We examined the surface expression of
IIb3 and v3
on platelets from patient Osaka-5 using flow cytometry. While the
GPIb-specific MoAb AP1 bound equivalently to Osaka-5 and control
platelets, the IIb3 complex-specific
MoAb AP2, the 3-specific MoAb AP3, and the
IIb-specific MoAb TP80 showed a marked reduction in the expression
of IIb3 on Osaka-5 platelets (Figure
1A). The amount of
IIb3 expressed on Osaka-5 platelet
surface was about 6% of control platelets (n = 11). On the other
hand, Alexa-conjugated goat F(ab')2 antimouse IgG clearly
showed that v3 complex-specific MoAb
LM609 reacted with Osaka-5 platelets as well as control platelets. The
mean fluorescence intensity (MFI) for LM609 bound to control platelets
was 1.56 ± 0.28 arbitrary units (mean ± SD, n = 11) and that to
Osaka-5 platelets was 0.64 (mean of duplicates). Thus, the amount of
v3 expressed on Osaka-5 platelet surface
appeared to be 41% of control platelets (Figure 1B). To further
examine the expression of v3 in Osaka-5
platelets, we measured the amounts of v3
in platelet lysates using sensitive ELISA. The amounts of
v3 in control platelets and in Osaka-5
platelets were 8.4 ± 2.1 ng/108 platelets (n = 11) and
4.3 ng/108 platelets, respectively (Figure 1C). These data
demonstrated that v3 expression in
Osaka-5 platelets was about half as much as that in control
platelets.
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| Figure 1.
Flow cytometric analysis and quantitative ELISA for
platelet 3 integrins on control and Osaka-5 platelets.
(A) Expression of IIb3. Washed
platelets obtained from a control subject and patient Osaka-5 were
incubated with 10 µg/mL AP1 (specific for GPIb), 10 µg/mL AP2
(specific for IIb3 complex), 10 µg/mL
AP3 (specific for 3), or 10 µg/mL TP80 (specific for
IIb) for 30 minutes at 22°C. After washing, bound MoAbs were
detected by FITC-conjugated goat F(ab')2 antimouse IgG.
MOPC21 (mouse IgG1) was used as a negative control (dotted
line). (B) Expression of v3. Control and
Osaka-5 platelets were incubated with 5 µg/mL LM609 (specific for
v3 complex) for 30 minutes at 22°C.
After washing, bound MoAbs were detected by Alexa-conjugated goat
F(ab')2 antimouse IgG. MOPC21 (mouse IgG1) was
used as a negative control (dotted line). The amounts of bound LM609
were expressed as MFI from 11 control subjects (mean ± SD) and MFI
from patient Osaka-5 (mean of duplicate). (C) The amounts of
v3 measured by quantitative ELISA; 100 µL platelet lysate (1 × 106 platelets/µL) was
applied to a sandwich ELISA. Standard curve was obtained using purified
v3. Data represents the mean ± SD from
11 control subjects.
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Nucleotide sequence analysis and allele-specific restriction
enzyme analysis
To identify the molecular defect in patient Osaka-5, the whole
coding regions of IIb and 3 cDNAs were amplified by
RT-PCR, as previously described.21 Examination of
nucleotide sequences of the PCR fragments revealed that the
3 cDNAs had a single A>C substitution at nucleotide 887 that leads to a His280Pro substitution of 3 (Figure
2A). Patient Osaka-5 appeared homozygous
for the 887A>C substitution, and no other nucleotide substitutions
were detected in the coding regions of either IIb or
3 cDNAs from patient Osaka-5. To confirm that patient
Osaka-5 was homozygous for the 887A>C substitution, exon 5 with their
flanking regions of the 3 gene were amplified
by PCR, followed by digestion with BspHI. A restriction site
for BspHI would be abolished by the 887A>C substitution.
Allele-specific restriction enzyme analysis showed that Osaka-5 was
homozygous for the AC substitution in exon 5 (Figure 2B). The
homozygosity of the substitution was also confirmed by nucleotide
sequence analysis of the PCR fragments from genomic DNA (data not
shown). Using allele-specific restriction enzyme analysis, we examined
the presence of the 887A>C substitution in 18 other unrelated Japanese
GT patients (type I, 8 cases; type II, 10 cases) and 20 control
subjects. This substitution was also present in 2 other type II GT
patients (1 homozygous, 1 heterozygous) (data not shown). No control
subjects had this substitution.
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| Figure 2.
Analysis of 3 cDNA and the
3 gene in patient Osaka-5.
(A) Nucleotide sequence analysis of 3 cDNA in patient
Osaka-5. The 3 cDNAs from control or Osaka-5 platelets
were amplified by RT-PCR. The amplified fragments were directly
examined using Taq DyeDeoxy Terminator Cycle Sequencing kit. Samples
were run and analyzed on an ABI 373A DNA sequencer. (B) Allele-specific
restriction enzyme analysis. The region around exon 5 of the
3 gene was amplified by PCR using primers IIIaE5 and
IIIaE6BspHI, followed by digestion with BspHI.
BspHI digestion of the PCR products yields 205-bp and 24-bp
fragments in normal allele. The AC substitution abolished a
restriction site for BspHI. The resulting fragments were
electrophoresed in a 6% polyacrylamide gel;  X174 digested with
HaeIII was used as a marker.
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Effect of His280Pro substitution (Pro2803) on the
expression of IIb3 and
v3
We constructed an expression vector that contained the wild-type
or the mutant Pro280 form of 3 and cotransfected each
3 construct with the wild-type IIb construct into 293 cells. When 100 ng GFP expression vector was cotransfected, the levels
of GFP expression were essentially the same between the wild-type IIb3 and the mutant
IIbPro2803-transfected cells (Figure
3A). Flow cytometric analysis using AP2
MoAb, AP3 MoAb, and TP80 MoAb showed that the level of the mutant
IIbPro2803 expression was markedly reduced compared
with the wild-type IIb3 expression (about
25% of wild-type, Figure 3A). Immunoprecipitation of the surface-labeled transfected cells using AP3 MoAb also showed that the
amount of IIbPro2803 complex was reduced and that the
molecular weight of the mutant 3 was the same as the
wild-type (Figure 3B). Immunoblot assay using polyclonal antisera
specific for IIb3 revealed that the
mature form of IIb was more markedly reduced than 3
in the mutant transfected cells, as observed in Osaka-5 platelets
(Figure 3C). To examine the effect of the His280Pro substitution in
3 on v3 expression, we
first transfected wild-type 3 or the mutant
Pro2803 construct into 293 cells. In these conditions, wild-type 3 or the mutant Pro2803 could
be associated with an endogenous human v of 293 cells. The 293 cells
transfected with empty vectors did not show any expression of
v3 (data not shown). Flow cytometric
analysis using LM609 MoAb and LM142 MoAb showed that the level of
surface expression of vPro2803 complex on the
transfected cells was almost the same as wild-type
v3 complex (Figure 3D). To rule out the
possibility that the normal expression of
v3 was due to the presence of an excess
amount of 3, we transfected wild-type 3
or the mutant Pro2803 construct with wild-type v
construct into 293 cells. The cotransfection of v and
3 cDNAs into 293 cells markedly increased the level of
v3 expression. However, the surface
expression of vPro2803 complex was almost the same as
wild-type v3 complex (Figure 3D). In addition, reduction in the amount of the transfected 3
construct did not make any difference between the expression level of
the wild-type and the mutant v3 (data not
shown). These data indicate that the 887A>C substitution leads to the
marked reduction in the amount of IIb3
without disturbing v3 expression at least in 293 cells.
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| Figure 3.
Effects of His280Pro 3 missense mutation
on the expression of IIb3 and
v3 in 293 cells.
(A) Flow cytometric analysis of IIb3 on
the transfected cell surface. Wild-type or His280Pro3
cDNA was cotransfected into 293 cells with wild-type IIb cDNA and
GFP expression vector pEGFP-C1. The binding of AP2, TP80, and PAC-1
with PT25-2 to the transfected cells was analyzed by flow cytometry 2 days after transfection. Results are representative of at least 3 separate experiments. (B) Immunoprecipitation analysis of biotin
surface-labeled transfected cells. The transfected cells were
surface-labeled with biotin 2 days after transfection.
Immunoprecipitation was then performed using AP3. Precipitates were
separated by 6% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under reducing conditions. After transfer to
a nitrocellulose membrane, precipitated proteins were detected by
chemiluminescence. (C) Immunoblot analysis of transfected cells. The
transfected cells were lysed and separated by 6% SDS-PAGE under
reducing conditions 2 days after transfection. After transfer to a
nitrocellulose membrane, IIb and 3 were detected with
a 1:10 000 dilution of rabbit polyclonal
anti-IIb3 antibodies. (D) Flow cytometric
analysis of v3 on the transfected cell
surface. Wild-type or His280Pro3 cDNA was transfected
into 293 cells (i) in the absence or (ii) in the presence of wild-type
v cDNA. The binding of LM609 (specific for
v3 complex) or LM142 (specific for v)
to the transfected cells was analyzed by flow cytometry 2 days after
transfection. MOPC21 was used as a negative control (dotted
line).
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To assess the ligand binding function of the mutant
IIbPro2803, we examined the binding of the
ligand-mimetic MoAb PAC-1 in the presence of the activating MoAb
PT25-2.25 PAC-1 could bind to the mutant
IIbPro2803 as well as the wild-type
IIb3 in the presence of PT25-2 (Figure
3A). The PAC-1 binding to IIb3 was
dependent on the PT25-2 binding, and the PAC-1/PT25-2 binding ratio for
IIbPro2803 was essentially the same as that for wild type (PAC-1/PT25-2 ratio: wild type, 0.31 ± 0.12; mutant,
0.36 ± 0.14; mean ± SD; n = 3).
Effect of missense mutations in 3 on the expression
of IIb3 and
v3
The different effects of the His280Pro3 mutation
between IIb3 and
v3 expression made us further examine the
effects of other missense mutations found in GT patients on
v3 expression. Previously reported 4 single amino acid mutations in 3 in GT were examined:
Leu117Trp, Ser162Leu, Arg216Gln, and Cys374Tyr.30-33 In
addition, we introduced a newly created Arg216Gln/Leu292Ser mutation
into 3. Each mutant 3 cDNA vector was
cotransfected with the wild-type IIb cDNA or wild-type v cDNA
vector into 293 cells. Again, cotransfection of the GFP expression
vector showed that the transfection efficiency was essentially the same between the wild-type and the mutant transfected cells (data not shown). As shown in Figure 4, we
confirmed that all 3 mutations examined markedly
impaired surface expression of IIb3.
Immunoprecipitation of the surface-labeled
IIb3-transfected cells using AP3 further showed that the amounts of the mutant
IIb3 were markedly reduced compared with
wild-type IIb3 (Figure 4B). As shown in
Figure 5, each of the Leu117Trp and
Cys374Tyr mutations resulted in the marked reduction in
v3 expression as well. In sharp contrast, none of Ser162Leu, Arg216Gln, or Arg216Gln/Leu292Ser mutations impaired
v3 expression. Of particular interest was
the effect of the Arg216Gln/Leu292Ser3 mutation.
Although the Arg216Gln/Leu292Ser mutation completely abolished
IIb3 expression, this mutation did not
impair v3 expression at all. Because 293 cells possess endogenous v1 and v5,34,35 LM142
(anti-v) may detect these v integrins. However, our previous
study showed that the expression of these endogenous v integrins
appeared to be low compared with exogenous
v3.34 Indeed, the Leu117Trp
3 mutation severely impaired the expression of v
subunits as well as v3, suggesting that
the bulk of expressed v integrins is
v3 in these conditions (Figure
5).
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| Figure 4.
Effects of 3 missense mutations on the
expression of IIb3.
(A) Flow cytometric analysis of IIb3
on the transfected cell surface. Wild-type 3 cDNA or
each mutant 3 was cotransfected into 293 cells with
wild-type IIb cDNA. The binding of AP2 or TP80 to the transfected
cells was analyzed by flow cytometry 2 days after transfection. Results
were mean ± SD from 3 separate experiments and expressed as percent
MFI relative to that of wild-type IIb3.
Two-tailed P values for paired samples were obtained by the
Student t test (*P < .01,
**P < .05). (B) Immunoprecipitation analysis of
biotin surface-labeled transfected cells. The transfected cells were
surface-labeled with biotin 2 days after transfection.
Immunoprecipitation was then performed using AP3. Precipitates were
separated by 6% SDS-PAGE under reducing conditions. After transfer to
a nitrocellulose membrane, precipitated proteins were detected by
chemiluminescence.
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| Figure 5.
Effects of 3 missense mutations on the
expression of v3.
(A) Flow cytometric analysis of v3 on the
transfected cell surface. Wild-type 3 cDNA or each
mutant 3 was cotransfected into 293 cells with wild-type
v. The binding of LM609 or LM142 to the transfected cells was
analyzed by flow cytometry 2 days after transfection. Results were mean
± SD from 3 separate experiments and expressed as percent MFI
relative to that of wild-type v3.
Two-tailed P values for paired samples were obtained by the
Student t test (*P < .01,
**P < .05). (B) Immunoprecipitation analysis of biotin
surface-labeled transfected cells. The transfected cells were
surface-labeled with biotin 2 days after transfection.
Immunoprecipitation was then performed using AP3. Precipitates were
separated by 6% SDS-PAGE under reducing conditions. After transfer to
a nitrocellulose membrane, precipitated proteins were detected by
chemiluminescence.
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To elucidate the mechanism of impaired expression of the mutant
IIb3, we performed pulse chase
experiments especially for His280Pro and Arg216Gln/Leu292Ser mutations.
Because 3 was synthesized in excess as compared with
IIb in wild-type transfected cells in our experimental
conditions,25 we used TP80 (anti-IIb) and LM142
(anti-v) for the precipitation of IIb3
and v3 complex, respectively. As shown in
Figure 6A, the association between
proIIb and Pro2803 or Gln216/Ser2923
was the same as that of wild-type 3 at 30 minutes after
chase. At 2 hours after chase, some of the wild-type
proIIb3 complex was transported to the
Golgi apparatus, where cleavage of proIIb into heavy and light
chains occurs. However, this process was impaired in His280Pro and
Arg216Gln/Leu292Ser mutants. Even at 22 hours after chase, mature
IIb3 was not detectable in
Arg216Gln/Leu292Ser mutant, while a small amount of mature IIb3 was observed in the His280Pro
mutant. In sharp contrast to IIb3
mutants, the kinetics of vHis280Pro3 and
vArg216Gln/Leu292Ser3 biosynthesis were the same as
that of wild type, and the normal amount of mature
v3 was synthesized at 22 hours after
chase (Figure 6B).
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| Figure 6.
Pulse chase analysis of 3 integrin
biosynthesis.
Wild-type, His280Pro, or Arg216Gln/Leu292Ser3 cDNA was
cotransfected into 293 cells with either (A) wild-type IIb cDNA
or (B) wild-type v cDNA. Cells were labeled one day after
transfection with 0.4 mCi/mL (14.8 MBq) [35S]methionine
for 30 minutes and chased with media containing 50 µg/mL of
nonradioactive methionine for various periods of time as indicated.
Immunoprecipitation was performed using either (A) TP80 or (B) LM142.
Precipitates were separated by 6% SDS-PAGE under reducing conditions.
Results are representative of 3 separate experiments.
|
|
Effect of missense mutations in 3 on the ligand
binding function of IIb3 and
v3
We then assessed the ligand binding function of the mutant
3 integrins. We measured the binding of FITC-fibrinogen
to mutant IIb3 and
v3. The
IIb3-transfected cells were treated with the IIb3-activating antibody, PT25-2,
while v3-transfected cells were treated
with 1 mM MnCl2, which induces a high-affinity state of
v3. Although 1 mM MnCl2 has
also been shown to result in fibrinogen binding to 51 in
endothelial cells,36 dot plots in Figure
7B show that fibrinogen binding to the
transfected 293 cells depend on the expression levels of
v3. In addition, the blockade of the
fibrinogen binding by IIb3-specific
antagonist FK633 at 10 µM and
v3-specific antagonist cyclo(RGDfV) at 50 µM indicated that the binding was specifically mediated by
IIb3 and
v3, respectively, in our experimental
conditions (Figure 7). Because the expression levels of
IIb3 and v3
were different in each mutation (Figures 4 and 5), we monitored
IIb3 and v3 expression by PT25-2 and LM142, respectively, and only analyzed the
cells expressing the same levels of IIb3
and v3 (Figure 7). Two variant type GT
mutations in 3, Asp119Tyr and Arg214Trp, were examined
in parallel as negative controls.37,38 As expected, Asp119Tyr and Arg214Trp mutations abolished the ligand binding function
of both 3 integrins. Neither His280Pro nor Cys374Tyr mutation impaired the ligand binding to both
IIb3 and
v3. Ser162Leu mutation markedly impaired
the ligand binding to IIb3 but not to
v3 at all (Figure 7B). Similarly,
Arg216Gln more severely impaired the ligand binding to
IIb3 than to
v3. Thus, Ser162Leu and Arg216Gln
mutations showed a different effect on ligand binding between 2 3 integrins.
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| Figure 7.
Soluble fibrinogen binding to mutant 3
integrins.
(A) Dot plots represent FITC-fibrinogen (horizontal) and PT25-2
(vertical) binding to IIb3-transfected
cells. The IIb3-transfected cells were
treated with 10 µg/mL PT25-2 (an
IIb3-activating antibody) for 30 minutes
on ice in the presence or absence of 10 µM FK633 (an
IIb3 antagonist). After washing, cells
were incubated with 150 µg/mL FITC-fibrinogen and
phycoerythrin-conjugated antimouse IgG for 25 minutes at room
temperature. Then, after 5 minutes incubation with propidium iodine,
cells were washed and analyzed by flow cytometry. Because the
expression levels of 3 integrins were different in each
mutation, we gated and analyzed cells showing the same expression
levels of IIb3 for fibrinogen binding.
(B) Dot plots represent FITC-fibrinogen (horizontal) and LM142
(vertical) binding to v3-transfected
cells. The v3-transfected cells were
treated with 1 mM MnCl2 for 30 minutes on ice in the
presence or absence of 1 mM RGDW or 50 µM c(RGDfV). LM142 (10 µg/mL) was added simultaneously to the tubes to monitor expression of
v3. The following procedures were the
same as described above. (C) Fibrinogen binding to
IIb3 mutants. Results were mean ± SD
from 3 separate experiments and expressed as percent MFI relative to
that of wild-type IIb3. Two-tailed
P values for paired samples were obtained by the
Student t test (*P < .01,
**P < .05). (D) Fibrinogen binding to
v3 mutants. Results were mean ± SD from
3 separate experiments and expressed as percent MFI relative to that of
wild-type v3. Two-tailed P
values for paired samples were obtained by the Student t
test (*P < .01, **P < .05).
|
|
| |
Discussion |
Among genetic defects responsible for GT phenotype, single amino
acid substitutions in each subunit have been especially informative in
defining precise structural domains of
IIb3 that play a role in the biosynthesis
and/or function.9-11 However, it remains elusive whether
missense mutations in 3 responsible for GT may induce the same defects in the other 3 integrin,
v3. In this study we investigated the
effects of 6 missense mutations in 3, including His280Pro mutation, on the expression and function of
IIb3 and v3
in 293 cells. Leu117Trp and Cys374Tyr3 mutations
impaired both IIb3 and
v3 expression, while His280Pro,
Ser162Leu, Arg216Gln, and Arg216Gln/Leu292Ser3 mutations
impaired IIb3 expression but not
v3 expression. With regard to ligand
binding, Ser162Leu and Arg216Gln mutations markedly impaired the ligand
binding to IIb3 but not to
v3. Our present data demonstrate that
some 3 missense mutations have a different impact on the
expression and function of IIb3 and
v3.
The IIb and v are homologous and 36% identical in primary amino
acid sequence.39 The IIb subunit has been found only in
combination with 3, while v is promiscuous and can
associate with at least 5 subunits (1, 3, 5,
6, and 8).2 As shown in Figure
8, our data show that missense mutations
at well-conserved Leu117 and Cys374 residues among 8 subunits
impaired the expression of both 3
integrins.40 In contrast, amino acid residues at positions
162, 216, 280, and 292 of subunits are rather diverse, and
mutations at these residues impaired only
IIb3 expression. Except for the Cys374Tyr
mutation, His280Pro, Ser162Leu, and Arg216Gln mutations responsible for
type II GT phenotype did not impair v3
expression in 293 cells, while the Leu117Trp mutation responsible for
type I GT phenotype disturbed v3
expression. From these data one could argue that different effects of
3 mutations on the biosynthesis of
v3 may reflect only the severity of
IIb3 deficiency. However, a newly created
double mutation, Arg216Gln/Leu292Ser, clearly denied this possibility,
because the mutation led to a severe IIb3
deficiency but normal v3 expression.
These findings strongly suggest that the expression of
IIb3 is more strictly regulated than
v3.
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| Figure 8.
Comparison of integrin 3 amino acid
sequences with other integrin subunits.
The boxed areas are well-conserved residues between several subunits. Missense mutations examined in this study are also indicated.
Leu117 and Cys374 in 3 are well-conserved residues,
while Ser162, Arg216, His280, and Leu292 in 3 are rather
diverse between 8 subunits.
|
|
We found a point mutation (887A>C) leading to His280Pro amino acid
substitution in 3 in 3 unrelated GT patients from 19 Japanese GT patients: 2 patients appeared homozygous, and 1 patient was
heterozygous. Thus, this mutation was found in 5 of the 38 possibly
mutant chromosomes. In addition to our patients, 3 other Japanese GT
patients with this mutation have been reported.41 Although
we could not rule out the possibility that patient Osaka-5 is
hemizygous for the mutation, the prevalence of the His280Pro mutation
in Japanese GT patients suggests that patient Osaka-5 is likely
homozygous rather than hemizygous. Cotransfection of wild-type IIb
and Pro2803 constructs into 293 cells resulted in an
impaired surface expression of IIb3
(about 25% of control). These data demonstrated that the His280Pro
mutation is responsible for GT phenotype. It has been demonstrated that
the hypothetical human 3 metal ion-dependent adhesion
site domain is critical for heterodimer assembly with human IIb and
ligand binding function.42,43 Moreover, the hexapeptide
sequence 275Val-Gly-Ser-Asp-Asn-His280 within the 3
metal ion-dependent adhesion site domain appears necessary for
species-restricted heterodimer formation.43 Our present
data demonstrated that the His280Pro mutation at the sixth residue of
the unique hexapeptide did not impair either assembly of proIIb and
3 or ligand binding function. In pulse chase studies, very little proIIb was processed into mature IIb, suggesting that
at least a portion of this mutant
proIIb3 was retained and degraded
within endoplasmic reticulum.
Because platelets express only a limited number of
v3 (about 100 per platelet), we carefully
examined the expression levels of v3 in
Osaka-5 platelets. In sharp contrast to the markedly impaired
IIb3 expression (about 6% of normal),
sensitive ELISA as well as flow cytometric analysis showed that Osaka-5
platelets possessed about 50% of the normal
v3 content, that is, an apparently higher
amount of v3 than was previously reported
in GT platelets due to 3 mutations (< 20% of
normal).12 Our transient transfection studies may
induce higher expression of the mutant 3 integrins in
293 cells than in the patient's platelets, probably due to pcDNA3-derived overexpression of these proteins in heterologous cells
(IIb3, about 6% in Osaka-5 platelets vs
about 25% in 293 cells; v3, about 50%
in Osaka-5 platelets vs about 100% in 293 cells). Nevertheless, our
data clearly showed the different impact of the
His280Pro3 mutation on the expression of the 2 3 integrins in Osaka-5 platelets as well as in 293 cells. Contrary to our findings, employing Chinese hamster ovary cells
Ambo et al41 demonstrated that this mutation impaired
the expression of 3 when complexed with endogenous
hamster v. The difference in the expression of the mutant
v3 between human 293 and Chinese hamster ovary cells is likely due to a difference between species.
There are some distinctive features between
IIb3 and
v3. Treatment of
IIb3 with ethylenediaminetetraacetic acid
at 37°C dissociates the complex into its individual subunits, while
v3 remains a heterodimer.44
This difference may reflect tighter cation binding to
v3 or additional cation-independent
interactions between v and 3. Divalent cations are
also required to support ligand binding functions of the
3 integrins. However, particular divalent cations affect
ligand binding to the 2 receptors differently. Namely, fibrinogen binds
to v3 in the presence of Mn2+
but not in Ca++, while it binds to
IIb3 in either cation.45 In
addition, we recently clarified the difference in the ligand binding
sites between IIb and v.34 In this study, we newly
demonstrate that Ser162Leu and Arg216Gln mutations show a different
effect on ligand binding between 2 3 integrins.
Consistent with the reports by Newman's group,32,33 we
showed that Ser162Leu and Arg216Gln mutations impaired the stability of
the complex between IIb and 3, as evidenced by the
fact that the binding of complex-specific MoAb AP2 was markedly
impaired compared with that of the IIb-specific MoAb TP80. In
contrast, neither mutation affected the stability of the complex
between v and 3, as evidenced by the normal binding of the v3 complex-specific MoAb LM609.
These findings further indicate structural differences between
IIb3 and
v3. Employing v/IIb chimeras, it
has been reported that ligand recognition specificity of
IIb3 is regulated by the amino-terminal
one third of the subunit that contains the amino-terminal 140 residues and first 2 divalent cation binding repeats of
IIb.46 Because missense mutations in 3
affect the expression and function of 3 integrins
differently, the key structure should lie in the subunits. Further
investigation of the structures in the subunits that regulate the
biosynthesis of the 3 integrins is underway.
Leukocyte adhesion deficiency is a genetic disease characterized by
abnormality of 2 integrins.47-49 In
leukocyte adhesion deficiency, missense mutations have been shown to
impair expression of all 2 integrins,
L2 (CD11a/CD18),
M2 (CD11b/CD18), and X2 (CD11c/CD18).50,51 There
is so far no example of a selective deficiency in only 1 or 2 of the
2 integrins.47 Because L, M, and X have been found only in
combination with 2, biosynthesis of
L2, M2, and
X2 may be regulated in a common
mechanism. It should also be interesting to carefully examine whether
some missense mutations in 2 may affect the expression
of these 2 integrins differently.
In conclusion, we demonstrate that IIb3
and v3 expression and function are
differently regulated by certain 3 missense mutations.
We also suggest that Ser162 and Arg216 residues regulate the stability
of IIb3 and
v3 differently. These findings would provide insights into the structural requirement for
IIb3 and v3
function as well as their expression.
| |
Acknowledgments |
We thank Dr Thomas J. Kunicki for the rabbit polyclonal antisera
specific for IIb3 and for MoAb AP2; Dr
Peter Newman for MoAb AP3 and the IIb and 3 cDNA
cloned into a mammalian expression vector pcDNA3; Dr David Cheresh for
MoAbs LM609 and LM142 and the v cDNA cloned into a mammalian
expression vector pcDNA1NEO; Dr Sanford Shattil for MoAb PAC-1; Drs
Makoto Handa and Yasuo Ikeda for MoAb PT25-2; Dr Jiro Seki for FK633;
and Dr P. Raddatz for cyclo(RGDfV).
| |
Footnotes |
Submitted August 8, 2000; accepted September 30, 2001.
Supported in part by grants from the Ministry of Education, Science,
and Culture; the Japan Society for the Promotion of Science; and
Welfide Medical Research Foundation, Osaka, Japan.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Yoshiaki Tomiyama, Dept of Internal Medicine and
Molecular Science, Graduate School of Medicine B5, Osaka University,
2-2 Yamadaoka, Suita Osaka 565-0871, Japan; e-mail:
yoshi{at}hp-blood.med.osaka-u.ac.jp.
| |
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Abstract
Introduction