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Rapid Purification and Characterization of Human Platelet Glycoprotein V:
The Amino Acid Sequence Contains Leucine-Rich Repetitive Modules
as in Glycoprotein Ib
By Takeshi Shimomura, Kingo Fujimura, Shuji Maehama, Motoyoshi Takemoto, Kenji Oda, Tetsuro Fujimoto,
Rieko Oyama, Masami Suzuki, Keiko Ichihara-Tanaka, Koiti Titani, and Atsushi Kuramoto
Glycoprotein V (GPV) is a membrane-associated, 82 Kd
platelet glycoprotein that is hydrolyzed during thrombin
activation to yield 69 Kd fragment. We have developed a
rapid and simple method for isolation of t h e protein from
platelet extracts using a combination of gel permeation,
anion-exchange. and lectin affinity chromatography. The
partial amino acid sequence was determined by analysis of
peptides generated by digestion of t h e S-carboxyamidomethylated protein with Achromobacter protease I or
cyanogen bromide. The sequence shows a remarkable
periodicity of leucine residues, which is homologous to t h e
consensus sequence of a highly diversified protein super-
family with a common repetitive module. Thrombin cleava g e site w a s determined to be located at t h e C-terminal
region of GPV by analysis of t h e products separated by
sizing and reversed-phase high performance liquid chromatography. By lectin blot analysis, t h e existence of mucintype carbohydrate chains w a s indicated, as well as t h e
existence of asparagine-linked carbohydrate chains shown
by the amino acid sequence analysis. From t h e s e data, we
report a structural model of GPV t h a t is analogous to
glycoprotein Ib.
0 1990 b y The American S o c i e t y of Hematology.
G
1,200g for 15 minutes to sediment platelets, which were subsequently washed three times in 10 mmol/L HEPES, pH 7.6 (1
mmol/L in EDTA, 0.15 mol/L in NaCI). GPV was extracted from
platelet
plasma membrane by incubating platelets (2 x lo9platelets/
Bernard-Soulier syndrome, a rare inherited bleeding disormL) at 37OC for 20 hours in 10 mmol/L HEPES, pH 7.6 (1 mmol/L
der, this glycoprotein is absent, as are GPIb and GPIX.3-6
in EDTA, 0.3 mol/L in NaCI, 1 mmol/L in p-aminophenylmethylPlatelets from these patients can be activated by thrombin,
sulphonyl fluoride). The extract from two donors' platelets was
but a long lag time is required to start the a g g r e g a t i ~ n . ~ brought to 40% saturation with solid ammonium sulfate, stirred for
Binding of a-thrombin to a high or moderate affinity site
30 minutes and centrifuged, and then brought to 60% saturation
located in the glycocalycin portion of G P I b is considered to
with ammonium sulfate and treated similarly. The pellet precipibe a step necessary for platelet a~tivation.'.~However,
tated in 60% saturation was taken up in a minimum volume (10 mL)
of 50 mmol/L potassium phosphate, pH 6.8 (1 mmol/L in EDTA)
measured equilibrium binding of a-thrombin to platelets
(buffer A), dialyzed against the same buffer for about 4 hours at
appears to be unrelated to platelet activation," and binding
4OC, concentrated with hygroscopic polymer (Aquacides, Calbioof thrombin inactivated by blocking the active site causes no
chem, USA) in a dialyzing bag, and clarified by centrifuging at
activation. GPV has been proposed to be a thrombin
10,OOOg for 30 minutes at 4OC for the subsequent purification
receptor." However, rate or extent of GPV hydrolysis seems
process.
not to be correlated directly with platelet activation.I2 In
We applied 500 pL of the dialyzed sample to a Superose 12
addition, blocking of GPV hydrolysis by anti-GPV antibodcolumn equilibrated with buffer A. The column was eluted with the
ies does not prevent thrombin tim mu la ti on.'^ Therefore, the
same buffer at a flow rate of 0.5 mL/min, and 0.5 mL fractions were
role of GPV in platelet activation by thrombin remains to be
collected. The whole sample was separated by multiple runs. The
elucidated.
GPV-rich fractionswere pooled and then directly applied to a Mono
Q column equilibrated with buffer A. The passed-through fraction
Knowledge of the structure of the GPV molecule is
was collected and incubated with wheat germ agglutinin Sepharose
essential for understanding the mechanisms involved in its
6MB (bed volume, 3 mL) for 3 hours at room temperature. The gels
interaction with thrombin and in disorders related to this
protein. In this regard, we established a rapid and simple
purification procedure by a modification of the method
From the Department of Internal Medicine. Research Institute
reported by Berndt and Phillips,14 Zafar and W a l ~ , ' ~or
.'~
for Nuclear Medicine and Biology, Hiroshima University. HiBienz et all3 and determined the partial amino acid sequence
roshima: and the Division of Biomedical Polymer Science, Institute
with the purified protein.
for Comprehensive Medical Science, School of Medicine. Fujita
Health University, Toyoake, Aichi, Japan.
MATERIALS AND METHODS
Submitted December 28.1989: accepted February 23,1990,
Columns and buffers. Superose 12 HR 10/30 gel permeation
Supported by Grants-in-Aid from the Japanese Ministry of
and Mono Q HR 5/5 anion exchange columns, and wheat germ
Education, Science and Culture for Scientific Research on Priority
agglutinin Sepharose 6MB were purchased from Pharmacia (UppAreas to A.K. and K.T. and from the Fujita Health University.
sala, Sweden). All the reagents used throughout the isolation
Address reprint requests to Kingo Fujimura. MD, Department of
procedure are of high performance liquid chromatography (HPLC)
Internal Medicine, Research Institute for Nuclear Medicine and
grade.
Biology, Hiroshima University. Kasumi 1-2-3. Minami-ku, HiPurification of GPK Fresh healthy donor platelets were preroshima 734. Japan.
pared using Hemonetics 30s (Haemonetics Corp, MA) and used
The publication costs of this article were defrayed in part by page
within 1 day of venipuncture. We obtained approximately 5 x 10"
charge payment. This article must therefore be hereby marked
platelets from a single donor. Donor platelets were centrifuged at
"advertisement" in accordance with 18 U.S.C. section I734 solely to
120 g for 15 minutes at rmm temperature to remove the contamiindicate this fact.
nated red blood cells. Acid-citrated dextrose (ACD) anticoagulant
0 1990 by The American Society of Hematologv.
(1/6 volume) was added to platelet-rich plasma and centrifuged at
0006-4971/90/7512-0014$3.00/0
LYCOPROTEIN (GP) V is the only platelet membrane glycoprotein so far identified that is specifically
hydrolyzed during activation by
In patients with
8/&,
Vol 7 5 , No 12 (June 15). 1990: pp 2349-2356
2349
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SHIMOMURA ET AL
2350
were packed in a column and washed with IO bed volumes (30 mL)
of buffer A. The retained glycoproteins were eluted from this column
with IO mL of buffer A containing 2.5% N-acetylglucosamine. The
eluted proteins were dialyied against 50 mmol/L NH,HCO, and
lyophilized.
An aliquot at each purification step was digested with human
thrombin (Green Cross. Japan) (5 U/mL) for I 5 minutes at 37°C
and, along with an undigest aliquot. was subjected tosodium dodecyl
sulfate-polyacrylamidegel electrophoresis (SDS-PAGE; 7.5%) according to the method of Laemmli." Monitoring of GPV was based
on susceptibility of GPV to thrombin. as assaKd using SDS-PAGE.
The amount of protein at each purification step was quantitated by
the method of Lowry et al." Isoelectric focusing was carried out
using the lmmobiline system (Pharmacia).
Amino acid sequcncc analysis o/GPV. The protein was d u c e d
with dithiothreitol and S-carboxyamidomethylated (CMA) with
iodoacetamide (Nacalai Tesque. Kyoto. Japan). The CMA-protein
wasdigested with Achromobacfer protease I(a gift of Dr T. Masaki.
Department or Agricultural Chemistry. lbaraki University, Mito.
Japan) for 3 hours at 37% in 3.1 mol/L urea/SO mmol/L Tris-HCI.
pH 9.0. using an enzyme:subslrate ratio of 1300 W
(W
/I .)I
Cleavage
at methionyl residues was carried out with 2% cyanogen bromide in
70% formic acid at room temperature as described by Gross." The
native GPV was digested with thrombin in 100 mmol/L sodium
phosphate. pH 7.4. at 37°C for 30 minutes. Peptides were partially
separated by HPLC on TSK gel columns connected in series
(G3000SW-G200OSW,each 7.5 x 600 mm.ToyoWa. Japan) in 6
mol/l. guanidine HCI/IO mmol/L sodium phosphate. pH 6.8.
Further purification of peptides was achieved by reversed-phase
HPLC on a column of Cosmosil 5C4-300 (4 x I50 mm. Nacalai
Tesque) using an acetonitrile gradient made in dilute aqueous
trifluoroacetic acid.
Amino acid compositions were determined with a Hitachi L-8500
Amino Acid Analyizr (Tokyo, Japan) or by the phenylisothiocyanate
Fdman degradations were done in an Applied
Biosystems 470A Protein Sequencer connected to an on-line PTH
Analym."
colicin
GPV
-88'
-
Prtpararion ofrabbi1 antibodits. A female New Zealand white
rabbit was immunized at 2-week intervals for 6 weeks with purified
GPV (0.2 mg/mL) in phosphatebuffered saline, mixed ( I : I by
volume) with complete Freund's adjuvant (Difco Laboratories.
Detroit. MI). lgG was prepared from the rabbit antisera by
treatment with DEAE-Sephacel.
k c f i n 6/01analysis. Aliquots (5 pg) of GPV and GPV treated
with thrombin as described above were incubated with or without
Arfhrohacrer neuraminidase (5 U/mL; Bochringer Mannheim Biochemicals. Yamanouchi. Tokyo. Japan) in 50 mmol/L sodium
acetate bulTer. pH 5.0. at 37OC for IS hours. They were then applied
to SDS-PAGE (8%). blotted onto a nitrocellulose membrane and
stained with horseradish peroxidase-conjugated peanut agglutinin
(PNA; Seikagaku-Kogyo. Tokyo, Japan) following manufacturer's
instructions. At the same time. the binding to the blot of the primary
rabbit antibody against GPV was detected with '-"l-protein A.
followed by autoradiography.
RESULTS
Puri/ication. At each purification step. existence of GPV
was analyzed by applying an aliquot of the collected fraction
to SDS-PAGE and could be recognized as a band stained
with periodic acid-SchitT (PAS), which was moved to a
smaller band by thrombin proteolysis. At the step of SuperOSE 12 gel permeation column chromatography, we obtained
GPV-rich fractions at the fractions 26 through 28 (Fig I ) .
After aninity chromatography on wheat germ agglutinin.
SDS-PAGE showed a single band by silver staining. and its
molecular weight (mol wt) was estimated at approximately
8 2 Kd. Purified GPV was hydrolyzed by thrombin to yield a
65 Kd protein. which was regarded as the large fragment of
GPV produced by thrombin proteolysis. previously reported
as GPVR' (Fig 2A). To examine homogeneity of purified
GPV. FPLC rcverscd-phase chromatography (PepRPC HR
5 / 5 . Pharmacia) was used by an acetonitrile gradient in
-200
-200
-116
- I 16
-92
qwg2
GPV- .
Y
[r
-66
-45
O.OE
0
0
m
a
C
IO
x > 3 0 4 0 5 0
FRACTION NUMBER
F b l . &pmr.tknofp(.t.kt extrm by gol porlnootkn
FPLC. Fr.crionr from Supuoso
12 eobnns were w b j m e d to
SDS-PAGE (7.6%)under reducing conditions end then to PAS
rteining. The PAS-steinad 82
Kd bend (GPV) in fractions 26
through 28 (tho loft i n u t ) was
ahifted to the 66 Kd bend
IGPVfl) by thrombin proteolysis
(the right inut: the right lane
sample of oech fraction wm an
eliquot of the fraction treated
with thrombin os described in
M e t r u l s and Methods). Glycocolicin was observed in frections 21 through 26.
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PURIFICATION AND CHARACTERIZATIONOf GPV
2351
kDo
-200
A
GPV-o
GPVfl
-
8
- I I6
-92
* 6.55
-66
4
0
5.85
Fig 2. Purified GPV o l t r
whoat g u m agglutinin (WGA).Qlinity chromatography. (A)
SDS-PAGE (7.5%) Of m
o
d
GPV undor roducingconditions.
Lano 1. sitwr staining of GPV;
l a n u 2 through 4. Coomasio
brilliant bluo staining of GPV,
GPV + thrombin. and thrombin. rospoctivoly. (61Iaooloctrk
focusing of purifiod GPV by Immobilino system.
-45
Ttmnnbin-
I ’ 4-4.
0
3.50
1 2 3 4
dilute aqueous trifiuoroacetic acid. A single sharp peak was
obtained at 45% of acetonitrile concentration (data not
shown). Purified GPV showed multiple forms, ranging from
5.6 to 6.6on isoelectric focusing (Fig 2B). All these characteristics. including 8 2 Kd carbohydrate-rich protein, thrombin susceptibility. and pl. are concomitant to those previously
reported“ for GPV. Yields of GPV at various purification
steps are summarized in Table 1.
Amino ucids composition. Amino acid composition of
CMA-GPV is shown in Table 2 on a molar basis. The
calculation was based on the data of Berndt and Phillips“
that polypeptide moiety comprises 52% of the total mass of
GPV (mol wt of 82 Kd). corresponding to a protein with mol
wt of 43 Kd. GPV appears to have some characteristic
enrichment of leucine residues.
Generution of peptides. A digest of the CMA-protein
(about 2 nmol each) with Achromohuctcr protease I (Fig 3A.
B. and C ) or cyanogen bromide (data not shown) was
separated by size exclusion HPLC. and pooled fractions were
further purified by reversed-phase HPLC. Two primary sets
of peptides, KI-K7 and MI-M8 (cyanogen bromide fragments). were thus isolated and subjected to amino acid and
sequence analyses. The digest of the intact protein with
thrombin was separated in a similar manner as described
above by a combination of size-exclusion (Fig 4) and
reversed-phase HPLC (data not shown). Two fragments.
Thl and Th2, were isolated. Fragment Thl seemed to be
identical to the previously reported GPVfl on the basis of the
apparent mol wt (69 Kd).
Sequence analysis. Sequence analysis of the intact protein (100 pmol) yielded no phenylthiohydantoins (PTHs) in
three cyclcs of Edman degradations. indicating that the
amino terminus of the protein is blocked. The aminoTable 2. Amino A d d compodtknof CMA-GW
CM-cya
Asx
Thr
7
31
16
28
37
34
30
16
sa
Glx
GC
Ala
Vd
Met
Ib
6
8
67
4
16
Lw
Platdot a r a c t from 10’’plat~
Ammonium Mate fractionation
(40% to 60%)
supaoso 12
Mono0
WGA-S@WOM
I5
TW
Phl3
100
26
18
2
0.2
LW
His
3
0.3
0.2
100
10
6.6
.Amounts of GPV prosant a dtrsrent isdatim stops w w roughly
esfimated from dansitomotricscan8 of SDS-PAGE gals.
tThe % y d d w w bwed on the a ” t
of GPV prosent in W - r i c h
fractions from !3mroae 12 column.
V
~
11
R O
2
22
27
Trp
NO
m
~
e
s
l
n
o
b mdof
l Gpv(mdwt.
~
~
~
82 Kdl. which her apromincontont of 52% carnpondno to43 Kd.
bawdon the data of &mdt md Phinii.’.
m
i
: NO. not dotorminod.
w
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2352
SHIMOMURA ET AL
K1
B
I
I
-
0
-
I
0
I
I
v
I
EC
E
(0
C
0
cu
8
cu
Q
I
Y
z
%
I
C
a
0
tl
, JJ
20
I
A
30
2
+-+-
3
4
c
I
40
50
1-0
VOLUMEImll
2-0
3b
RETENTION TIME lminl
Fig 3. Separationof peptides generated by digestion of the CMA-protein with Achromobacferprotease 1. (A) Primary separation of the
digest (2 nmol) on tandem TSK columns (G3000SW-G2000SW, each 7.5 x 600 mm) equilibrated in 6 mol/L guanidine HCI/10 mmollL
sodium phosphate, pH 6.8, at a flow rate of 0.5 mL/min. Peptides were monitored at 230 nm (0.16 AUFS) and collected manually. (6 and C)
Purification of pools 1 and 3 by reversed-phase HPLC on a Cosmosil 5C4-300column (4 x 150 mm) using a trifluoroacetic acid-an
acetonitrile system. Purified peptides are identified by prefix K.
0.1
E
C
8
cv
a
OW/
20
30
40
50
VOLUMElmll
Fig 4. Primary separation of thrombin digest of intact GPV by
sizing HPLC, as described in Fig 3. Void volume (V0),total elution
volume (Vt), and the elution positions of standard proteins with
known molecular weights are shown by vertical arrows.
terminal sequences of seven K peptides (Kl-7), eight M
peptides (Ml-8), and two Th peptides (Thl and 2) are shown
in Table 3. The link of K5 to K6 was established by analysis
of M2. Peptide M8 was considered to be included within the
sequence of K5. Peptides M3 and M4 were initially isolated
as a mixture, but could be differentiated by referring to the
sequence of Th2 that provided one of the two sequences. The
cause of unexpected cleavage between Asp-Ser within the
Th2 peptide is unknown. The amino terminus of T h l was
blocked, clearly indicating that GPVfl (69 Kd fragment of
GPV) was derived from the N-terminal region of GPV, and
the thrombin cleavage site was located in a close proximity to
the C-terminus of GPV.
We have not yet obtained the entire sequence of GPV, but
most of peptides ( K l , K2, K4, K5-K6, M1, M6, and M7)
show a remarkable periodicity of leucine residues (Fig 5).
This periodicity appears to be homologous to that observed
with a group of proteins, including integral membrane or
membrane-associated proteins (GP Ib,22-24
adenylate cyclase
of yeast,2s toll gene product,26 ~haoptin,~’)
proteoglycans or
connective tissue proteins (PGI,28 PGII,29 fibrom~dulin,~~),
and plasma proteins (LRP,3’ RA13*) (Fig 6). Aligning the
peptides so as to correspond to the consensus sequences
reported in this article made it possible to obtain highly
homologous tandem repeats and to propose consensus sequence of GPV. GPV is likely to contain 10 or more of these
repeats (Fig 5 ) .
Lectin blot analysis. After treatment of purified GPV
with neuraminidase, the apparent mol wt was reduced to 70
Kd (Fig 7) and it bound PNA, clearly showing the presence
of sialic acid and mucin-type carbohydrate chain. Thrombin-
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2353
PURIFICATION AND CHARACTERIZATION OF GPV
Table 3. Amino Acid Sequence of Three Primary Sets of Peptide
Fragments Generated by Digestion With Achromobacter
Protease 1, Cyanogen Bromide, or Thrombin
Peotide
Sequence
K1
K2
K3
K4
K5
K6
KJ
LRQVSLRRNRLRALPRALFRILSSLESVQLD~NGLLGAQAKLERLLLHSNRLVSlDsgCVFRDAAQCSGGDVARlSALGLPT.LT
LLDLSGN*LTHLPK
MVLLEQLFLDHNALRGIDQNMFQK
LVNLQELALNQNQLDFLPASFTNLENL-
M1
GGLQEL~r RTQLRnPAAAFRrLSRLRMGVTLSPRI-a-
M2
M3
M4
M5
M6
MJ
M8
pqgafw
FQKLVNLQELaLNQNQLDFLPAslfSS-EAPVHPALAP-S-EP-V
Y NTPDReLAtYGGFN-blocked
ISDSHlSAVAPGTFSDLlKLKTLRLsrNTGRGVLQSQSFSGTkVLQrVLL
Th 1
Th2
N-blocked
GPPRPAADSS-EAPVHPALAP-S-EP-V-AQD
Three sets of peptides are prefixed by K, M, and Th. respectively.
Sequences written in lower-case letters indicate tentative identification.
Those not identified are shown by dashes. Asterisks indicate potential
asparginelinked glycosylation sites.
cleavaged fragment, GPVfl, seems to bind P N A less intensively than GPV after neuraminidase treatment, in comparison with the intensity of immunoblotted GPVfl, suggesting
that the mucin-type carbohydrate chains mostly exist in the
C-terminal region of GPV (Fig 8).
DISCUSSION
In the present study, we attempted to purify the platelet
membrane glycoprotein V to homogeneity by modification of
the method reported by Berndt and P h i l l i p ~ or
’ ~ Zafar and
W a l ~ . ” The
~ ’ ~modification included use of FPLC system at
the size-exclusion and anion-exchange chromatography steps
and a new lectin-affinity chromatography at the final step,
and eliminated the hydroxyapatite and cation-exchange
chromatography steps. The previous method^'^“^ required
many dialysis steps using various buffers before each chromatography. W e used only one buffer system through all
chromatographies, and all the procedures can be easily done
within 4 days. As to the yield, we can easily purify 200 pg of
GPV from 10l2platelets. This high yield also seems to have
an advantage over the method reported by Berndt and
Phillips or Zafar and Walz (100 pg from 10l2 platelets),
although the yield seems to be dependent on freshness of
platelet samples (data not shown) as reported by Berndt and
Phillips.I4 The apparent molecular weight, thrombin sensitivity, PI, and amino acid composition of GPV purified in the
present study are very similar to those of GPV isolated by the
method previously reported.
Bienz et all3 have purified GPVs, a fragment of GPV
cleaved by calpain, from platelets sonicated in a buffer
containing calcium ion, using wheat germ agglutinin and
anion-exchange chromatography.” GPVs seems to have
almost the same mol wt and PI as GPV purified in the present
study. In our purification, there remains a possibility that
purified GPV is a fragment generated by calpain. However,
in order to activate calpain, it appears that their method
consumes a half amount of platelets. Therefore, our method
should have an advantage over theirs in terms of the final
yield.
Although we have not yet obtained the entire sequence of
GPV, most of the peptides analyzed contained homologous
leucine-rich repetitive sequences, which are observed with a
number of proteins. Schneider et
have proposed that
these repetitive modules should be common structural features of a novel protein superfamily whose members exert
their functions by highly specific protein-protein interactions. For example, PGII and fibromodulin bind to a specific
type of collagen and cause inhibitory effects on collagen
fibril f~rmation.~’-~’
RAI binds to angiogenin or RNase and
abolishes both the angiogenic and ribonucleolytic a ~ t i v i t i e s . ~ ~
Yeast adenylate cyclase has been suggested to bind to the
cytosolic side of the cell membrane with the repeat^.^'
Drosophila toll gene product plays a role in embryonic
dorsal-ventral patterning, hypothetically using the extracellular repeats for the cell adhesion.26 Drosophila chaoptin is
an intercellular cytoadhesive protein for photoreceptor cells.27
K1
K L R Q
R A L O R N a S S I E s
v s
9’
V Q
K2
K4
K5/6
Fig 5. Internal homology
observed with peptides of GPV.
Partial amino acid sequences
so far determined are tentatively aligned to achieve maximum homology. Highly conserved residues (identified with
a more than 40% frequency)
are boxed. The tentative consensus sequence is indicated at
the bottom; X is used where no
clear homology is observed.
K G L L G A Q A K L E R
S G
K L
K
K M V L L E Q
L A
M1
M6
M7
Tentative
consensus
L W L
V
M I
L R D
U
J
T
S
S
N
L
D
R
R
S
S
O
M
T
P
H
T
G
Q ~ R T
R L I S A V A
R G V D Q S Q S l F ( S G T K V u Q R
L X L X X N X L X X L P X X X F X X L X X L X X
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2354
ilj,s
SHIMOMURA ET AL
GP V
L X
X L X X L P X X X
r x x
G P Ibd
L X
X
L S X
G P IbB
L V
N
GP I X
L L L A N N S
LRG
L D L O G N X
PG I
L X L X N N
K
L X X
PG I1
L X L X N N
K
4 X X H K X L X X
FM
L X L X H N
Q
L X X
RAI
L X
X
L X E
YAdCY
L X L S X N X
L X X
Toll
L X L X X N X
L X X
Chaoptin
L, I) , O X 2 X
L X X
"["I"
L R T
L Q T
L X X
Fig 6. Consonsus soquo" of OPlba//3, GPlX and othw
proteins. LRO, Ieudnarkh u2-gfycoprotein: W I. protoogtycnn
biglycan: PO II. proteogtycan dscarin: FM. 69 Kd connective t i u u a protein fibromodulin: RAI. ribonudwselangiogonin inhibitor module A:
yAdcy, v s t adenylate cyclase: Toll, Drosophila Toll gene product: Chaoptin. Drosophila cytoadhesive protein of R coils. The Consensus
sequences shown are defined by residues appearing with a more than 40% frequency. Lowercase a and b indicate hydrophobic amino acid
residues (1. V. M. L, F. and AI and hydroxy amino acid residues IT and S).respectively. Boxes show identities or consorvotive repbcements
within homologous amino acid residues: 1, V, M, L. F, and A, or T and S. and indicate deletion and gap, respectiveiy.
-
Neuraminidase
- + -+ - + - +
kDa
11697-
F i g 7 . LoctInbbtano)y.h
of GPV. Aliquotr of p u r h d OPV
66'
43-
-1
31
2
3 4
5
6
7
8
llanos 1 through 4) and OPV
treated with thrombin llanos 6
through 8 )were incubeted with
( + ) or without ( - 1 neuraminic k ~ WbjWed
,
t o SDS-PAGE
(8%).and blotted onto nitrocellulose membranr. The membranes were either incubated
with rabbit anti-GPV antibody
followed by "%protein A treatment and autoradiographic imaging (lanes 1, 2, 6. and 8). or
stained with horseradishperoxidase-conjugeted PNA using 4chloro-1-naphthol as a substrate lbnes 3. 4. 7, and 8).
Molecular weights
- of merker
proteins are indicated on the
lamargin.
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2355
PURIFICATION AND CHARACTERIZATION OF GPV
Thrombin
N
IIr
m
l
t,, ,#
C
Fig 8. Schematic representation of the structural organization
of GPV. It remains to be clarified whether or not GPV has a
transmembrane domain. Aspargine-linked and mucin-type carbohydrate chains are shown by Y and I shape, respectively. Five
aspargine-linked glycosylation sites have been identified so far by
protein sequence analysis. Their numbers and locations are
tentative. The leucine-rich repeats are shown by boxes. Arrow
shows thrombin cleavage site.
The N-terminal tryptic fragment of GPIba contains seven of
these repeats, which appear to contain the binding sites for
von Willebrand factor and t h r ~ m b i n . ~However,
'.~~
Tanoue et
a134have recently reported that the precise binding sites are
located in the C-terminal region of the fragment without the
repeats. GPV may also bind to other macromolecules.
GPlbP, a major protein phosphorylated under the conditions
that increase cytoplasmic concentration of CAMP in
platelet^,^^.^' is linked to GPIba by disulfide bonds' and
contains one leucine-rich module.24 GPIX forms a complex
with GPIb38-40
and has been recently reported to contain one
such m o d ~ l e . Therefore,
~'
the platelet membrane glycoproteins, which are simultaneously defective in Bernard-Soulier
syndrome, all contain more or less the leucine-rich modules.
They must have evolved from the same ancestral gene and
have some correlations in their functions.
The apparent molecular weight of cyanogen bromide
fragment M1 was estimated to be 36 Kd (almost half the
mass of GPV) from the elution volume by size-exclusion
chromatography (data not shown). The fragment appears to
be derived from the C-terminal region of GPV on the basis of
the amino acid composition, which contains no homoserine
and which contains only one lysyl residue. Therefore, other
cyanogen bromide fragments and most K peptides containing
more or less the leucine-rich repeats were probably derived
from the amino-terminal half of GPV. The sequence of
fragment Th2 is very similar to that reported as the Nterminal sequence of GPVfl by Zafar and Walz.I6 However,
our data clearly indicate that the N-terminus of the large
fragment ( T h l ) generated by cleavage with thrombin is
blocked. We assumed, therefore, that thrombin cleavage site
is located in a close proximity to the C-terminus of GPV.
From the data described herein, we propose the structural
organization of GPV as shown in Fig 8, which shows a
remarkable similarity to the structure of GPIbu reported by
Titani et a122and Lopez et aLz3These findings should be
useful for understanding the pathogenesis of Bernard-Soulier
syndrome and also the mechanism of thrombin interaction
with platelets.
ACKNOWLEDGMENT
We thank H. Sumida, Y. Oto, K. Yamamoto, S. Ohishi, and H.
Ohta for their excellent secretarial assistance and typing of the
manuscript.
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1990 75: 2349-2356
Rapid purification and characterization of human platelet glycoprotein
V: the amino acid sequence contains leucine-rich repetitive modules
as in glycoprotein Ib
T Shimomura, K Fujimura, S Maehama, M Takemoto, K Oda, T Fujimoto, R Oyama, M Suzuki, K
Ichihara-Tanaka and K Titani
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