From the Center for Advanced Research in
Biotechnology of the University of Maryland Biotechnology Institute
and the National Institute of Standards and Technology, Rockville,
Maryland 20850, the § Instituto de Química,
Universidad Nacional Autónoma de México, Circuito Exterior,
Ciudad Universitaria, Coyoacán México, Distrito Federal
04510, and
Otsuka Pharmaceutical, Inc., Rockville, Maryland
20850
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The murine monoclonal IgG1 antibody 7A9 binds
specifically to the endothelial leukocyte adhesion molecule-1
(E-selectin), inhibiting the attachment of neutrophils to endothelial
cells. The primary and three-dimensional structures of the Fab fragment of 7A9 are reported. The amino acid sequence was determined by automated Edman degradation analysis of proteolytic fragments of both
the heavy and light chains of the Fab. The sequences of the two chains
are consistent with that of the IgG1 class with an associated light
chain with two intrachain disulfide bridges in each of the heavy and
light chains. The tertiary structure of the antibody fragment was
determined by x-ray crystallographic methods at 2.8 Å resolution. The
F(ab')2 molecule, treated with dithiothreitol,
crystallizes in the space group
P212121 with unit cell parameters
a = 44.5 Å, b = 83.8 Å, and
c = 132.5 Å with one Fab molecule in the asymmetric
unit. The structure was solved by the molecular replacement method and
subsequently refined using simulated annealing followed by conventional
least squares optimization of the coordinates. The resulting model has
reasonable stereochemistry with an R factor of 0.195. The
7A9 Fab structure has an elbow bend of 162° and is remarkably similar
to that of the monoclonal anti-intercellular adhesion molecule-1
(ICAM-1) antibody Fab fragment. The 7A9 antigen combining site presents
a groove resembling the structure of the anti-ICAM-1 antibody, and
other antibodies raised against surface receptors and peptides.
Residues from the six complementary determining regions (CDRs) and
framework residues form the floor and walls of the groove that is
approximately 22 Å wide and 8 Å deep and that is lined with many
aromatic residues. The groove is large enough to accommodate the loop
between
-strands
4 and
5 of the lectin
domain of E-selectin that has been implicated in neutrophil adhesion
(1).
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neutrophils are the major type of cell involved in the early stages of many forms of acute inflammation. The neutrophil accumulation in the lung can cause disease by damaging normal host tissue as in the case of adult respiratory distress syndrome (ARDS)1 (2), in the inflammation response (asthma, and graft rejection), or in insult as a result of trauma or bacterial infection. Cell surface receptors such as E-selectin (also called ELAM-1; endothelial leukocyte adhesion molecule) belong to a subclass of the IgG superfamily, including VCAM (vascular cell adhesion molecule) and ICAM (intercellular adhesion molecules 1, 2, and 3). E-selectin reacts with a fucosylated carbohydrate residue on the neutrophil (3), whereas the leukocyte integrins containing the CD18 antigen react with ICAM-1. The expression of E-selectin and ICAM-1 is induced by cytokines produced at the site of inflammation. This serves to enhance neutrophil adherence to endothelial cells and migration from the circulation into extracellular tissues at these sites. Neutrophils contribute further to the inflammatory process by releasing tissue-damaging mediators.
E-selectin is a member of the selectin gene family. Each of these
proteins is composed of an amino-terminal lectin domain followed by an
epidermal growth factor-like (EGF) domain, five complement regulatory
repeat units, a single membrane-spanning region and a carboxyl-terminal
cytoplasmic domain. The lectin and the EGF domains are both necessary
and sufficient to mediate neutrophil adhesion (4). The
three-dimensional structure of the lectin/EGF domains that includes the
ligand-binding region has been reported for this molecule (1). A
specific surface region of the lectin domain of E-selectin that
contains a loop between -strands
4 and
5 with exposed Tyr-94 and Arg-97 side chains has been
implicated in ligand binding based on the structural and related
biochemical studies (1).
Antibodies and antibody fragments specific for the cell-surface receptors have the potential for modulating the interaction of neutrophils with endothelial cells, and hence, the inflammatory response. A monoclonal anti-ICAM-1 antibody R6.5, that inhibits the attachment of neutrophils to endothelium and also prevents the attachment of major group human rhinovirus (HRV) to ICAM-1, has been reported (5). Recently, the structure of the Fab fragment of this antibody was reported at 2.8 Å resolution (6). The surface contour of the antigen-combining site of this molecule has a groove that resembles more the structure of an antipolypeptide antibody than the structure of an antiprotein antibody.
Another potent blocking antibody directed at a cell surface receptor, E-selectin in this case, has been raised. This antibody binds to the lectin/EGF domain of E-selectin.2 We report here the primary and three-dimensional structures of the Fab fragment determined by automated Edman degradation analysis of proteolytic fragments of the heavy and light chains of the antibody and by x-ray crystallographic techniques at 2.8 Å resolution, respectively. The analysis of the structural studies of the anti-E-selectin 7A9 Fab presented here focuses on the complementary determining regions and the comparison with the three-dimensional structures of an anti-ICAM Fab and the anti-human rhinovirus Fab.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibody Production-- The perfusion system used was a 50-liter stirred tank bioreactor utilizing an external rotating filter. SP-20 fusion mouse-mouse hybridoma cells were cultured in a medium with a protein-free formulation, and 0.1% Pluronic F-63 (Sigma) was added for shear protection. The cell densities were maintained at 1 × 107 viable cell counts/ml and perfusion-adjusted to keep nutrients (glucose, etc.) and metabolites at the appropriate levels. Ion exchange and affinity chromatography were used to purify the antibody.
Generation and Purification of F(ab')2-- The conversion of 7A9 antibody to F(ab')2 was accomplished by incubating the IgG1 with pepsin (E:S of 1:100) for 2 h at 37 °C, after adjusting pH to 3.5 using 1.0 M citrate buffer. The digestion products were subjected to cation-exchange chromatography in an S-Sephacryl fast flow column, using a linear sodium chloride gradient (0.1-0.5 M) in 20 mM acetate buffer, pH 5.0, as eluant. F(ab')2 eluted as a single peak with purity greater than 97%, as judged from SDS-polyacrylamide gel electrophoresis analysis (results not shown).
Flow Cytometry Analysis--
Titration analysis of the intact
antibody and of the F(ab')2 was performed using a Coulter
FACS Elite Analyzer. Samples for the flow cytometry analysis of
endothelial cells (human umbilical vein endothelial cell, HUVEC)
included stimulation with and without interleukin-1 (IL-1
). This
assay measures the number of cells that shift to greater fluorescence
intensity after specific binding (positive response). In this analysis,
the intact 7A9 antibody and the F(ab')2 fragment showed
comparable binding efficiency to the surface of HUVEC cells on a molar
basis, indicating that the digestion with pepsin did not affect its
binding ability (Fig. 1A).
|
Biological Activity--
Biological activity was based on
inhibition of HL60 (a myeloid leukemia line from ATCC) binding to
soluble E-selectin assay (7). Ten µg/ml of the 7A9 antibody
F(ab')2 fragment were added in wells, followed by
51Cr-labeled HL60 cells (105/well) and
incubated for 30 min at 22 °C in a static adhesion assay.
Nonadherent cells were removed by rinsing with 1% bovine serum albumin
in 0.1 M phosphate-buffered saline, and the residual activity in the wells was measured in a counter. The details of the
assay have been previously described (8). Fig. 1B shows that
the anti-E-selectin antibody F(ab')2 fragment is inhibitory to granulocyte binding to HL60 cells in a dose-dependent
manner.
Primary Structure Determination--
The amino acid sequences of
the F(ab')2 heavy and light chains were determined after
separating both chains of the reduced, alkylated intact 7A9. The
samples were reduced with dithiotreitol, alkylated with vinylpyridine
or iodoacetamide, and purified by reverse-phase high performance liquid
chromatography. Each purified chain was subjected to automated Edman
degradation analysis. Additionally, a reduced alkylated sample from
purified F(ab')2 was digested with Lys-C protease, and the
resulting fragments were separated by reverse-phase high performance
liquid chromatography using a C-18 column. The amino acid sequences of
the purified peptides were determined by using a protein microsequencer
(ABI, Model 477-A) coupled to an on-line PTH-Analyzer (Model 120-A) and
a 900-A data reduction module. Digestion with Lys-C protease resulted in 26 peaks for the heavy and light chains of an Fab fragment (chromatogram not shown). Fig. 2 shows
the complete amino acid sequence for each of the peaks in the heavy and
light chains of the fragment. The sequences of the two chains are
consistent with that of the IgG1 class with an associated light
chain. Both the heavy and light chains have two intra-chain disulfide
bridges. The sequences of the six CDRs are indicated in Fig. 2.
|
Crystallization-- The F(ab')2 was incubated before the crystallization trials with dithiotreitol for 19 h at a ratio of 2:1 (w/w). The initial crystallization trials were performed using a "fast screen" approach (9) employing the Hampton Research Crystal Screen kit (10). Experiments were set up using the hanging drop vapor diffusion method (11). For the screen, the crystallization drops were prepared by mixing 3 µl of a solution containing 9.6 mg/ml protein in 10 mM HEPES/HCl at pH 7.0 with an equal volume of the reservoir solution. Initially, small crystals appeared in droplets equilibrated against well solutions containing 0.2 M (NH4)2SO4, 0.1 M sodium cacodylate, 30% (w/v) PEG8k at pH 6.5. Refinement of the crystallization conditions was performed using sitting drop vapor diffusion experiments (12). The final reservoir solution consisted of 0.2 M (NH4)2SO4, 0.1 M sodium cacodylate, and 20% (w/v) PEG8k at pH 6.5. Crystals appeared within 3 days at room temperature and grew to full size within 2 weeks (typical dimensions of 0.2 × 0.3 × 0.7 mm3).
X-ray Data Collection and Processing--
The diffraction data
were collected at room temperature using a Siemens electronic area
detector. This detector was mounted on a Rigaku RU-200 generator
operated at 40 kV, 60 mA with a 0.3-mm focal spot. A graphite
monochromator followed by a 0.5-mm collimator was used. During data
collection, the area detector was mounted 16 cm from the crystal and
2 was 16°. Diffraction data collected with the area detector were
electronic images, each comprising a 0.2° oscillation counted for 3 min. The determination of unit cell parameters, crystal orientation,
and the integration of the reflection intensities were carried out with
the XENGEN program system (13). The crystals are orthorhombic,
belonging to space group P212121
with unit cell constants a = 44.5 Å, b = 83.8 Å, and c = 132.5 Å. The volume of this cell
indicates one Fab molecule in the asymmetric unit with a
Vm (14) of 2.66 Å3/dalton,
corresponding to a solvent content of 53%. From a total of 42,928 observations extending to 2.8 Å resolution, a unique data set of
10,993 reflections (86.5% complete; I > 2.1
(I)) was obtained with a merging R-factor of
0.068.
X-ray Structure Determination and Refinement-- The structure of the 7A9 Fab fragment was determined by molecular replacement (15) followed by iterations of expanding and adjusting the model to fit the electron density map and refining the structure. The molecular replacement and refinement calculations employed the X-PLOR program package (16). All the model building and correction procedures were carried out using the program QUANTA Version 4.1.1 (Molecular Simulations Inc., Burlington, MA).
The search model used in the molecular replacement calculations was based on the crystal structure of the Fab 8F5 that neutralizes human rhinovirus serotype 2 (Protein Data Bank code 1BBD) (17). This Fab has a high percentage of sequence identity with the 7A9 Fab, 96% for the light chain and 83% for the heavy chain, although its elbow bend angle is the smallest reported to date (127°). The search probe was constructed by truncating the side chains at the CB or CA atoms for all corresponding residues that were different between the 8F5 and the 7A9 Fabs. The rotation search was done using the intact probe with variable "elbow bend" angles between variable (V) and constant (C) domains ranging from 127° to 167° (16). The largest peak in the rotational function search was 6
|
Structure Analysis and Comparison-- Analysis of the stereochemistry of the intermediate and final models was done using the PROCHECK program package (20). The molecular comparisons were carried out with the ALIGN program (21) using only the CA atom positions of the polypeptide backbone. Surface curvature and electrostatic calculations were performed using the program GRASP (22).
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular Structure--
The tertiary and quaternary structure of
this molecule is consistent with all the crystallographically
determined Fab structures. It presents an elbow angle (between the
pseudodyad axes relating the V and C domains of
the two chains (23)) of 162°, 35° larger than the search model
(Fig. 3). The four domains of the Fab are characterized by two -sheets packed closely against each other with
a disulfide bridge connecting them (VL,
Cys23-Cys94; VH,
Cys22-Cys96; CL,
Cys140-Cys200; and CH,
Cys147-Cys199).
|
|
The Antigen Combining Site-- The most interesting feature of the structure is the surface of the antigen combining site that presents an irregular concave surface (Fig. 5). Currently it is known that five out of the six hypervariable loops that form the antigen-binding site are limited to a few main-chain conformations (canonical) (26). These conformations can be predicted by the size of the CDR and the occurrence of a specific set of residues that produce a known conformation. Recent studies (27) indicate that antibodies exhibit a surprisingly small number of combinations of canonical structures. Moreover, it has been shown (28) that the shapes allowed by these combinations correlate, in some cases, with the type of antigens with which the antibody interacts. Four different groups of antibody-antigen topographies have been distinguished: concave (for small haptens), moderately concave, grooves (for peptides and carbohydrates), and planar (for proteins).
|
Comparison of Anti-E-selectin, Anti-ICAM, and Anti-human Rhinovirus Fabs-- Fig. 5 shows the CA superposition for the variable fragments (Fv) of the three antibodies, 7A9, and R6.5 (Protein Data Bank code 1RMF) (6) directed against cell receptors E-selectin and ICAM-1, and the Fv of the anti-human rhinovirus 85F (Protein Data Bank code 1BBD) (17), used for the molecular replacement analysis. The combining sites of 7A9 and 8F5 belong to the same canonical class, while R6.5 has an extra residue in the L1 CDR and belongs to class 1-2-4-1-1. Structurally it is clear that these Fv fragments are remarkably similar. For the VL domain, the main differences can be appreciated in the L1 CDR loops, due mainly to differences in length. However, in the three structures, this loop is pointing into the solvent. A greater variation is observed for the VH domain. At this stage of the refinement, we cannot conclude anything concerning H3 CDR because we could not find clear density for the five residues at the tip of the loop. On the other hand, this is the most variable loop among antibodies.
The conformation of H2 CDR is very similar for 7A9 and 8F5; however, R6.5 shows a conformation not observed before for this type of region. It is possible that this loop had been incorrectly built (31). Evidently, the topology of the binding site is approximately the same, despite some slight differences in the conformation of some loops. To stress this fact, the molecular surface of 7A9 and R6.5 Fabs is shown in Fig. 6. Clearly, both antibodies present a groove at the antigen binding site.
|
Conclusions--
The feature most frequently observed in
antibody-peptide complex structures is a -turn in the peptide that
is embedded in the antibody-antigen interface. Several examples of this
recognition motif have been described to date and include a type I
-turn, reported for the VP2 peptide in complex with 8F5 (32) and a type II
-turn for the HIV-1 peptide (gp120) in the 59.1 complex (33). Shoham (30) had suggested that antipeptide antibodies seemed to
induce a
-turn conformation in the bound peptide, irrespective of
the peptide sequences. However secondary structure predictions for the
sequences have shown that they have a tendency to form the turn types
found in the Fab-peptide structures. Therefore, it would be expected
for these peptides to mimic their protein counterparts (30, 34, 35). In
1995, Tormo et al. (36) reported a model for 8F5 IgG docked
onto the viral surface through the peptide in VP2, which adopted the
same conformation. On the other hand, Jedrzejas et al. (6)
proposed that R6.5 could bind ICAM-1 through a
-turn in domains D1
or D2.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Patricia Hughes, James Wilkins as well as Tom Rohrer, Maurice Guertin, Cathy Kletke, Bob Dunn, and Magdalena Bystricka for performing fermentation, purification, and characterization work, Norma Gabor and Dawson Beall for the contribution in performing bioassay, and Dr. Juan Carlos Almagro for valuable comments. Dr. Adela Rodríguez-Romero thanks the CONACyT and the Dirección General de Asuntos del Personal Academico, UNAM, for support for sabbatical leave at CARB.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (codes 1a5f and r1a5fsf) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶ To whom correspondence should be addressed: The National Institute of Standards and Technology, 9600 Gudelsky Dr., Rockville, MD 20850.
1
The abbreviations used are: ARDS, adult
respiratory distress syndrome; E-selectin, endothelial leukocyte
adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; EGF,
epidermal growth factor; FACS, fluorescence-activated cell sorter;
Fv, variable domain fragment; HUVEC, human umbilical
vein endothelial cell; IL-1, interleukin-1
; PEG8k, polyethylene
glycol with an average molecular weight of 8,000; CDR, complementary
determining regions.
2 W. Newman, unpublished observation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|