From the
CD28 and CTLA-4 are homologous T cell receptors of the
immunoglobulin (Ig) superfamily, which bind B7 molecules (CD80 and
CD86) on antigen-presenting cells and transmit important costimulatory
signals during T cell activation. Here we have investigated the subunit
structure of CTLA-4 and the stoichiometry of its binding to B7
molecules. We demonstrate CTLA-4 is a homodimer interconnected by one
disulfide bond in the extracellular domain at cysteine residue 120.
Each monomeric polypeptide chain of CTLA-4 contains a high affinity
binding site for B7 molecules; soluble CTLA-4 and CD86 form complexes
containing equimolar amounts of monomeric CTLA-4 and CD86 (i.e. a 2:2 molecular complex). Thus, CTLA-4 and probably CD28 have a
receptor structure consisting of preexisting covalent homodimers with
two binding sites. Dimerization of CTLA-4 and CD28 is not required for
B7 binding, nor is it sufficient to trigger signaling.
Intercellular interactions between T lymphocytes and
antigen-presenting cells (APC)
Binding interactions between CD28, CTLA-4, and
B7 molecules have been studied with recombinant-soluble immunoglobulin
fusion proteins of these molecules. CTLA4Ig bound to CD80 with
A fundamental uncertainty regarding the binding interactions of
CTLA-4
These fragments of CTLA-4 and CD86 were characterized by SDS-PAGE (Fig. 3A). Under nonreducing conditions, CTLA4Ig gave a
major M
An
intermolecular disulfide bond was also present in CTLA4t. CTLA4t
(prepared from CTLA4Ig analyzed in lane 1) contained a major dimeric M
Fragments of CTLA-4 and CD86 were further characterized by gel
permeation chromatography (Fig. 3B). The elution times
of CTLA-4 fragments corresponded closely to M
The disulfide
bonding pattern of CTLA4t was determined (). The position
of the intermolecular disulfide bridge in CTLA4t was determined by
further analysis of fragment Ch-(114-128). Mass spectral analysis
of the peptide was performed in the presence or absence of
dithiothreitol. This analysis revealed that the mass ((M +
H)
The intramolecular disulfide
bonding pattern was determined by Edman degradation of
cystine-containing peptides. Sequence analysis located the positions of
the purified CNBr peptides in the CTLA-4 sequence. Edman degradation of
a large molecular weight CNBr fragment gave simultaneously three
amino-terminal sequences beginning at Met
The composition of the CD86t
It was important to determine if
some of the CD86t present in the complex peak was due to contamination
with free CD86, which was present in excess. Extrapolation of the free
CD86t A214 peak (C; see also Fig. 3B) indicated
that free CD86t in the 19-20-min fraction contributed <
CTLA-4 expressed in transfected COS cells or in activated T
cells exists primarily as a protein of M
Residue Cys
Each chain of a CTLA-4 homodimer has a binding site for CD80
and CD86. Monomeric CTLA-4X has binding activity for CD80Ig and CD86Ig
in immunoprecipitation analysis and monomeric CTLA4X
This
difference may be due to differences in binding kinetics of CD80 and
CD86. We previously showed that the equilibrium dissociation constants
for CTLA4Ig binding to CD80 and CD86 were very similar (within 2-fold)
but that CTLA4Ig dissociated more rapidly from CD86 than from CD80. We
proposed that CD86 is a faster on/faster off counter receptor for
CTLA-4 than is CD80. Since monomeric CTLA-4 binds less avidly than
dimeric CTLA-4, dissociation differences between CD80 and CD86 may be
more pronounced with monomeric CTLA-4. In vivo, this
difference may be important where CD80 or CD86 expression levels are a
limiting factor for interaction with CTLA-4. Occupancy of CTLA-4 would
be suboptimal, and on average, less than two binding sites per molecule
occupied. Since complexes of CD86 with a single CTLA-4 binding site
would dissociate faster, the time of CTLA-4 receptor occupancy by CD86
would be less, and hence, effects on T cell costimulation would also be
less. Thus, differences in costimulatory signals by CD80 and CD86 would
be exaggerated at low expression levels. We previously noted that CHO
cells transfected with CD80 and CD86 differed more in their adhesion to
CD28 and in T cell costimulation when added at low numbers (14).
The
complex of CD86t
The Ig domains of CD28 and
CTLA-4 display little sequence similarity with other members of the
IgSF, limited to a few conserved residues characteristic of the Ig
fold(24) . Despite sharing a common structural scaffold, members
of the IgSF have diverse structures, utilize different combinations of
Ig domains to form binding sites, and have distinct binding
stoichiometries per molecule. In antibodies, variable regions from two
different Ig domains combine to form an antigen binding
site(25) , and on per molecule basis, each Ig molecule contains
two antigen binding sites (a 2:2 stoichiometry). With human growth
hormone receptor, two molecules (two Ig domains) bind a single growth
hormone molecule (human growth hormone receptor); the complex has a 2:1
stoichiometry(26) . Thus, the number of Ig domains per binding
site of human growth hormone receptor is the same as a Fab fragment,
but the stoichiometries of the intact molecules is different. A single
domain of CD2 binds its CD58 ligand and the binding stoichiometry on a
per molecule basis is thought to be 1:1(27, 28) .
Finally, with CD8, a single Ig domain of each chain of a homodimer is
believed to bind a major histocompatibility complex class I molecule;
thus, on a per molecule basis, CD8/MHC class II complexes have a 2:2
stoichiometry (29, 30). The stoichiometry of CD8 has not been directly
demonstrated, but is inferred from mutagenesis studies(29) .
The binding stoichiometries of CTLA-4 and CD28 most closely resemble
CD8. With both molecules each chain of a homodimer contains a binding
site and the intact molecule has a 2:2 stoichiometry. However, there
are significant differences in the way CD8 and CTLA-4
Ligand-induced receptor dimerization is a common theme
of many signaling pathways. Dimerization of growth factor receptors
permits receptor transphosphorylation (31) and facilitates
recruitment of SH2-containing effector
molecules(32, 33) . The CD28 signaling pathway also
involves tyrosine kinase activation and binding of SH2-containing
effector molecules(9) . However, CTLA-4 or CD28 signaling differ
from growth factor receptor signaling in two ways: their dimeric states
are preformed and they are inactive. Thus, with CTLA-4
Why then are CTLA-4 and CD28 covalent homodimers
having two binding sites? One possible clue to the answer is the fast
kinetic off rates of B7 binding(14) . Recent data
It is currently
unknown whether signal transduction requires occupancy of one or both
subunits of CTLA-4 and/or CD28 by B7 molecules. If occupancy of both
subunits is required for signal transduction, monomeric B7 molecules on
the APC must be brought into proximity such that both subunits of
CTLA-4
It is also possible that
signaling through CTLA-4
CTLA4t
was fragmented with CNBr and peptides were isolated by gel permeation
chromatography and identified by amino acid sequence analysis. CNBr
peptide M-(98-128) was subfragmented with chymotrypsin (Ch) and
the peptides were purified by reversed-phase HPLC. An intermolecular
disulfide bond (C120-C120) in one of these fragments was confirmed by
mass spectral analysis as described in the text. A high molecular
weight CNBr fragment was subfragmented with trypsin (T),
cystine-containing peptides were isolated and identified by amino acid
sequence analysis. Peptide T-(55-83) was sequenced only as
indicated. These analyses permitted determination of two intramolecular
disulfide bonds (Cys
We thank Gary R. Matsueda for valuable discussions in
preparing CTLA4t, William Fenderson for his excellent technical
assistance, and Drs. J. Ledbetter and A. Aruffo for comments on the
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)generate T cell
costimulatory signals which regulate T cell responses to antigen in an
antigen nonspecific manner(1) . Costimulatory signals determine
the magnitude of T cell responses, and whether an encounter with
antigen activates or inactivates subsequent responses to
antigen(2) . T cell activation in the absence of costimulation
results in aborted or anergic T cell responses(3) . A key
costimulatory signal(s) is provided by engagement of T cell receptors
CD28 and CTLA-4 with B7 molecules on APC(4) . CD28 and CTLA-4
are both members of the immunoglobulin superfamily (IgSF) having single
variable-like domains in their extracellular regions(5) . These
molecules share sequence identity, most notably clustered in their
CDR3-like regions(6) . Both CD28 and CTLA-4 bind the same
natural ligands, CD80 (formerly known as B7-1) and CD86 (formerly known
as B7-2 or B70)(7, 8) . Engagement of CD28 by B7
molecules delivers a strong costimulatory signal(4, 9) ,
whereas engagement of CTLA-4 in different systems triggers either a
weak costimulatory signal (10, 11) or a negative
signal(12) .
20-fold higher avidity than did the analogous CD28Ig(13) .
Amino acid residues responsible for the increased binding activity of
CTLA4Ig to CD80 were localized to the CDR-1 and COOH-terminally
extended CDR3-like regions(6) . CD80 and CD86 share only
25% amino acid sequence identity in their extracellular domains,
but show very similar equilibrium binding to CD28Ig or CTLA4Ig (14).
CD28 and B7 molecules is their binding stoichiometry. CD28
is a disulfide-linked homodimer, whereas the subunit structure of
CTLA-4 has been controversial. Some studies have suggested that CTLA-4
is also a disulfide-linked
homodimer(10, 13, 15) , but another suggested
that it exists as a monomer(16) . It is currently unknown
whether dimerization is required for binding activity of CD28 and or
CTLA-4, as it is for many IgSF members(17) . In this study, we
have determined the binding stoichiometry of CTLA-4 for B7 molecules.
We show that CTLA-4 is a disulfide-linked homodimer containing two
binding sites for its ligands.
mAbs, Ig Fusion Proteins, and Flow
Cytometry
Murine mAbs 9.3 (anti-CD28), G10-1 (anti-CD8), and
G17-2 (anti-CD4) and F(ab`) fragments of mAb G19-4
(anti-CD3) were provided by Dr. J. Ledbetter of Bristol-Myers Squibb
Pharmaceutical Research Institute, Seattle. Murine mAb L6 (anti-human
lung tumor antigen) and its chimeric derivative (human Fc) were from
Drs. K. E. and I. Hellström also of Bristol-Myers Squibb
Pharmaceutical Research Institute, Seattle. mAb 11D4 (anti-CTLA-4) has
been described previously(10) . mAb 7H4 (anti-CTLA-4) was
prepared in the same manner as other anti-CTLA-4 mAbs(10) ; mAb
7H4 binds CTLA4Ig, but does not block its binding to CD80. Human
CTLA4Ig, CD28Ig, CD80Ig, CD86Ig, CD80 mIg, CD86 mIg fusion proteins
were prepared as described(14) . For immunostaining, cells were
removed from their culture vessels by incubation in PBS containing 10
mM EDTA. CHO cells (1-10
10
) were
first incubated with mAbs or Ig fusion proteins in DMEM containing 10%
fetal bovine serum (FBS), then washed and incubated with fluorescein
isothiocyanate-conjugated goat anti-mouse or anti-human Ig second step
reagents (Tago, Burlingame, CA). Cells were washed and analyzed on a
FACScan (Becton Dickinson).
Cell Culture
Anti-CD3-activated T cell blasts were
prepared essentially as described(10) , except that culture
flasks were coated with F(ab`) fragments (10 µg/ml) of
anti-CD3 mAb G19-4 instead of intact mAb. Peripheral blood mononuclear
cells were isolated from heparinized blood and cultured on anti-CD3
F(ab`)
-coated flasks for 3 days at 37 °C in RPMI 1640
containing 10% FCS, 2 mM glutamine, and pen-strep (100
units/ml penicillin, 75 units/ml streptomycin). Retroviral vectors
encoding CD28 and C4-28 (extracellular and transmembrane regions
of human CTLA-4 fused to the cytoplasmic domain of CD28) will be
described elsewhere.
(
)3T3 cells were transduced
with these vectors, selected for neomycin resistance, and cultured in
DMEM containing 10% FBS, 2 mML-glutamine, 50
µM 2-mercaptoethanol, and pen-strep. COS cells were
maintained in DMEM containing 10% FBS.
Iodination and Immunoprecipitation
Cells were
harvested and washed with PBS. Cell surface iodination using
lactoperoxidase was performed as described(18) . Surface-labeled
cells were washed three times with PBS and solubilized in lysis buffer
(150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1% Nonidet P-40, 0.25% deoxycholate, 10 µg/ml each of
aprotinin and leupeptin) for 30 min at 4 °C. Detergent-insoluble
material was then removed by centrifugation (13,000 g for 15 min at 4 °C) and the supernatant was used for
immunoprecipitation. Solubilized membrane proteins were also iodinated
using lactoperoxidase. Cells (2
10
for COS cells
and 150
10
for peripheral blood lymphocytes) were
resuspended in (5 or 10 ml, respectively) hypotonic buffer (10 mM Tris-HCl, pH 7.5, 0.5 mM MgCl
, 10 µg/ml
each of aprotinin and leupeptin) and incubated on ice for 10 min. Cells
were then lysed (>95% lysis) using a tight fitting Dounce
homogenizer. The homogenate was transferred to a clean tube containing
0.1 volume of 10 mM Tris-HCl, pH 7.5, 0.5 mM
MgCl
, 1.5 M NaCl, and debris was removed by
centrifugation at 800
g for 5 min. The supernatant was
removed, and EDTA was added to a final concentration of 5 mM.
Membranes were collected by centrifugation at 125,000
g for 45 min. The membrane pellet was solubilized in lysis buffer on
ice for 30 min, and detergent-insoluble material was removed by
centrifugation. Soluble proteins were then iodinated using
lactoperoxidase, and unreacted
I was removed by passage
over a column of Sephadex G25 (Pharmacia Biotech Inc.), equilibrated
with lysis buffer. The labeled protein extract was then used for
immunoprecipitation analysis.
Immunoprecipitation
Analysis
I-Labeled lysates were incubated at 4
°C with an irrelevant antibody (2 µg) and 50-µl packed
volume immobilized protein A (IPA, r-protein A-Sepharose, Repligen,
Cambridge, MA). IPA was incubated before use for
1 h in a solution
of 5% bovine serum albumin in PBS and washed in lysis buffer. Sepharose
beads were removed by centrifugation, and aliquots of the supernatant
(1-2
10
cpm) were mixed with mAbs or Ig
fusion proteins and incubated at 4 °C for 1-2 h. Bovine serum
albumin-blocked IPA was added (50 µl packed volume), and incubation
at 4 °C was continued for an additional 1-2 h. Samples
treated with mAb 11D4 or its isotype controls (mouse IgG1) were
incubated with a ``bridging'' antibody (rabbit anti-mouse
IgG, Sigma) at 50 µg/ml for 1-2 h at 4 °C before addition
of protein A-Sepharose. Beads were collected by centrifugation, washed
four times with PBS containing 0.5% Nonidet P-40, twice with PBS alone,
and immunoprecipitated proteins solubilized in SDS-PAGE sample buffer.
When labeled lysates from anti-CD3-stimulated T lymphocytes were used,
CD28 was removed from solution by immunoprecipitation with anti-CD28
mAb 9.3 before addition of CD80Ig. For immunoblotting analysis,
aliquots of conditioned medium or samples of purified protein (1
µg) were incubated with 5 µg of human Ig (negative control),
CD80Ig, or CD86Ig for 1 h at 4 °C. Ig-containing proteins were then
precipitated with 50 µl of IPA. Precipitates were washed three
times with PBS, bound proteins were eluted by addition of concentrated
SDS-PAGE sample buffer, and samples were analyzed by SDS-PAGE.
Preparation of Recombinant Fragments of
CTLA-4
Soluble extracellular domain of CTLA-4 was expressed in
COS cells by introducing a stop codon into CTLA-4 cDNA after
Asp by polymerase chain reaction-directed
mutagenesis(6) . For CTLA4X, the OMCTLA-4 (13) expression
plasmid was used as template, the forward primer
GAGGTGATAAAGCTTCACCAATGGGTGTACTGCTCACACAG was chosen to match sequences
in the vector, and the reverse primer,
GTGGTGTATTGGTCTAGATCAATCAGAATCTGGGCACGGTTC, corresponded to the last
seven amino acids in the extracellular domain of CTLA-4 followed by a
stop codon (TGA). CTLA4X
was constructed in similar
fashion, except that the reverse primer specified a C120S mutation.
Polymerase chain reaction products were digested with HindIII/XbaI and directionally subcloned into the
expression vector
LN (a gift of Dr. A. Aruffo, Bristol-Myers
Squibb Pharmaceutical Research Institute, Seattle). Each construct was
verified by DNA sequencing.
Expression and Purification of CTLA-4
Fragments
CTLA4X and CTLA4X were produced by
transiently transfected COS cells(18) . Recombinant proteins
were purified by CTLA-4 mAb affinity chromatography from transfected
COS cell conditioned medium. Anti-CTLA-4 mAb 11D4 was coupled to
cyanogen bromide (CNBr)-activated Sepharose 4B (8 mg of mAb/ml of
resin) as suggested by the manufacturer (Pharmacia Biotech Inc.). COS
cell conditioned medium was collected, filter-sterilized, and applied
to the affinity resin by gravity flow. Following application of
conditioned medium, the resin was washed with 5 bed volumes of PBS.
Bound CTLA-4 fragments were eluted with 0.05 M trisodium
phosphate, pH 11.5. Fractions (0.5 ml) were collected and immediately
neutralized by addition of 0.2 ml of 2 M Tris-HCl, pH 6.8.
CTLA-4-containing fractions were identified by capture immunoassay (10) and/or A
. Peak fractions were
pooled, concentrated using a Centriprep 10 (Amicon, Beverly, MA), and
filter-sterilized. Recoveries of recombinant proteins were
50-90% based on immunoreactivity in the capture immunoassay.
Extinction coefficients of 1.0 and 1.1 ml/mg were experimentally
determined by amino acid analysis for purified CTLA4X and
CTLA4X
preparations, respectively. M
= 13,400, corresponding to the predicted peptide mass of
CTLA4X, was assumed for calculation of molar concentrations. Where
indicated, concentrated conditioned medium from transfected COS cells
was used as a source of recombinant CTLA-4X. This was concentrated
30-fold using a Centriprep 10 before fractionation.
Preparation of Thrombin Fragments
CTLA4Ig or
CD86Ig fusion proteins (0.5 mg/ml in Tris-HCl buffer, pH 7.4-7.5)
were digested with purified bovine thrombin (Armour Pharmaceutical
Corp., Kankakee, IL) at a final concentration of 100 units/ml, at 37
°C for 16-24 h. After cleavage, thrombin was removed by twice
incubating the protein solution with 0.10 packed bed volume of washed
Affi-Gel Blue (Bio-Rad). Ig Fc-containing fragments were removed by
twice incubating the protein solution with 0.20 packed bed volume of
washed IPA. The resulting solution contained protein fragments CTLA4t
and CD86t. The final recovery of purified fragments was
10-40%. Extinction coefficients of 1.3 and 1.2 ml/mg were
experimentally determined by amino acid analysis for purified CTLA4t
and CD86t preparations, respectively. M
values of
13,900 and 25,800, corresponding to the predicted peptide mass of a
single chain of CTLA4t and CD86t, respectively, were assumed for
calculation of molar concentrations.
Gel Permeation Chromatography
Two columns were
used for gel permeation chromatography. CTLA4X preparations were
analyzed using a TSK-GEL G2000 SW column (7.8
300
mm, Tosohaas, Montgomeryville, PA). The column was equilibrated in PBS
containing 0.02% NaN
at a flow rate of 1 ml/min.
Concentrated COS cell medium containing CTLA4X (0.2 ml) was
fractionated by gel permeation chromatography, and fractions of 0.5 ml
were collected and used for analysis. In other experiments, purified
protein fragments were analyzed on a Waters 300 SW column equilibrated
with PBS, containing 10 mM Tris-HCl, pH 7.4, and 0.01%
NaN
, at a flow rate of 0.35 ml/min. Essentially the same
results were obtained with both systems.
Protein Sequencing
Protein sequencing methods have
been described previously(21) . CTLA4t was first cleaved with
cyanogen bromide (CNBr), and the peptides were purified. CNBr peptides
were further digested with trypsin or chymotrypsin in the presence of 1
mM iodoacetamide and purified by reversed-phase HPLC.
Automated sequence analysis was performed in a pulsed-liquid protein
sequencer, and mass spectra were recorded on a Biolon 20 plasma
desorption mass spectrometer (Applied Biosystems, Inc., Foster City,
CA).
SDS-PAGE and Immunoblotting
SDS-PAGE was performed
on Tris/glycine gels. In some cases, performed gels (Novex, San Diego,
CA) were used. Analytical gels were stained with Coomassie Blue, and
images of wet gels were obtained by digital scanning. Gels containing I-labeled proteins were dried and subjected to
autoradiography. For immunoblotting, separated proteins were
electrophoretically transferred to nitrocellulose membranes. Membranes
were blocked with Specimen Diluent (Genetic Systems Corp., Redmond, WA)
and probed with anti-CTLA-4 mAb 7H4 diluted in Specimen Diluent. Bound
mAb was detected using horseradish peroxidase-conjugated goat
anti-mouse Ig (Biosource International, Camarillo, CA) and ECL reagents
(Amersham Corp.).
Competitive Binding Assays
Binding assays were
performed on immobilized recombinant B7Ig fusion proteins(14) .
Individual wells in 96-well plates (Immulon 2; Dynatech laboratories,
Inc., Chantilly, VA) were coated for 16-24 h with 50 ng/well goat
F(ab`) anti-mouse immunoglobulin antibodies (Tago). Wells
were then washed, blocked with Ig binding medium (150 mM NaCl,
50 mM Tris-HCl, pH 7.5, 10% FBS, 0.1% NaN
), and
coated with CD80 mIg or CD86 mIg at 1 µg/ml. CTLA4Ig was
radiolabeled with
I using Bolton-Hunter
reagent(14) , diluted in Ig binding medium, and mixed with
solutions of competitor proteins diluted in PBS. Aliquots of the
mixtures (50 µl) were then added to duplicate B7Ig-coated wells.
Binding was allowed to proceed for
4 h at 23 °C, wells were
washed, and bound radioactivity was measured by
counting.
Cell Surface CTLA-4 Is a Disulfide-linked
Homodimer
Various studies on the subunit structure of CTLA-4
have employed different serological reagents and different cell
types(10, 13, 15, 16) . It was unclear
how these differences contributed to the reported variation in the
subunit structure of CTLA-4. We wished to determine the subunit
structure of biologically active CTLA-4 transfected COS cells and on
activated T cells. As shown in Fig. 1, both specific anti-CTLA-4
mAb and CD80Ig immunoprecipitated from surface-labeled COS cells a M
46,000 protein under nonreducing
conditions and a M
30,000 protein under
reducing conditions. Similar results were obtained with activated T
cells; a M
50,000 protein was obtained under
nonreducing conditions and a M
33,000
protein under reducing conditions. Thus, regardless of the cell source,
CTLA-4, as recognized by specific mAb or soluble natural ligand,
migrated more rapidly under reducing conditions. This was consistent
with CTLA-4 being a disulfide-linked homodimer or being linked to an
unlabeled protein. Evidence supporting the first of these possibilities
was provided in subsequent experiments (see ).
Figure 1:
CTLA-4 contains an intermolecular
disulfide bond. Left panel, immunoprecipitation from COS
cells. COS cells were transfected with the OMCTLA-4 expression plasmid.
Forty-eight h post-transfection, cells were removed from their culture
dishes and cell surface-labeled with I using
lactoperoxidase.
I-Labeled cells were then solubilized
with non-ionic detergent solution, subjected to immunoprecipitation
analyses with: CD5Ig (4 µg) (lanes 1), control mAb
G17-2 (anti-CD4, 2 µg) (lanes 2), control mAb
G10-1 (anti-CD5, 2 µg) (lanes 3), CD80Ig (4 µg) (lanes 4), and mAb 11D4 (anti-CTLA-4, 2 µg) (lanes
5). Samples were analyzed by SDS-PAGE (10% gel) under nonreducing
(-
-mercaptoethanol (
ME)) or reducing
+
-mercaptoethanol (+
ME) conditions.
Migration positions of standards are indicated. Right panel,
immunoprecipitation from anti-CD3-activated peripheral blood T cells.
Membranes were prepared from anti-CD3-activated human mononuclear
peripheral blood cells. Detergent extracts of these membranes were
labeled with
I and subjected to immunoprecipitation
analysis. Labeled extracts were precleared of CD28 by
immunoprecipitation with mAb 9.3 before addition of the following (4
µg each): control mAb L20 (lanes 1); mAb 11D4
(anti-CTLA-4) (lanes 2); control Ig, chimeric mAb (lanes 3);
CD80Ig (lanes 4). SDS-PAGE (10% gel) was run under nonreducing
(-
ME) or reducing (+
ME)
conditions.
Preparation and Characterization of Soluble Recombinant
Fragments of the Extracellular Domains of CTLA-4 and CD86
Since
biologically active CTLA-4 was a homodimer, we wished to determine
whether one or two CTLA-4 chains comprise a binding site for B7
molecules. We therefore prepared soluble fragments of CTLA-4 and CD86
extracellular domains. Two strategies were employed, as shown
schematically in Fig. 2. The characterization of these fragments
is presented below ( Fig. 3and ).
Figure 2:
Schematic representation of Ig fusions and
proteolytic and recombinant fragments used in this study. Shown are
cartoon structures of CTLA4Ig, CD86Ig, CTLA4t, CD86t, CTLA4X, and
CTLA4X. Preparation of these molecules and elucidation
of their subunit structures are discussed in the text. The relative
positions of inter- and intracellular disulfide bonds in CTLA-4-related
structures was determined as described in Table I; the disulfide bonds
in CD86 were inferred from homology to other members of the IgSF. The
Fc domains of CTLA4Ig and CD86Ig are indicated. The arrow indicates the site of thrombin cleavage in CTLA4Ig. The relative
proportions of monomeric and dimeric CTLA4X were estimated from gel
permeation chromatography profiles of purified
preparations.
Figure 3:
Characterization of Ig fusions and
proteolytic and recombinant fragments. Purified recombinant Ig fusion
proteins, thrombin fragments and soluble receptors were analyzed by
SDS-PAGE (A) or gel permeation chromatography (B). A, SDS-PAGE analysis. Samples (1 µg) of each of the
indicated proteins were analyzed on 4-20% (lanes
1-9) or 5-15% (lanes 10-12) acrylamide
gels under nonreducing (lanes 1-7, 10-12) or
reducing (lanes 8, 9) condition: lane 1, CTLA4Ig; lane 2, CD86Ig; lane 3, CTLA4t; lane 4,
CTLA4X; lane 5, CTLA4X, which had been enriched for monomeric
protein by gel permeation chromatography; lane 6, CD86t; lane 7, molecular weight markers; lane 8, CTLA4t; lane 9, CTLA4X; lane 10, molecular weight markers; lane 11, CTLA4t (a different preparation from that shown in lane 1); and lane 12, CTLA4X
. B, gel permeation chromatography. Samples (
3-4
µg each) of the indicated proteins were analyzed by gel permeation
chromatography on a Waters 300 SW column. Panel a1, CTLA4t; panel a2, CTLA4t, following reduction with 10 mM dithiothreitol at 23 °C for 30 min; panel a3, CTLA4X; panel a4, CD86t. Panel b1, CTLA4t (a different
preparation than panel a1); panel b2, CTLA4t
following reduction with dithiothreitol; panel b3,
CTLA4X
; panel b4, CTLA4X
following reduction. Arrows indicate elution positions
of standards having the indicated molecular weights: V
, void volume; catalase, M
= 232, 000; aldolase, M
=
158,000; bovine serum albumin, M
= 67,000;
ovalbumin, M
= 43,000; carbonic anhydrase, M
= 31,000.
We first
prepared proteolytic fragments of CTLA4Ig and CD86Ig which retained
binding activity. Preliminary examination of the Ig hinge sequence
showed that it contained the sequence 126E-P-K128 which is similar to a
thrombin cleavage site in actin(22) . We determined that this
nonclassical thrombin site was suitable for producing active fragments
of CTLA-4, CD86, CTLA4t, and CD86t. As a second strategy, we introduced
an in frame stop codon into a cDNA construct encoding CTLA-4, proximal
to the transmembrane domain. The resulting cDNA was then expressed in
COS cells and, yielding a mixture of monomeric and dimeric proteins,
CTLA4X. These proteins were purified on an anti-CTLA-4 mAb affinity
column as described under ``Materials and Methods.'' Analysis
of purified preparations by gel permeation chromatography indicated
that purified preparations contained 80% monomeric CTLA4X and
20% dimeric CTLA4X. To improve the yield of monomeric CTLA4X, we
later mutated the cysteine residue which mediates the interchain
disulfide bond in CTLA-4 to a serine (see below). This mutant protein,
CTLA4X
, was expressed in COS cells and purified.
100,000 protein and a minor protein
of M
50,000 (lane 1). The minor
protein was not seen in all preparations and comigrated with the M
50,000 protein found under reducing
conditions(13) . CD86Ig migrated as a diffuse M
75,000 protein (lane 2) which was not affected by
reduction (data not shown). Thus, as shown previously, CTLA4Ig migrated
during SDS-PAGE as a disulfide-linked homodimer of M
50,000 subunits, whereas CD86Ig did not contain an
interchain disulfide bond. Since the Ig hinge disulfides were mutated
to serine residues, dimerization of CTLA4Ig results from a disulfide
bond in CTLA-4(13) . CD86 does not contain an interchain
disulfide bond, hence, CD86Ig was not disulfide linked.
38,000 protein and a minor monomeric M
22,000 protein (lane 3). The
presence of monomeric CTLA4t reflects the presence of monomeric CTLA4Ig
in the starting preparation (lane 1). Another preparation of
CTLA4t contained only the dimeric M
38,000
protein when analyzed under nonreducing conditions (lane 11).
Analysis of CTLA4t preparations after reduction revealed only the
monomeric M
22, 000 protein (lane
8). CTLA4X preparations contained a minor dimeric M
38,000 protein and a major monomeric M
22,000 protein (lane 4); reduction of this
preparation gave a M
22,000 protein (lane 9). The proportion of monomeric CTLA4X in these
preparations could be enriched by gel permeation chromatography (lane 5). This enriched preparation was stable for several
weeks when reanalyzed, indicating that monomeric CTLA4X did not readily
reform dimeric molecules. CTLA4X
migrated as a
monomeric M
22,000 protein (lane
12). CD86t migrated as a M
57,000
protein (lane 6); as with the parental molecule, CD86Ig, the
mobility of CD86t was unaffected by reduction (data not shown).
values obtained by SDS-PAGE. The CTLA4t preparation analyzed by
SDS-PAGE in Fig. 3A, lane 1, gave a major protein of M
40,000 and a minor high molecular weight
protein when analyzed by gel permeation chromatography (Fig. 3B, panel a1). The minor high molecular weight
protein was not observed in another preparation which had not been
concentrated after final purification (panel b1); reduction of
both preparations before chromatography gave a M
30,000 protein (panels a2 and b2). CTLA4X
eluted primarily as a M
30,000 peak, with a
shoulder (panel a3). CTLA4X
eluted as a M
30,000 protein (panel b3) whose
elution time was unaffected by reduction (panel b4). Thus,
CTLA4t behaved as a disulfide-linked dimer in solution, whereas
CTLA4X
and the major protein in CTLA4X were monomeric.
As shown in panel a4, CD86t eluted larger (M
140,000) than its M
determined by
SDS-PAGE (M
57,000). The elution time of
CD86t was unaffected by reduction and denaturation with 4 M
guanidine HCl (data not shown and Fig. 6). This treatment
disrupted the high avidity interaction between CTLA-4 and CD86,
suggesting that it likewise would have disrupted interactions between
subunits of CD86. Therefore, the larger apparent size of CD86t in
solution is probably not due to oligomerization, but to aberrant
migration during gel permeation chromatography, possibly resulting from
its high degree of glycosylation(19, 20) .
Figure 6:
CTLA-4 homodimer binds two CD86 molecules. A, formation of complexes between monomeric CTLA4X and CD86t.
Aliquots (3 µg,
200 pmol) of CTLA4X, which had been
enriched for monomeric protein by preparative gel permeation
chromatography (see Fig. 3A), were incubated in a final volume
of 0.1 ml with CD86t (7.4 µg,
300 pmol, dotted line,
or 15 µg,
600 pmol, solid line) for 30 min at 23
°C before fractionation by gel permeation chromatography as in Fig.
3. The arrow indicates the elution position of CD86t and the vertical dotted line the elution position of CTLA4t/CD86t
complexes. Monomeric CTLA4X elutes at 26 min. B, formation of
complexes between CTLA4t and CD86t. Aliquots of CTLA4t (3.4 µg,
250 pmol) were incubated in a final volume of 0.1 ml with CD86t
(3.7 µg,
140 pmol, dotted line, or 7.4 µg,
300 pmol, solid line) for 30 min at 23 °C before
fractionation by gel permeation chromatography. CTLA4t elutes at 25
min. C, preparative chromatography of complexes between CTLA4t
and CD86t. Aliquots of CTLA4t (22 µg,
1600 pmol) were
incubated in a final volume of 0.2 ml with CD86t (44 µg,
1700
pmol) for 30 min at 23 °C before fractionation by gel permeation
chromatography. Complexes eluting with the horizontal bar (19-20 min) were collected and used in D. The arrow indicates the elution position of CD86t. The peak
eluting at >26 min was not observed in other experiments in which
lower concentrations were used. D, composition of complexes
between CTLA4t and CD86t. An aliquot (
the total) of complexes
eluting at the position of the horizontal bar in C was dissociated by treatment for
30 min at 23 °C with 4 M guanidine HCl and 10 mM dithiothreitol and
reanalyzed as in B. This experiment was repeated twice with
identical results.
Determination of the Disulfide Bonding Pattern in
CTLA4t
CTLA4t was further characterized by protein sequencing to
identify amino and carboxyl termini and disulfide bonding patterns. The
amino terminus of CTLA4t was determined by automated Edman degradation
(21). Sequence analysis revealed an amino terminus corresponding to M1
(M27 of the precursor sequence), in agreement with the predicted site
of signal peptide processing(13) . Carboxyl-terminal sequence
analysis was performed to determine the site of thrombin cleavage.
CTLA4t was first fragmented with CNBr, and the peptides were purified
by gel permeation chromatography. The carboxyl-terminal CNBr fragment
was identified by sequence analysis. This peptide CNBr-(98-X)
was subsequently subfragmented with chymotrypsin (Ch), and the amino
acid sequence of the most carboxyl-terminal fragment
(Ch-(114-X)) was determined. The sequence of this peptide
terminated at residue Lys, suggesting that this residue
was the carboxyl terminus. Fragment Ch-(114-128) was subjected to
mass spectral analysis which confirmed the carboxyl-terminal residue as
K128. This was the predicted thrombin cleavage site.
) of Ch-(114-128) under nonreducing and
reducing conditions was 3336.4 and 1669.2, respectively. The change in
mass of the peptide following reduction demonstrates that this peptide
contained one intermolecular bond at Cys
. The
experimental mass values were in good agreement with the predicted
masses of this peptide under nonreducing and reducing conditions,
3336.6 and, 1668.8, respectively.
(or
His
), Gly
, and Asp
in equimolar
amounts. These results suggested that the large molecular weight CNBr
fragment consisted of three peptides interlinked by two disulfide
bonds: CNBr-(1(2)-53)-CNBr-(55-85)-CNBr-(86-97). To confirm
this, the large molecular weight peptide was subfragmented with
trypsin, and the disulfide-containing peptides were purified by
reversed-phase HPLC and identified by Edman degradation. Two major
cystine-containing tryptic peptides (T) were obtained:
T-(15-28)-T-(86-97) and T-(39-53)-T-(55-83).
These results demonstrate the existence of two intramolecular disulfide
bonds between Cys
-Cys
and
Cys
-Cys
. The first of these pairs is
the disulfide bond characteristic of the Ig fold. The C48-C66 disulfide
bond is unusual in the Ig superfamily, but did not appear to be
artifactual, since disulfide interchange during purification of
fragments could be excluded by the lack of carboxamidomethylated
cysteines in the fragments. This bond was also predicted in molecular
modeling studies of CTLA-4, since these residues were adjacent in the
model(6) .
Monomeric CTLA-4 Binds CD80 and CD86
To determine
if monomeric CTLA-4 contained an intact binding site for B7 molecules,
we compared B7 binding properties of monomeric and dimeric forms of
CTLA-4. Concentrated conditioned medium from CTLA4X-transfected COS
cells was subjected to immunoprecipitation analysis with CD80Ig and
CD86Ig. Immunoprecipitated proteins were separated by SDS-PAGE and
different forms of CTLA-4 were identified by immunoblotting analysis.
As shown in Fig. 4, CD80Ig and CD86Ig immunoprecipitated both
monomeric and dimeric forms of CTLA-4 from solution. Fractionation of
CTLA4X preparations by gel permeation chromatography separated
monomeric and dimeric forms of CTLA-4. Both forms were
immunoprecipitated with CD80Ig and CD86Ig. Identical results were
obtained when purified CTLA4X was used for analysis. Thus, both
monomeric and dimeric forms of CTLA-4 bound CD80 and CD86. However, the
relative amounts of monomeric and dimeric forms of CTLA-4 precipitated
by CD80Ig and CD86Ig were different (Fig. 4). CD80Ig precipitated
more monomeric CTLA-4, whereas CD86Ig precipitated more dimeric CTLA-4.
Figure 4:
Monomeric extracellular domain of CTLA-4
binds CD80 and CD86. COS cells were transfected with the CTLA4x
expression plasmid, conditioned medium was collected and concentrated
approximately 30-fold. A sample of concentrated conditioned medium (0.2
ml) was fractionated by gel permeation chromatography on a TSK-Gel
G2000 SW column at 1 ml/min and fractions (0.5 ml) were
collected. In parallel column runs, aldolase, M
= 158,000, eluted at 6.5 min. (fraction 13);
bovine serum albumin, M
= 67,000, at 6.8
min. (fraction 14); and ovalbumin, M
= 43,000, at 7.4 min (fraction 15). Aliquots of
concentrated conditioned medium (0.02 ml) or the indicated fractions
(0.25 ml) were then subjected to immunoprecipitation analysis with
human Ig (HIg, negative control), CD80Ig, or CD86Ig.
Precipitates were analyzed by SDS-PAGE (8-16% gel) under
nonreducing conditions and CTLA-4-related proteins were detected by
immunoblotting analysis. Migration positions of molecular weight (M
) standards are indicated on the left and on the right, migration positions of HIg, CD80Ig
and/or CD86Ig, and CTLA4x dimer and monomer. HIg, CD80Ig, and CD86Ig
reactivities in this assay represent cross-reaction of the horseradish
peroxidase conjugate with human Ig. This figure is representative of
three experiments using concentrated culture medium and two experiments
using affinity-purified CTLA4x.
The relative avidities of monomeric and dimeric CTLA-4 were compared
by competitive binding assay in the experiment shown in Fig. 5.
CTLA4Ig was labeled with I and bound to CD80Ig and CD86Ig
which had been immobilized on plastic wells. CTLA4t competed similar to
CTLA4Ig (EC
4 versus
8 nM for
CTLA4Ig versus CTLA-t inhibition of CD80Ig binding and
8 versus
10 nM for CD86Ig). CTLA4X
competed for
I-CTLA4Ig binding with an EC
300 nM for CD80Ig and
900 nM for
CD86Ig. Monomeric CTLA-4 was a more effective competitor than
multimeric (18) CD28Ig (EC
>
10,000
nM). The apparent plateau in binding inhibition by CD28Ig was
not seen in other experiments. Control Ig did not show significant
competition in this assay at concentrations >13,000 nM.
Thus, monomeric CTLA-4 was a less avid competitor for
I-CTLA4Ig binding than was dimeric CTLA-4, but was more
effective than multivalent CD28Ig.
Figure 5:
Comparison of the avidities of dimeric and
monomeric CTLA-4. Ninety-six-well plates were coated with CD80Ig (A) or CD86Ig (B). I-Labeled CTLA4Ig
(final concentration, 0.6 nM) was mixed with unlabeled CTLA4Ig (circles), CTLA4t (squares), CTLA4X
(triangles), or CD28Ig (inverted triangles), to
the indicated final concentrations. The mixtures were then added to
B7-coated wells and binding was measured. Binding in the presence of
competitor is expressed as a percentage of binding in wells containing
no competitor (30,000 cpm for CD80Ig and 16,000 cpm for CD86Ig).
Concentrations of competitors were determined assuming the following M
values per chain or binding site): CTLA4Ig, M
= 39,000; CTLA4t, M
= 13,900; CTLA4X
, 13,400; CD28Ig, M
= 42,000; and chimeric L6 mAb, M
= 75,000. Addition of chimeric mAb L6
inhibited
I-CTLA4Ig binding by <10% over a
concentration range of 5-13,000 nM. This experiment was
repeated twice with identical results, and similar results were also
observed using partially purified monomeric preparations of
CTLA4X.
CTLA-4 Homodimer Binds Two CD86 Molecules
To
determine the stoichiometry of CTLA-4B7 complexes, we analyzed
the size of complexes formed with monomeric and dimeric CTLA-4 and the
composition of the complex formed with dimeric CTLA-4 (Fig. 6).
Addition of increasing concentrations of CD86t to a fixed concentration
of CTLA4X monomer (A) or CTLA4t (B) resulted in
formation of larger complexes. Higher concentrations of CD86t were
required to force complete complex formation with monomeric CTLA-4X
than with dimeric CTLA4t, consistent with the lower avidity of
monomeric CTLA-4 (Fig. 5). Complexes formed with monomeric
CTLA-4X eluted
1 min later than complexes formed with dimeric
CTLA4t. The elution times of these complexes were faster than the
largest molecular weight standard, so it was not possible to accurately
estimate their molecular mass; however, a change in elution time of 1
min in the range of standards analyzed corresponded to M
70,000. To determine the composition of complexes between
dimeric CTLA4t and CD86t, larger amounts of these molecules were mixed
and complexes were subjected to preparative gel permeation
chromatography (C). The elution profile of this mixture gave a
more clearly resolved complex peak than seen in the experiment shown in B, and in addition, another late eluting peak. The identity of
the late eluting peak was not determined (it was not seen when smaller
amounts of these proteins were analyzed, see Fig. 3), although it
eluted similar to monomeric CTLA4t. A peak corresponding to free CD86t
was observed, indicating that it was present in excess. The leading
edge (19-20 min elution time) of the high molecular weight
complex peak was collected and analyzed for the relative amounts of
CD86t and CTLA4t in two ways. In the first, an aliquot of the complex
was dissociated with 4 M guanidine HCl and 10 mM dithiothreitol and reanalyzed by gel permeation chromatography (D). Peaks corresponding to CD86t and monomeric CTLA-4 were
clearly resolved. Absorbance at 214 nm is proportional to the number of
peptide bonds, and hence, to peptide mass. Thus, comparison of
integrated peak areas divided by calculated molecular masses permits an
estimate of the relative molecular ratios of CD86t and CTLA4t in the
complex. This analysis gave a ratio of CD86t to CTLA4t of 1.2:1. The
same value was obtained when a separate aliquot of the complex was
fractionated and analyzed.
CTLA-4
complex was also determined by subjecting it to NH
-terminal
amino acid sequencing. An aliquot corresponding to
of the total
isolated CD86t
CTLA4t complex was subjected to automated Edman
degradation. Two sequences were obtained simultaneously, corresponding
to the NH
termini of CD86 and CTLA-4. The initial yield of
M1 from CTLA-4 was 38 pmol and L1 from CD86, 41 pmol. At position 3, 34
pmol of valine (CTLA-4) and 37 pmol of isoleucine (CD86) were
determined. Thus, the average ratio of CD86t to CTLA4t determined by
amino acid sequence analysis was 1.1:1, similar to the ratio determined
by comparison of absorbance peaks.
3%
of initial CD86t. Since
1700 pmol of CD86t were used, then
<50 pmol were present in fraction 19-20. The total amount
of CD86t recovered was
320 pmol (40 pmol at position 1 in
1/8
total sample). Thus, <
16% (<50/320) of recovered CD86t could
be attributed to excess free CD86t based on sequence analysis. This was
similar to the 10-20% of CD86t recovered in excess of a 1:1
ratio, as measured by both absorbance and NH
-terminal
sequencing.
45,000-50,000 under nonreducing conditions and M
30,000-33,000 under reducing conditions. The native
dimeric form of CTLA-4 is preserved in soluble fragments of its
extracellular domain by an intermolecular disulfide bond at C120.
Mutation of this cysteine to serine abolished residual dimeric protein
in CTLA4X preparations. Our data thus show that CTLA-4, like its
homologue CD28, exists primarily as a disulfide linked homodimer.
is conserved in most CTLA-4
CD28
family homologues (6) but a recent report (23) showed
that chicken CD28 lacks this cysteine residue and does not contain an
intermolecular disulfide bond. This led to the proposal that chicken
CD28 differs from other family members in being monomeric. However, it
cannot be ruled out that chicken CD28 exists as a non-disulfide-linked
dimer. Our data show that although full-length CTLA-4 is predominantly
homodimeric, removal of the transmembrane and cytoplasmic domains
(CTLA4X) leads to predominantly monomeric protein. Thus, the presence
of C120 does not ensure dimerization. This suggests that dimerization
of CTLA-4 and formation of the disulfide bond at Cys
requires the transmembrane and/or cytoplasmic domains of the two
chains.
competes for binding of
I-labeled CTLA4Ig to CD80Ig
and CD86Ig. Monomeric CTLA-4 had reduced binding activity when compared
with dimeric CTLA-4. CTLA4X
was 30-90-fold less
active at competing for
I-CTLA4Ig than dimeric CTLA4t.
CD80Ig and CD86Ig differed in their preferences for monomeric and
dimeric forms of CTLA-4. Monomeric CTLA-4 was preferentially
immunoprecipitated by CD80Ig, whereas dimeric CTLA-4 was preferentially
precipitated by CD86Ig. In competitive binding assays, dimeric CTLA4t
was approximately equivalent at competing for
I-CTLA4Ig
binding to CD80Ig and CD86Ig (EC
8 versus 10
nM, respectively), whereas monomeric CTLA4X
was
3-fold less effective at competing for binding to CD86Ig
than to CD80Ig (EC
300 versus 900
nM). Thus, CD86 binds less well to monomeric CTLA-4.
CTLA4t formed in the presence of excess CD86t was
isolated and determined to contain 1.1-1.2 mol of CD86t/mol of
CTLA-4. Thus, one molecule of CD86t fragment bound per chain of CTLA4t
or two molecules of CD86t per CTLA4t homodimer (i.e. a 2:2
stoichiometry). Since CD28 is homologous to CTLA-4, and is homodimeric,
it likely also binds two B7 molecules.
CD28 bind
ligand. Different areas of the V(ariable)-like domains are utilized for
binding, with the CDR-1 and CDR-2-like loops and
-strands A and B
being involved in the binding site of each CD8 subunit(29) , and
the CDR-1 and CDR-3-like loops and
-strand G, at least in part,
being involved in binding by CTLA-4(26) . Also, CD8 forms an
Fv-like homodimer, which is stable even in the absence of a disulfide
bond (30). In contrast, the two subunits of a homodimer of CTLA-4 have
no particular affinity for each other, and monomeric CTLA-4 is stable
in solution.
CD28,
dimerization per se is not sufficient to trigger signal
transduction.
(
)indicate that the t of CTLA4Ig occupancy by
CD80Ig and CD86Ig is
11 and
3 s, respectively, and that the t of CD28Ig occupancy is also very short. These off rates are
rapid compared with those known for growth factor/growth factor
receptor interactions(34, 35, 36) . Rapid
receptor kinetic off rates favor disengagement of CD28/CTLA-4/B7
intercellular interactions, but discourage productive intracellular
signaling. Homodimerization of CTLA-4 and CD28 should enhance signaling
by increasing the time of receptor occupancy by ligand. Homodimeric
CTLA-4 has increased binding avidity for B7 molecules, since the
probability of rebinding ligand after its dissociation is increased.
The effective time of receptor-ligand interactions would thereby be
prolonged and receptor activation may be increased.
CD28 can be occupied. However, unlike CTLA-4 and CD28, B7
molecules are not disulfide-linked. Thus for full occupancy of
CTLA-4
CD28 to occur, B7 molecules must either preexist as
noncovalent oligomers on the APC, or they must oligomerize as the
result of binding to CTLA-4
CD28. Evidence for the former
possibility was provided by immunofluorescence microscopy studies
showing that that B7 molecules on human Langerhans cells assume a
non-uniform distribution(37) .
CD28 requires occupancy of only one
receptor subunit by a B7 molecule. If so, oligomers of B7 on APC (37) could cross-link different CTLA-4
CD28 homodimers.
Early studies (4) showed that CD28 receptor cross-linking with
mAbs enhanced signaling through this receptor, but that Fab fragments
did not, suggesting that cross-linking of CTLA-4
CD28 receptors is
an essential feature of their signaling pathways. The binding
stoichiometry of CTLA-4
CD28 receptors may enhance
CTLA-4
CD28 receptor cross-linking by B7 aggregates on APC.
Table: Disulfide-linked peptides from CTLA4t
-Cys
and
Cys
-Cys
) as described in the text.
Numbers are from the amino terminus of mature CTLA4t.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.