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Expression in a Cell-Free System of Normal and Variant Forms of Human
Antithrombin 111. Ability to Bind Heparin and React With a-Thrombin
By Richard C. Austin, Richard A. Rachubinski, Francoise Fernandez-Rachubinski, and Morris A. Blajchman
Human antithrombin 111 (AT-Ill) cDNA was cloned into the
cell-free expression phagemid vector pGEM-BZf( ) and
site-directed mutagenesis was used t o remove nucleotides
encoding the signal peptide. AT-Ill messenger RNA (mRNA)
transcripts derived from this construct were translated in
an mRNA-dependent rabbit reticulocyte lysate (RRL) system containing (36S)methionine. Immunoprecipitation of
the cell-free translation mixture with rabbit polyclonal
antibodies to AT-Ill showed, by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), a 47-Kd
polypeptide which is the non-glycosylated mature form of
plasma AT-Ill. Densitometric scanning showed that this
polypeptide constitutes greater than 90% of the radiolabeled polypeptides produced in this system. HeparinSepharose chromatography resulted in the elution of cellfree derived AT-Ill as a broad peak between 0.2 and 0.7
mol/L NaCI. The cell-free derived AT-Ill also reacted with
human a-thrombin. In 2 minutes approximately 20% of the
AT-Ill was found associated with a higher molecular weight
species, consistent with the formation of a 1:1 stoichiometric covalent complex between a-thrombin and AT-Ill.
Unfractionated heparin accelerated the rate of formation
of such complexes. When Ser394 was mutated t o Leu t o
form the AT-Ill Denver mutant, the cell-free translation
product of this mutation did not show any significant
complex formation when reacted with a-thrombin. A
truncated form of AT-Ill (Met251-Lys432), containing only
the putative thrombin-binding domain, was synthesized
independently. This 21-Kd polypeptide did not bind heparin: however, it was cleaved by a-thrombin presumably at
the reactive center Arg393-Ser394. When Ser394 was
mutated to Leu the cell-free translation product of this
truncated AT-Ill mutation did not react with a-thrombin at
the reactive center. This simple cell-free approach, along
with site-directed mutagenesis, should allow for the rapid
and accurate mapping of the functional domains of human
AT-Ill.
0 1990 b y T h e American S o c i e t y of Hematology.
H
either Pharmacia LKB Biotechnology (Baie d’Urf6, Quebec, Canada) or Bethesda Research Laboratories (Burlington, Ontario,
Canada). T4 DNA ligase, T7 RNA polymerase, ribonucleotides,
and 7-methylguanosylguanosinetriphosphate (7-mGpppG Cap analog) were purchased from Pharmacia LKB Biotechnology. RNase
inhibitor, RNase-free DNase, and the cell-free expression vectors
pSP64, pSP65, and pGEM-3Zf( +) were purchased from PromegaBiotec (Toronto, Ontario, Canada). D-phenylalanyl-L-propyl-Larginine chloromethyl ketone (PPACK) was purchased from Calbiochem (Mississauga, Ontario, Canada). Protein molecular weight
markers were from Sigma (St Louis, MO), a-(”P) dATP (3,000
Ci/mmol) from Amersham (Oakville, Ontario, Canada), and
L-(35S)methionine ( 9 0 0 Ci/mmol) from New England Nuclear
(Lachine, Quebec, Canada). All other chemicals and reagents were
of the highest quality available.
+
UMAN antithrombin I11 (AT-111) is a single-chain
plasma glycoprotein consisting of 432 amino-acid
residues and four N-linked carbohydrate side chain^."^ It is a
member of a family of serine protease inhibitors that are
referred to as the Serpins.’ AT-I11 is the major physiologic
inhibitor of thrombin as well as other clotting factors of the
intrinsic coagulation p a t h ~ a y . ~Protease
‘~
inactivation by
AT-I11 is through the formation of a 1:l stoichiometric
complex with the serine protease at the AT-I11 reactive site
Arg393-Ser394 (designated P,-Pi ). With the formation of
this complex, the serine protease is inactivated and the
complex rapidly removed from the circulation. The rate of
complex formation between AT-I11 and the serine protease is
greatly enhanced in the presence of heparin.”*”
The physiologic importance of AT-111 is demonstrated
clearly by individuals with inherited or acquired AT-I11
deficiency who often suffer from recurrent thromboembolic
disease.’*.” Characterization of the genetic defects in patients with congenital deficiency of AT-I11 indicates that
certain deficiencies can be attributed to mutations which lie
in the AT-I11 gene.I4-I6
Recently, the cDNA encoding human AT-I11 has been
used to express AT-I11 in bacteria, yeast, and mammalian
system^.^."-^^ In this article, we report the cell-free expression
of normal and variant forms of human AT-I11 and their
ability to interact with heparin and a-thrombin. This simple
cell-free approach, coupled with site-directed mutagenesis,
should allow for a better understanding of the structural
requirements for AT-I11 activity.
MATERIALS AND METHODS
Materials
The cDNA encoding human AT-I11 was generously provided by
Dr E.V. Prochownik (University of Michigan, Ann Arbor).’**’
Human a-thrombin (>3,000 NIH units/mg; >99% active) was
kindly provided by Dr J. Fenton (New York State Division of
Biologicals, Albany). All restriction enzymes were purchased from
Blood, Vol 76, No 8 (October 15). 1990: pp 1521-1529
From the Canadian Red Cross Blood Transfusion Service and the
Departments of Biochemistry and Pathology, McMaster University, Hamilton, Ontario, Canada.
Submitted January 16. 1990; accepted June 11, 1990.
Supported in part by Canadian Red Cross Society Grant No.
HA21.995. R.C.A. is a recipient of a Research Fellowship Award
from the Heart & Stroke Foundation of Canada. F.F.-R. is a
recipient of a postdoctoral fellowship from the Medical Research
Council of Canada. R.A.R. is a recipient of a scholarship from the
Medical Research Council of Canada.
A preliminary report of this work was presented at the Thirtieth
Annual Meeting of the American Society of Hematology. San
Antonio, TX, December 1988 and has appeared in abstract form in
Blood 72:362a, 1988 (suppl 1).
Address reprint requests to M.A. Blajchman, Room 2N31.
Department of Pathology, McMaster University Medical Centre.
I200 Main St W.Hamilton, Ontario, Canada L8N 3 W .
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 I734 solely to
indicate this fact.
8 1990 by The American Society of Hematology.
0006-4971/90/7608-0006$3.00/0
1521
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1522
AUSTIN ET AL
Met hods
Insertion of the AT-III cDNA into cell-free expression vectors.
The AT-Ill cDNA was initially isolated from the Psi I site of the
plasmid ~ K T 2 1 8 ~and
’ ligated into the Pst I site of pSP64. The
ligation mixture was used to transform the Escherichia coli strain
DH5a to ampicillin resistance. To enhance cell-free expression, the
AT-I11 cDNA was subcloned from pSP64 into the Sma I-Psr I site of
pSP65 following Bal 31 exonuclease digestion of the 5’-poly dC-dG
tail region according to the method of Davis et al.” The AT-I11
cDNA insert was subcloned from pSP65 into the EcoRI-HindlII site
of pGEM-3Zf( +) for cell-free expression and site-directed mutagenesis. The resultant construct. containing the AT-Ill cDNA in the
correct orientation with respect to the T7 promoter, was designated
pGEM-3Zf( +)-AT-IIIL32.432.
Site-directed mutagenesis of the full-length A T-III cDNA.
Site-directed mutagenesis was performed by the method of Taylor et
a1 using the reagents and protocol as outlined in the Amersham
mutagenesis kit.*’ All oligodeoxyribonucleotideswere synthesized at
the Institute for Molecular Biology and Biotechnology, McMaster
University, Hamilton, Ontario, Canada. The cDNA encoding human AT-Ill was cloned into the cell-free expression phagemid vector
pGEM-3Zf( + ) to yield single-stranded DNA templates for sitedirected mutagenesis. A 28-mer oligodeoxyribonucleotide primer
5’-GGTAGCGGCCATGCACGGGAGCCCTGTG-3’was used to
delete the nucleotides encoding the signal peptide. Thus, the resultant AT-I11 cDNA consisted of an open-reading frame beginning
with Met-32 followed by His1 to Lys432. The resultant construct
was designated pGEM-3Zf( +)-AT-I11,,12 and is shown in Fig 1.
The AT-I11 Denver mutation was produced with the oligodeoxyribonucleotideprimer 5’-GCTGGCCGTTTGCTAAAC-3’,which contains a single-base substitution that converts Ser394 of the reactive
center into a TTG or Leu codon. AT-Ill-Denver is a natural variant
of AT-Ill that has lost thrombin-inhibitory a~tivity.~’
Authenticity
of these mutations was confirmed by double-stranded DNA
sequencing24using the dideoxy-chain termination method.2s
Construction of the cell-free expression vector containing truncated AT-III cDNA. The cDNA encoding AT-I11 was digested to
completion with HinfI and the resultant fragments were separated
Amp-r
ori
Fig 1. The cell-free expression vector pGEM-fZfI +l-AT-lll,e.The direction of transcription is indicated by the arrow.
Abbreviations: Amp-r, ampicillin-resistance gene: 11 ori, origin of
replicstion for filamentous phage f l ; ori, origin of replication for
the plasmid.
by polyacrylamide gel electrophoresis.26The largest fragment, which
contained approximately 700-bp coding for the thrombin-binding
region (Met251-Lys432). was isolated. blunt-ended with T4 DNA
polymerase, and subcloned into the Sma I site of pGEM3Zf( +).
The resultant construct, which contained the 700-bp fragment in the
correct orientation with respect to the T7 promoter, was designated
pGEM-3Zf( +)-AT-III,,,.,,, because translation was initiated from
Met25 I.
Cell-free transcription of the AT-III eDNA. Cell-free transcrip
tion using T7 RNA polymerase was performed essentially as
described by Krieg and Melton.,’ Plasmid DNA (4 pg) was
linearized downstream from the AT-Ill insert with Hind111 and
transcribed in a total volume of 100 pL containing 40 mmol/L
Tris-HCI (pH 7.5). 6 mmol/L MgCI,, 2 mmol/L spermidine, 10
mmol/L NaCI, 10 mmol/L dithiothreitol (DTT), 100 pg/mL bovine
serum albumin (BSA), 0.5 mmol/L ATP, CTP, UTP, and
7-mGpppG, 0.1 mmol/L GTP supplemented with 100 U of RNase
inhibitor, and 60 U of T7 RNA polymerase. Transcription was
performed at 37OC for I hour, after which template DNA was
removed by incubating with 4 U of RNase-free DNase at 37OC for
15 minutes. The mixture was immediately centrifuged through a
(3-50 Sephadex (Pharmacia) column previously equilibrated with
0.02 mol/L Tris-HCI (pH 8.0). 0.001 mol/L EDTA and then was
extracted once with phenol and twice with ch1oroform:isoamyl
alcohol (24:l vol/vol). The RNA was recovered by the addition of
0.25 vol of 10 mol/L ammonium acetate (pH 5.0) and 2.5 vol of
absolute ethanol, followed by storage overnight at -20°C. The RNA
was recovered by centrifugation, washed once with 70% ethanol. and
dried under vacuum. The RNA was resuspended at a concentration
of 1 mg/mL in DEPC-treated water and stored at -7oOC.
Cell-free translation of AT-III mRNA transcripts. AT-I11
mRNA was translated in a micrococcal nuclease-treated rabbit
reticulocyte lysate (RRL) system according to the method of Pelham
and Jackson.28The translation mixture contained, in a final volume
of 20 pL, 14 r L of micrococcal nuclease-treated RRL (PromegaBiotec), 1 pL of transcribed AT-Ill mRNA, 1 pL of amino acids (1
mmol/L) minus methionine, 50 mmol/L potassium acetate. 1.0
mmol/L magnesium acetate, 7 mmol/L creatine phosphate, creatine phosphokinase (35 rg/mL), 20 U RNase inhibitor, 1.4
mmol/L DTT, calf liver tRNA (35 pg/mL), and 20 pCi of
L-(%)methionine. Translations were performed for 60 minutes at
3OOC. Incorporation of (’5S)methionine into trichloroacetic-acidinsoluble material was measured according to Mans and Novelli.29
AT-I11 translation products were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)M under
reducing or nonreducing conditions.” followed by autoradiography.
Immunoprecipitation of AT-III translation products.
Immunoprecipitation was performed by the method of Fujiki et all2
using a rabbit polyclonal antibody to human AT-I11 (Diagnostica
Stago, Asnieres, France). Immunoreactive material was analyzed by
SDS-PAGE followed by autoradiography.
Heparin-sepharose chromatography of AT-III translation products. The ability ofcefl-free-derived AT-I11 to bind to heparin was
assessed by heparin-Sepharose chromatography according to the
method of Miller-Andersson et ai.” Standard porcine heparin (>I 50
USP U/mg; Sigma) was coupled to cyanogen bromide-Sepharose
(Pharmacia). Translation mixtures were diluted twofold in 0.04
mol/L Tris-HCI (pH 7.5). 0.2 mol/L NaCI, and centrifuged at
65,000 rpm for 1 hour at 4OC to pellet insoluble material. The
translation mixture was then dialyzed against heparin-binding
buffer (0.02 mol/L Tris-HCI, pH 7.5.0.1 mol/L NaCI) overnight at
4OC and applied to heparin-Sepharose equilibrated in the same
buffer. Binding was performed for 60 minutes at room temperature.
The translation products were prewashed with 3 column vol of
heparin-binding buffer and then with the same buffer containing
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1523
CELL-FREE ANTITHROMBIN 111 EXPRESSION
increasingconcentrations of NaCl (0.1 to 2.0 mol/L). The resultant
fractions were analy7ed by SDS-PAGE followed by autoradiograPb.
Formorion ojrhromhin-AT-///complcxc.c. The translation mix-
ture was diluted twofold with thrombin-binding buffer (0.12 mol/l.
NaCI. 0.02 mol/L Tris-HCI. pH 7.5). centrifuged as described
previously. dialy7ed against the same buRr. and mixed with human
n-thrombin diluted in the same bufkr. The molar ratio of human
a-thrombin to cell-free-derived AT-Ill was approximately 1OO:l.
The rcaction mixture was incubated at 37% and terminated at
specific time p i n t s by the addition of PPACK to a final concentration of S pmnl/l.. The radiolabeled protein bands were analyzed by
SDS-PACE and visuali7ed by autoradiography.
Dcmciromerric sconninR. Densitometricscanningof the rutoradioprams was performed using the tioefcr CSZOO transmittance/
reflectance scanning densitometer and the GS3S0 data system
(Ilocfer Scientific Inztruments. San Francisco. C h ) .
RESULTS
Expression of Human A 7-111 in a Cell-Free System
Initially. cDNA encoding human AT-Ill was inserted into
the cell-free expression vcctor pSP64. However. cell-free
synthesis of human AT-Ill from this construct was low;
approximately twofold to threefold over background. This
problem of inefficient synthesis of AT-Ill was overcome with
the partial deletion of the 5’-homopolymeric dC-dG tail
region of the AT-Ill cDNA. Subscqucntly. cell-free synthcsis of AT-Ill using the pGEM-3%f( )-AT-Ill ,2.,12 construct increased to approximatcly 50-fold over background.
The yield of human AT-Ill, in this cell-free system. was
approximately 400 to 500 ng/ml. RRL. asdetermined by the
incorporation of (“S)methionine. Deletion of thc signal
peptide by site-directed mutagenesis resulted in the cell-free
expression of the unglycosylntcd mature form of human
AT-Ill. The construct used for the cell-free expression of
mature AT-Ill was designated KJEM-3Zf( A )-AT-III,.,,~
and is shown in Fig I .
The mRNA obtained from the cell-free transcription of
fl~E%f-3%f(A ) - A T - I I I , , , ~was
~ translated in RRI, in the
prcsence of (“S)mcthionine. The major polypeptide synthesized had an apparent molecular mass of 47 Kd as dctermined by SDS-PAGE (Fig 2. lane 4) and migrated faster
than prc-AT-Ill, which included the signal peptide (Fig 2.
lane 3). The mature (1-432) 47-Kd polypeptide could be
immunoprecipitated with rabbit polyclanal antibodies to
Fig 2. A ~ ) y r of
h cell-freo-derhnd AT-NI trambtion products. Total translation mixtures labeled
with f”S)methionine were analyzed by SDS-PAGE
on a 12% polyacrylamide gel under reducing condtrims. followed by autoradiography. Lane 1 contains Bromo mosaic virus mRNA. Lane 2 contains
no mRNA. Lane 3 contains pre-AT-Ill
mRNA.
Lane 4 contains AT-Ill,
mRNA. Lana 6 contams
the total translation mixture, translated with AT111,
mRNA and immunoprecipitated with rabbit
polydonal antibodies to human AT-Ill. M W M represents molecular weight protein markers (Kdl: 66.
bovine serum albumin: 46, egg albumin; 38. rabbit
muscle glyc~aldehydc3-phosphate dehydrogenase: 29. bovine erythrocyte carbonic anhydrase:
20. soybean trypsin inhibitor: 14. bovine milk
o-lactalbumin.
m
~~
~
29
1
human AT-Ill (Fig 2. lane 5 ) . Densitometric scanningof the
autoradiograms showed that this cell-frce-dcrived AT-Ill
polypeptide constituted greater than 907 of the total translated products synthesi7ed in this cell-free-translation systcm. The additional plypeptides seen, which were of lower
molecular mass. did not result from proteolysis of the 47-Kd
polypeptide (data not shown). and arc most likely due to
truncated translation products initiated on in-phase AUG
codons within the mRNA transcripts as suggested by the
amino acid squcncc of the human AT-Ill protein. ;ISwell as
previous work by others.“ *‘
RindinR of Cell-Frep-Derived A T-IllIO
If eparin-Sepharost-
Dcnsitomctric scanning of the autoradiograms showed
that approximately 70’7 of the cell-free-dcrivcd AT-Ill
bound to the heparin-Scpharosc column and eluted as a
major peak with buffers containing between 0.2 and 0.7
mol/L SaCI (Fig 3A). The addition of plasma AT-III at a
concentration of 100 nmol/I. did not affect heparin affinityof
thccell-frcc-dcrived AT-Ill. Cell-free-derivcd AT-Ill. which
did not bind initially to the heparin-Scpharosc column, did
not bind when chromatographcd a second time (data not
shown). On clution from thc heparin-Scpharose column,
cell-frce-derived AT-Ill migrated, on SIX-PAGE. like
cell-free-derivcd AT-I I I unexposed to heparin. The ccll-freederived AT-Ill-Denver variant (Scr 394 Leu: see Fig 4)
had an elution pattern very similar to that of cell-freederived normal AT-Ill (Fig 3A). However. purified plasma
AT-Ill eluted from the heparin-Sepharose column as a .
major peak between 0.6 and 1.0 mol/I. NaCl (Fig 3R). The
results also indicated that the presence of RRL lowers the
binding affinity of plasma AT-111 to heparin-Scpharose and
may account for some of the differences noted in heparin
affinity between plasma AT-Ill and cell-frce-derivcd AT-
-
111.
To determine whether aberrant folding due to incomplete
disulfide bond formation was related to heparin afiinity.
different affinity fractions of cell-free4crivcd AT-Ill. which
eluted off heparin-Scpharose at increasing S a C l conccntrations. were analyzed by SDS-PAGEunder both reducing and
nonreducing conditions (Fig 5 ) . Cell-free-derived AT-Ill
polypeptides eluting off heparin Sepharose with 0.1 mol/l,
SaCl showed no difference in migration in the reduced and
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1524
AUSnN ET AL
-A
0
02
04
08
08
I2
I
1 4
18
18
Normal
Denver
A C G T
A C G T
2
N.CI W O I i O n (u)
A
B
00
01
0.
os
OI
10
I?
16
1.
NICl c o n C " r 8 t h
*a
Fig 4. Partial nudootido uquonco JIowhrg tho mutation in
AT-Ill,,-Denver
in codon 394. In tho normal AT-I,,
cDNA.
codon 394 is TCG. which codes for Ser (A). In AT-Ill, ,-Denver.
codon 394 h8s boon mutated t o TTO. whkh codes for Lou (El.
20
0
nonreduced forms (Fig 5 , l a n a I and 2). In contrast. the
majority of the cell-frec-derived AT-Ill polypeptides eluting
otT heparin S e p h a r w with 0.7 mol/L NaCl migrated faster
in the nonreduced form than in the reduced form (Fig 5.
compare lancs S and 6). The results obtained with this
fraction were comparable with those observed with plasma
AT-Ill eluted otT heparin-Sepharose (Fig 5. l a n a 7 and 8).
The results indicate that the cell-frdcrived AT-Ill polypeptides with increased heparin aftinity may represent AT111 moieties with correct disulfide bonding.
c
00
02
04
06
08
IO
12
14
18
18
PO
N.CIConcwn.lion0
chronutography of dfroo-deFig 3. H.p.rin-&pIuroso
rived and pbsma-drivod AT-111w i d - . C o l t - f r n transhtion
mixtures containing ("Slmothionine-labeled AT-Ill polypoptidea
were diluted twofold in heparin-binding buffor. dialvzod overnight
at 4'c in the same buffer, and applied to heparin-Sopharose
followed by incubation for 60 minutes at room temperature.
Cell-freo-dsrived AT-Ill wai eluted off hoperin-Sopharose in
heparin-binding buffer containing 0.1 to 2.0 mol/L NaCI. and the
fractions were analyzed by SDS-PAGE on a 12% polyacrylamide
gel under reducing conditions. followed by autorodiography. Tho
percontaw of bound and unbound AT-Ill polypeptides chronutographed on heparin-Sepharosewas determined by densitometric
scanning of the autoradiograms. Pbsma A T 4 was analyzed as
described above except chat the polyacry(smide gel was stained
with Coonussia blue. (A) Heparin affinity of cell-free-darived
nornul AT-Ill,
( 0 )and AT-Ill, ,-Denver
(0);(9) heparin effinity
of plasma AT-Ill vrith
or without RRL (W); (C) heparin affinity of
trunuced AT-Ill,,,
(A).
(a)
,
Formation of Complexes of Cell-Free- Derived A T-Ill
Wirh Human a-Thrombin
The results in Fig 6A show that. when incubated with
human a-thrombin. cell-frecderived normal AT-Ill forms a
complex of higher molecular m a s of approximately 76 Kd.
This 76-Kd complex was immunoreactivewith rabbit antibodies to both human AT-Ill and human prothrombin (data not
shown). Densitometric scanning of the autoradiograms indicated that I S 7 to 20% of the cell-frderived AT-Ill could
form a complex with n-thrombin after 2 minutes of incubation. Beyond this time point. the amount of complex declined
slowly, either through degradation or dissociation. This
pattern of complex formation followed by dissociation was
also observed with plasma AT-Ill and a-thrombin at a I:I
molar ratio. However. when PPACK was added at a final
concentration of 5 mmol/L after 2 minutes. the complex did
not decline appreciably. even after 40 minutes (data not
shown). Complex formation between cell-free-derived AT111 and a-thrombin occurred only with AT-Ill that had the
ability also to bind heparin-Sepharw. Importantly. unfrac-
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1525
CELL-FREE ANTITHROMBIN 111 EXPRESSION
Cdl-1-
umr1
Fig 6. A M W S of dl-froo-dutvod AT-Ill polypoptides eluted off heparin-Sopharose. Cell-freederived AT-Ill polypeptides eluting off haparinSepharose at 0.1 mollL Ilanes 1 and 2). 0.4 mol/L
(lanes 3 and 4). and 0.7 mol/L NaCI (lanes 6 and 61.
raspoctivaly. were ~ ~ l y by
~ e
SOS-PAGE
d
on a
12% polyocrylamido gal under reducing (R) or
nonreducing (N) conditions. followed by autoradiography. Plasma AT-111previously purified ofl heparinSepharose (lanes 7 end 8 ) was analyzed in the
soma way. except that the polyacrylamide gel was
stained w i t h Coomassie blue.
uwu
1
2
3
4
2
3
4
5
66
45
36
R
6
7
8
3s
29
A
5
6
7
0
66
4
5
6
7
8
N
R
N
R
N
R
N
tionated heparin accelerated the rate of thrombin-AT-Ill
complex formation. as thc 76-Kd complex could easily be
seen after I5 seconds of incubation in the presence of heparin
(data not shown). We also observed the n-thrombindependent generation of a 43-Kd AT-Ill plypeptide in these
reactions (Fig 6: highlighted by dot).
When the cell-free-derived AT-Ill,,,,?-Denver variant
was incubated with n-thrombin, only a very small amount of
the 76-Kd complex ('c 1'7 at 2 m i n u t e ) could be identified in
the autoradiogram (Fig 6R). In addition. the 43-Kd polypeptide was not detected until 20 m i n u t e after the start of
incubation. indicating minimal reactivity between this ATIll,,,?-Denvcr variant and a-thrombin.
When plasma AT-Ill was added to the reaction mixture at
a 2:l molar ratio toa-thrombin. thecell-free-derived AT-Ill
was still able to form complex with a-thrombin (Fig 7).
However. in contrast to the results shown in Fig 6 h . the
complex formed between cell-freederived AT-Ill and
n-thrombin was stable even after 40 minutes.
Proteolysis of the cell-frce-derived AT-Ill normal and
Denver polypeptides was ruled out by the fact that control
incubations without human n-thrombin did not result in any
degradation. even after 80 minutes.
Expressionof a TrtincatedForm of AT-Ill Containing the
Thromhin-Riding Region
45
36
29
B
3
29
45
1
2
-
06
Llwy
Plasma AT-Ill
AT-Ill
&hd
0
15.'
1.
2'
5'
10'
20'
40'
Fig 6. Intraction botwnn h w n u-thrombin and ofthor
coll-froo-derived normal AT-III,e
or AT-lll,,-Omer
polypeptides. (~%)methionine-lebeled RRL transletion mixtura containing
a i t h r call-free-derived ( A ) normal AT-III,UI
or ( 8 ) AT-I1l,eD e n v u potypeptides wera diluted twofold in thrombin-binding
buffer. dialyzed overnight a t 4 ° C in tho same M e r . end incubated
w i t h human 0-thrombin at 37'C. The samples were then analyzed
by SOS-PAGE on a 10%polyacrylamide gel under reducing conditions. followed by autoradiography. Lane 1 contains no thrombin
(control). Lanes 2 through 8 contain u-thrombin and incubated for
16 seconds, 1, 2. 6, 10, 20. end 40 minutes, respectively. Tho
arrowhead indkates the migration position of the complex betwoen d-thrombin end cell-free-dwhred AT-II. The dot ( . I ropresents the migration position of the 43-Kd form of AT-Ill formed
after incubation w i t h u-thrombin.
A truncated form of human AT-Ill, containing amino
acids Met2S1 to Lys432. was syntheized to study the
putative thrombin-binding region. Authenticity of the *EM3737 + ) - A T - l l l ~ ~ , construct
.,,~
was confirmed by restriction
mapping and didmxy sequencing of the cDNA. Cell-free
translation of AT-III?~,.,,?mRNA and subsequent immunoprecipitation with rabbit plyclonal antibodies to human
AT-Ill showed a 21-Kd polypcptide by SDS-PAGE (Fig 8 ) .
which is consistent with its cxpcctcd molecular mass.
Inrwacrion of Cell-Free-Derived Truncated A T-Ill With
Heparin and n-Thrombin
The ccll-free4erivcd 21-Kd AT-Ill polypeptide did not
bind to heparin-Sepharose (Fig 3C). Densitometric scanning
showed that more than 94% of the 21-Kd plypeptide precnt
in the translation mixture had no affinity for heparin.
suggesting that the heparin-binding domain(s) of AT-Ill lie
between !lis1 andScr250. When mixed with humana-throm-
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AUSTIN ET
1526
MWM
1
2
3
4
5
6
7
AL
8
66
45
36
29
0
15"
1'
2'
5'
SO'
20'
10'
bin. the cell-free-derived 21-Kd polypeptide did not form a
covalent complex of higher molecular mass. Instead. it was
cleaved into two products of relative molecular mass of 17
and 4 Kd. respectively (Fig 9A). These smaller polypeptides
could be immunoprecipitated with rabbit polyclonal antibodies to human AT-Ill. suggesting that they had originated
from the cleaved 21-Kd polypeptide (data not shown). These
two polypeptides thus had a molecular mass which would
correspond to products derived from cleavage at the reactive
center of AT-Ill (Arg393-Scr394). Autoprotcolysis of the
21-Kd polypeptide was ruled out in control experiments in
which incubation without thrombin did not result in any
degradation. even after 80 minutes.
To confirm that the site of cleavage was the reactive
center. a 21-Kd AT-Ill-Denver polypeptide was synthesi7ed
using site-directed mutagenesis. When mixed with human
n-thrombin. this 21-Kd polypeptide cleaved much more
slowly than truncated normal 21-Kd AT-Ill polypeptide.
even after 10 minutes (Fig 9R). In addition. this 21-Kd
polypeptide appeared to undergo cleavage at a site different
from that observed with the 21-Kd normal AT-Ill p l y p e p tide.
DISCUSSION
In this study. WE report the cell-free expression of normal
and variant forms of AT-Ill. The yield of cell-free-dcrived
AT-Ill was approximately 400 to 5 0 0 ng/mL of R R L and is
comparable with that obtained using tr-interferon analogs
similarly expressed.'" A large portion of this cell-frccderived AT-Ill binds heparin. and forms a I : I stoichiometric
complex with n-thrombin. The formation of complexes
between cell-frcc-dcrived AT-Ill and n-thrombin is accclerated in the presence of heparin. The present data alsosupport
previous evidence for both the location of the hcparinbinding domain toward the amino-terminus of the AT-Ill
molecule" " and that the reactive site Arg393-Ser394 of
h u m a n AT-111 is essential f o r thrombin-AT-111
interaction.'-'"'
The cell-free-derived AT-Ill differs from plasma AT-Ill
in that it is not glycosylatcd. Previous results from other
laboratories have shown that the quantitative removal of the
carbohydrate side-chains from plasma AT-Ill has no cffcct
on heparin binding or complex formation between AT-Ill
and thrombin."" Thus. the lack of glycosylation of the
cell-free-derived AT-Ill probably docs not affect the results
obtained in the present study. tlowever, it is interesting to
note that the beta form of plasma AT-Ill, which lacks
Carbohydrate at position 135. has higher afinity for heparin
than fully glycosylatcd AT-Ill.'"
Cell-frec-derivcd human AT-I I I displayed heterogeneity
for heparin binding. Approximately 70'7 bound to hcparinSepharose, a significant increase over that reported using
-
m '1
I-
Fig 8. Anotyoh of cell-free-drived t r u n a t o d
AT-Ill,,,.
Totel tronslation mixtures labeled with
(%)methionine (lanes 1 through 3)were immune
precipitated with rabbil polyclonal antibodies to
human AT-Ill (lanos 4 through 6 ) and analyzed by
SDS-PAGE on e 12% polyacrylamide gel under
reducing conditions. followed by autorodiography.
Lanes 1 and 4 contain Bromo mosaic virus mRNA.
Lanes 2 and 6 contain no mRNA. Lones 3 and 6
contain AT-Ill,,,
mRNA.
45
38
28
20
A.
Fig 7. Interaction betweon hunun o-thrombin,
plasma AT-Ill, and cell-free-derived normal AT-Ill,
4x2 polypeptides. Tha procedure was performed as
described in Fig 6 except that plasma AT-Ill was
added to the reaction mixture at a 2:l molar ratio
with respect to humen 0-thrombin. The arrowhead indicates the migretion position of the complex between n-thrombin and cell-free-dorived
AT-Ill. The dot ( - 1 represents the migretion position d the 43-Kd form of AT-Ill formed after
incubation with u-thrombin.
TOtd
2
3'
'4
--
5
6'
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
CELL-FREE ANTITHROMBIN 111 EXPRESSION
MWM
1
1527
2
3
4
5
6
7
15'
1
5
10
20
40
2
3
4
5
15-
1'
5'
10
20.
40
6e
4s
36
29
20
14
-
A
MWM
0
1
6e
4s
3e
29
20
14
B
0
Fie 9. Intormionbetween human o-thrombin with cdl-fmod r i v e d truncated AT-IIIy5,
polypeptides. ('%)methioninelabeled translation mixtures containing either (A) cell-freederived truncated AT-IIIyB,
or (El AT-III,s, .,,-Denver
p o m i d e s were incubated with human a-thrombin at 37% and
analyzed by SDS-PAGE on a 10% to 20% polyacrylamide gradient
gel under reduclng conditions. followed by autoradiography. Lane
1 contains no n-thrombin (control). Lanes 2 through 7 show
incubation with a-thrombin for 16 seconds. 1. 6. 10. 20. and 40
minutes. respectively. The .ymbols u and B indicate, respectively.
the migration positions of 17-Kd and 4-Kd polypeptides.
,,,
mammalian cells." However. cell-frec-dcrived AT-Ill eluted
from heparin-Sepharosc at lower S a c 1 concentrations than
observed with plasma-derived AT-Ill. This decrease in
heparin binding may be due to: (I ) the presence of factors in
thc RRL which affect hcparin binding. which would be
consistent with the decrease in heparin binding observed for
plasma AT-Ill in the presence of RRL: and/or (2) incomplete disulfide bond formation of a portion of the cell-frecderived AT-Ill produced in this system. This is in agreement
with the results of Longas et a!'. in which they showed that
under mild reducing conditions. which disrupt disulfide bond
formation. AT-Ill eluted at approximately half the NaCl
concentration required for elution in the absence of reducing
agent. Recause the interaction of AT-Ill with heparin is at
least partially ionic. the present data suggest that cell-frccderived AT-Ill. which is incompletely disulfide bonded. most
likely results in reduced afinity for hcparin. In addition. the
independently expressed truncated 21-Kd AT-Ill polypcp
tide from amino acids Met251 to Lys432 produced by
pGEM-3Zf( -+ )-AT-lll?<,.,,? did not bind to hcparinSepharosc. This latter result indicates that the hcparinbinding domain of AT-Ill docs not extend beyond Mct251.
This observation is consistent with previous observations
indicating that the heparin-binding domain of AT-Ill lies in
the amino-terminal part of the molecule.'""
Approximately 20': of the ccll-frcc4erivcd human AT111 was capableof forming a stablecomplex with a-thrombin
after 2 minutes of incubation. The stability of the complex to
boiling in the presence of S D S and 1)TT strongly suggests
that ;I covalent bond was formed between AT-Ill and
a-thrombin. In addition. AT-Ill that did not have heparin
binding ability did not react with thrombin. These obscrvations indicate that a functional heparin-binding domain may
be necessary for complex formation between AT-Ill and
a-thrombin. The inability of a significant portion of ccll-frecderived AT-Ill to react with a-thrombin could be due toonc
or a combination of the following: ( a ) the formation of an
aberrantly-folded inactive subpopulation of AT-Ill molecules; (b) the presence of factors in the RRL that inhibit the
formation of complexes between cell-frcc-dcrived AT-Ill
and a-thrombin: or (c) the dissociation of some of the
AT-lll-cr-thrombin complexes during electrophoresis. as has
been described elsewhere." Recause the molar ratio of
a-thrombin to the ccll-frce4erivcd human AT-Ill was
approximately 1OO:l in our expcriments. dissociation of the
complex may also have been favored and may be related to
the presence of the 43-Kd polypeptide seen after the addition
of a-thrombin." The 43-Kd polypeptide may also represent
AT-Ill cleaved by. but not complexcd to a-thrombin. Nonetheless. human AT-Ill expressed in mammalian cells or
yeast similarly had a large fraction of expressed protein
unable to form complex with a-thrombin.'"."'
The cxact mechanism of thrombin interaction with AT-Ill
is not completely understood. nor has the exact region
responsible for complex formation been ascertained. However. certain natural AT-Ill variants have indicated that the
hydrolysis of the Arg393-Ser394 bond is essential for thrombin inactivation by AT-! I I. Site-directed mutants. involving
the Ser394 residue of AT-Ill, expressed in the grccn monkey
cell line COS provided similar information." Thus. mutations at, or near. the thrombin-binding domain of AT-Ill
may result in an abnormal conformation of the AT-Ill
molecule. causing impaired thrombin binding. AT-IIIDenver. characterized by impaired ability to inhibit thrombin. has been identified as a reactive site variant of AT-Ill
containing a Scr to Leu subtitution at position 394." When
synthcsi7ed in a cell-free system. the hT-III,.,,~-Denver
variant also displayed impaired ability to form a covalent
complex with a-thrombin. In addition. the 43-Kd p l y p c p
tide product was not evident after the addition of a-thrombin, suggesting that complex formation. followed by cleavage
of the complcx by a-thrombin. or initial cleavage of AT-Ill
by thrombin is inhibited.
To better understand the region responsible for thrombin
binding. we synthesi7cd a truncated form of human AT-Ill
which consisted of amino acids Met251 to Lys432. This
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
AUSTIN ET AL
1528
truncated 21-Kd AT-I11 polypeptide did not form complex
when incubated with a-thrombin, but was cleaved into two
distinct products of molecular mass 17 and 4 Kd, respectively. The size of these cleavage products provided further
evidence for the reactive site of human AT-I11 to be the bond
between Arg393 and Ser394. The lack of complex formation
between a-thrombin and this truncated polypeptide suggests
that other amino-terminal domains of the AT-I11 molecule,
which contain the heparin binding region, are required for
complex formation with a-thrombin to occur. It is also
possible that the truncated cell-free-produced AT-I11 molecule may not be folded in the stressed conformation deemed
necessary for complex formation between AT-111 and
a-thrombin to occur.5o However, the minimum structure of
human AT-I11 required for complex formation with a-thrombin has yet to be defined. Further evidence confirming the
a-thrombin cleavage site of AT-I11 a t the reactive center was
shown by the experiment involving site-directed mutagenesis
of the truncated AT-III,,,,,, a t Ser394 to Leu; thus forming
the AT-III,,,,,,-Denver
mutation.,, This truncated polypeptide showed little cleavage with thrombin. Such data provide
direct evidence that Ser394 is essential for the cleavage of
AT-111 by a-thrombin.
The establishment of a cell-free system for the expression
of AT-I11 will allow for the rapid production of a number of
variant AT-I11 polypeptides to study the interaction of
AT-I11 with heparin and a-thrombin. In addition, use of a
cell-free expression system allows us to focus on the structure
and function of AT-I11 variants independent of their cellular
stability and of the constraints of the cellular secretory
machinery. For instance, the human mutant Z-allele protein
of the related Serpin, a,-antitrypsin, fails to be secreted from
hepatocytes.” Thus, this cell-free expression system for
human AT-111, coupled with site-directed mutagenesis, should
enable us to have a better understanding of the functional
domains of AT-111.
ACKNOWLEDGMENT
We thank Drs Fred A. Ofosu and William P. Sheffield for helpful
discussions. This work is dedicated to the memory of Jed B.
Matthews.
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1990 76: 1521-1529
Expression in a cell-free system of normal and variant forms of human
antithrombin III. Ability to bind heparin and react with alpha-thrombin
RC Austin, RA Rachubinski, F Fernandez-Rachubinski and MA Blajchman
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