From the
We established monoclonal antibodies (mAbs) against the mouse
macrophage galactose/N-acetylgalactosamine-specific lectin
(MMGL) that is a 42-kDa calcium-dependent lectin, using a solid phase
carbohydrate binding assay as a novel strategy for screening mAbs. The
specificity of six mAbs were investigated by antibody binding to native
or recombinant forms (rML) of MMGL, flow cytometry, and
immunoprecipitation using a macrophage cell line RAW264.7. Four of
these mAbs strongly inhibited the binding of fluorescein
5-isothiocyanate-labeled galactosylated polylysine to immobilized rML,
one inhibited moderately, and one did not inhibit binding. The
competitive binding study revealed that the binding sites of these four
blocking mAbs were closely related to each other but were different
from the rest of these mAbs. A non-blocking mAb having a unique binding
specificity (LOM-11) exhibited calcium-dependent binding to rML,
suggesting that calcium-dependent epitope was not situated in the
vicinity of the ligand binding site. Furthermore, pretreatment of rML
with the mAb LOM-11 preserved ligand binding activity, especially in a
low calcium environment. The four blocking mAbs mentioned above
facilitated the binding of the mAb LOM-11 to rML. These results
indicate that there is a positive cooperativity between the
lectin's ligand binding site and its physically distinct
calcium-dependent epitope.
Diverse structures of cell surface glycoconjugates on a variety
of cell types have recently been postulated to be a biological basis
for the specificity of cell to cell interactions. Evidence for this has
been provided by investigations of selectins and their specific
carbohydrate
ligands(1, 2, 3, 4, 5, 6, 7) .
Such interactions play an essential role in the process of
leukocyte-vascular endothelial cell-adhesive interaction during
inflammation and in other physiological mechanisms such as lymphocyte
homing. In addition to the physiological standpoints, pathological
studies revealed that certain carbohydrate determinants are recognized
as tumor markers with prognostic and diagnostic
values(8, 9, 10) . To understand the biological
roles of the expression of diverse carbohydrate structures, the study
of intrinsic carbohydrate recognition molecules (or animal lectins) is
one of the most important subjects to elucidate. In mammals, two major
categories of lectins have been recognized(11) . One such group
is calcium-dependent (or calcium-type) lectins, which includes
selectins and a variety of cell surface molecules found on macrophages
(12-15) and NK cells(16, 17, 18) . Some of
the lectins on macrophages and NK cells have been reported to
participate in recognition mechanisms involved in natural immunity such
as immunosurveillance against tumor
cells(19, 20, 21) . However, the way in which
the ability of calcium-type lectins to recognize specific carbohydrates
relates to their biological roles on macrophages and NK cells has not
been determined.
Recent studies have revealed a considerable
diversity of macrophages depending on their tissue location and
differentiation stage. Clear evidence for such diversity has been
provided by the development of a variety of monoclonal antibodies
(mAbs)
To gain further insight into the biological functions of MMGL, it
would be of great importance to produce mAbs whose binding sites are
related to the carbohydrate binding activity of the calcium-type
lectin. Toward this end, we have previously established a simple solid
phase enzyme-linked immunosorbent assay (ELISA)-based method for
quantitatively measuring lectin activity of MMGL (ligand binding assay)
(25). In the present report, we describe development of mAbs directed
against MMGL and their characterization by means of the ligand binding
assay, which represents a novel strategy for screening mAbs against
calcium-type lectins. We also discovered a distinct calcium-dependent
epitope on MMGL, physically separated from the carbohydrate binding
site. The significance of this epitope in the regulation of the
molecular function of this molecule was also investigated.
In the case of the ligand
binding assay(25) , rML-coated wells received 75 µl of a
solution containing hybridoma cell culture supernatant or varying
concentrations of purified mAbs diluted in buffer D-1% BSA containing
glutathione (buffer D (+glutathione)-1% BSA). For an EGTA control,
75 µl of 10 mM EGTA in 20 mM MOPS (pH 7.0), 0.15 M NaCl, 0.1% Tween 20 (buffer E) containing glutathione and 1%
BSA (buffer E (+glutathione)-1% BSA) were added to an ELISA plate
well. After incubation for 30 min at 4 °C, each well received 25
µl of FITC-Gal-PLL (2 µg/ml in buffer E with glutathione and 1%
BSA) solution and was then incubated for 1 h at 4 °C. The binding
of FITC-Gal-PLL was detected by 1 h of incubation of ELISA plates at
room temperature with 100 µl of peroxidase-conjugated rabbit
anti-FITC (diluted 1/500 in buffer D-1% BSA) in each well. In some
experiments, rML-coated wells received 100 µl of buffer D
(+glutathione)-1% BSA with or without varying concentrations of
purified mAb LOM-11 or LOM-14 and were incubated for 1 h at room
temperature. The wells were washed three times in buffer D and then
three times in an appropriate calcium-EDTA buffer modified from buffer
E (specified in the legend for Fig. 7) or buffer D to remove
unbound antibodies. Then, each well was incubated for 1 h at room
temperature with 100 µl of varying concentrations of FITC-Gal-PLL
in the corresponding calcium-EDTA buffer (+glutathione)-1% BSA to
measure ligand binding or with calcium-EDTA buffer
(+glutathione)-1% BSA or buffer D (+glutathione)-1% BSA alone
to measure the effect of exposure to calcium-EDTA buffer on mAb
binding. The wells were washed three times in the corresponding
calcium-EDTA buffer or buffer D, followed by washing in buffer D three
more times. The binding of ligands or mAbs was detected as described
above.
Carbohydrate-protein interaction at cell surfaces is an
important recognition event in multicellular organisms. Cell surface
lectins are believed to recognize carbohydrate chains on the surfaces
of other cells and to potentially transduce extracellular signals.
Efforts to elucidate such possibilities, however, have been hampered by
the absence of adequate reagents to identify the functional status of
cell surface lectins in vitro or in vivo. We focused
on the macrophage cell surface calcium-type lectin specific for
galactose and N-acetylgalactosamine. The major findings in
this study are as follows. 1) By utilizing an ELISA-based ligand
binding assay, we succeeded in obtaining mAbs against MMGL (macrophage
Gal/GalNAc-specific calcium-type lectin) with different characteristics
including those capable of inhibiting ligand binding. 2) We discovered
a calcium-dependent epitope, defined by a non-blocking mAb (LOM-11)
described in this study, that is not in the vicinity of the ligand
binding site. This suggests the presence of a calcium-dependent
conformation. 3) Binding of antibodies to the calcium-dependent epitope
stabilized the rML conformation into an active one, especially in
environments where free calcium concentration is low.
We used native
MMGL rather than rML as an immunogen because we did not want to provoke
synthesis of antibodies that recognize epitopes only displayed on rML
as accessible forms. To obtain a sufficient quantity of native MMGL, we
used a macrophage cell line RAW264.7 instead of peritoneal macrophages.
The choice of this cell line was based on our preliminary
experiments,
The reactivity of mAbs to cell surface MMGL on
RAW264.7 cells was demonstrated by flow cytometric analysis (Fig. 3). This was consistent with the reactivity against rML
that contained only extracellular portions of MMGL. In addition, all
mAbs described here clearly immunoprecipitated a 42-kDa component from
RAW264.7 cell lysate as a single band, except that LOM-8.7 and LOM-8.2
also immunoprecipitated an additional minor band of approximately 80
kDa (Fig. 2). This characteristic was shared with only the
LOM-8.7 and LOM-8.2 subclones and was not true for LOM-4.9 or LOM-4.7,
even though these mAbs had closely related binding sites (Fig. 5). The additional 80-kDa minor band could be a dimeric
form of MMGL whose epitopes were only available to mAbs of the LOM-8
series. Alternatively, this band may represent a minor glycoprotein
that had a cross-reactive epitope.
Calcium has been reported to play
critical roles in the formation of a network of coordination and
hydrogen bonds that stabilize the ternary complex of protein, calcium,
and carbohydrate as revealed by crystallographic studies on
mannose-binding proteins(31) . In the present immunochemical
study, we provided evidence showing that calcium affected overall
conformation of MMGL. The change in the conformation is detected by an
antibody termed LOM-11. The binding of LOM-11 mAb to rML was greatly
inhibited by the presence of EGTA (Fig. 6). The detection of a
conformation representing calcium-bound state by a mAb LOM-11 is
consistent with the observation from x-ray crystal structure of the rat
mannose-binding protein that two calcium in the carbohydrate
recognition domain help to organize a portion of the molecule with no
regular secondary structure(31) . Such a characteristic is
unique to LOM-11 and was not shared with other mAbs, including those
capable of inhibiting ligand binding. Interestingly, the binding site
of LOM-11 appeared to be physically separated from the carbohydrate
binding site. This conclusion was based on the following observations.
1) Ligand (FITC-Gal-PLL) binding to rML was not inhibited by LOM-11 (Fig. 4). 2) LOM-4.9, LOM-4.7, LOM-8.7, and LOM-8.2 did not
inhibit binding of biotinylated LOM-11 (Fig. 5b). In
other words, four different mAbs capable of inhibiting ligand binding
to rML did not inhibit binding of biotinylated LOM-11. 3) LOM-11 did
not affect binding of biotinylated LOM-8.7 (Fig. 5a) and
biotinylated LOM-4.7. This also supports the case for physical
distinction between LOM-11 binding sites and epitopes that are closely
associated with the carbohydrate binding site. Thus, the removal of
calcium resulted in a dynamic change in the conformation of rML, and
this change was detected by the mAb LOM-11, whose binding site was
distinct from the carbohydrate binding site.
Although remarkable
reduction in the binding of LOM-11 was demonstrated in the presence of
10 mM EGTA, absolute abolishment was not seen under this
condition (Fig. 6). Considering the loss of carbohydrate binding
activity of rML in 7.5 mM EGTA (Fig. 4), the
EGTA-resistant binding of LOM-11 at high concentrations (about 100-fold
difference) might represent weak affinity between LOM-11 and
calcium-free rML. An interesting possibility was that the binding of
LOM-11 to calcium-free rML might enhance an affinity between calcium
and rML. Measurements of calcium binding to rML would be necessary to
clarify this issue.
LOM-11 binding sites not only represented a
calcium-dependent conformation but also were involved in the
carbohydrate binding activity of rML. As already reported(25) ,
rML lost its carbohydrate binding activity at a free calcium
concentration of less than 10
In conclusion, we established a series of mAbs with
different characteristics against a macrophage calcium-type lectin
using carbohydrate ligand binding assay as a novel strategy for
screening. We found a calcium-dependent epitope on this lectin that was
defined by mAb LOM-11. Binding of antibody to this epitope stabilized
the carbohydrate binding activity of this lectin, especially in low
calcium environments. The practical strategy we employed will be useful
as a general method for screening mAbs against cell surface
calcium-type lectins. It remains to be elucidated 1) whether the effect
of the mAb LOM-11 is seen for MMGL on macrophages, 2) whether a further
cooperative relationship between the carbohydrate binding site and the
calcium-dependent epitope exists, 3) whether the binding of mAb LOM-11
enhances an affinity between calcium and rML, and 4) where the epitope
of the mAb LOM-11 that detects the calcium-dependent conformation of
MMGL is physically located.
We thank Dr. Takuya Tamatani (Japan Tobacco Inc.,
Yokohama, Japan) for useful suggestion on cell surface biotinylation
and Dr. David M. Wildrick for editorial assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)specific for certain tissue macrophages
(reviewed in Ref. 22). In addition to the studies with mAbs, tissue
type-selective expression of macrophage calcium-type lectins has also
been recognized(23, 24) . Among these molecules, a
galactose/N-acetylgalactosamine-specific calcium-type lectin
(termed MMGL) has been characterized from mouse and rat peritoneal
exudate macrophages(13, 15) . This molecule has been
suggested to be involved in tumor cell recognition by macrophages (19, 21) and in the receptor-mediated endocytosis
pathway of galactose-terminated glycoproteins(13, 21) .
Reagents
MOPS, phenylmethylsulfonyl
fluoride, pepstatin A, aprotinin, Triton X-100, polyethylene glycol
(Hybrimax), oxaloacetate/pyruvate/bovine insulin-media supplement
(OPI-Media supplement), biotinamidocaproate N-hydroxysuccinimide ester, N-hydroxysuccinimidobiotin, and biotin-conjugated monoclonal
anti-rat and
light chains were purchased from Sigma; EDTA,
CHAPS, Tween 20, EGTA, HEPES, and GIT serum-free medium were from Wako
Pure Chemical (Tokyo); recombinant protein G-Sepharose 4B (20 mg of
human IgG/ml of beads capacity), peroxidase-conjugated goat anti-rat
IgG (H+L), Rat MonoAB ID/SP kit, and peroxidase-conjugated
streptavidin were from Zymed Laboratories Inc. (South San Francisco,
CA); hypoxanthine aminopterin thymidine media supplement and
hypoxanthine thymidine media supplement were from Boehringer Mannheim
Biochemica (Mannheim, Germany); bovine serum albumin (BSA), fraction V,
was from Seikagaku (Tokyo); peroxidase-conjugated rabbit
anti-fluorescein 5-isothiocyanate (FITC) was from Dakopatts (Denmark);
2,2`-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) diammonium
salt was from Nacalai Tesque (Kyoto, Japan); FITC-avidin DCS was from
Vector Laboratories, Inc. (Burlingame, CA). Galactose-Sepharose 4B was
prepared by coupling of lactose to amino-derivatized Sepharose 4B beads
by reductive amination according to published
methods(26, 27) . A soluble recombinant form of MMGL
(rML) was purified from Escherichia coli transformed with cDNA
containing extracellular domains of MMGL as described by Sato et
al. (15). A rabbit anti-rML polyclonal antiserum and FITC-labeled
galactosylated polylysine (FITC-Gal-PLL) were produced as previously
described(25) . A penicillin-treated, lyophilized preparation of
the Su-strain of Streptococcus pyogenes, OK-432, was kindly
provided from Chugai Pharmaceuticals (Tokyo).
Cells and Monoclonal Antibodies
A mouse
macrophage cell line RAW264.7, a rat myeloma cell line Y3-Ag 1.2.3
(28), and a hybridoma cell line M1/9.3.4.HL.2 (anti-CD45, or
anti-leukocyte common antigen, IgG2a) were cultured in Dulbecco's
modified Eagle's medium (Nissui Pharmaceutical, Tokyo) containing
4.5 g/liter glucose, 10% heat-inactivated fetal bovine serum
(Bioproducts, Inc., Walkersville, MD), 100 units/ml benzylpenicillin
potassium (Wako Pure Chemical, Tokyo), and 100 µg/ml streptomycin
sulfate (Sigma) in a humidified atmosphere of 5% CO, 95%
air at 37 °C. Peritoneal exudate macrophages elicited by OK-432
were prepared from ICR mice (Charles River Japan Inc., Tokyo) as
described(19) .
Purification of MMGL
RAW264.7 cells
(10) were extracted with 30 ml of lysis buffer (20 mM MOPS (pH 7.0), 0.15 M NaCl, 20 mM CaCl
, 1 mM MgCl
, 0.02%
NaN
, 1% Triton X-100, 0.3 µM aprotinin, 3
µM pepstatin A, 1 mM phenylmethylsulfonyl
fluoride). The extract, precleared with 20 ml of Bio-Gel P-100
(Bio-Rad), was added to a 20-ml galactose-Sepharose 4B beads
equilibrated in buffer A (20 mM MOPS (pH 7.0), 0.5 M NaCl, 20 mM CaCl
, 0.02% NaN
)
containing 0.1% Triton X-100 (buffer A-Triton), and the suspension was
continuously mixed for 18 h at 4 °C. The beads were washed in a
column with 400 ml of buffer A-Triton, and the bound material was
eluted in 100 ml of 20 mM MOPS (pH 7.0), 0.5 M NaCl,
10 mM EDTA (buffer B) containing 0.1% Triton X-100. After
restoration of calcium (final concentration of 20 mM), the
eluted fraction was subjected to a second cycle of affinity
purification (10 ml of packed galactose-Sepharose 4B beads). The column
was washed with 200 ml of buffer A-Triton and then with 50 ml of buffer
A (containing 10 mM CHAPS) for buffer change. The bound
material was eluted in 50 ml of buffer B containing 10 mM
CHAPS and 0.02% NaN
. After calcium restoration,
concentration (to 100 µl) and buffer change (to 20 mM MOPS, 0.15 M NaCl, 20 mM CaCl
, 10
mM CHAPS) of the eluates were carried out using the
Centriprep-30 and Centricon-30 (Amicon, Beverly, MA). The concentrated
preparation was divided into aliquots and stored at -80 °C. 5
µl of the preparation (5
10
cell equivalents)
were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 10%
acrylamide) under nonreducing conditions (Fig. 1).
Figure 1:
SDS-PAGE profile of purified MMGL. MMGL
was purified from a detergent extract of RAW264.7 mouse macrophage
cells by affinity chromatography on a galactose-Sepharose 4B column,
electrophoretically separated by SDS-PAGE (10% gel) under non-reducing
conditions, and visualized by silver staining. The positions and
molecular masses (in kDa) of standards are shown on the left.
Standards are phosphorylase b (97 kDa), BSA (66 kDa), aldolase
(42 kDa), and carbonic anhydrase (30 kDa). The arrow indicates
the band of purified MMGL.
Generation of Monoclonal
Antibodies
F344/DuCrj (Fischer) rat (Charles River Japan
Inc., Tokyo) was subcutaneously immunized with 2.4 µg of purified
MMGL in complete Freund's adjuvant. 1 month later, a second
immunization with 3.8 µg of purified MMGL in incomplete
Freund's adjuvant was given to the same rat, followed by an
intraperitoneal booster injection of 100 µg of rML 4 days before
fusion. Splenocytes of the immunized rat were fused with rat myeloma
cells (Y3-Ag 1.2.3) at a 2:1 cell ratio. Cell suspensions were
distributed into wells of 96-well plates (8.7 10
spleen cell equivalents/well) in hypoxanthine aminopterin
thymidine-containing Dulbecco's modified Eagle's medium
supplemented with 20% fetal bovine serum and OPI-Media supplement and
were cultured for 8 days. Cells in wells of interest were subjected to
limiting dilution for cloning in Dulbecco's modified
Eagle's medium containing 20% fetal bovine serum and OPI-Media
supplement. The screening of hybridoma cells and clones was carried out
based on four criteria used to evaluate the supernatant of each well:
1) its binding activity to immobilized native MMGL purified from RAW
264.7 cells, 2) its binding activity to immobilized rML, 3) its
inhibitory activity against the binding of FITC-Gal-PLL (a synthetic
multivalent ligand) to immobilized rML (ligand binding assay) as
previously described(25) , and 4) flow-cytometric analyses of
antibody binding to RAW264.7 cells.
ELISA Assay
Native MMGL was adsorbed
overnight at 4 °C on wells of an ELISA plate (655061, Greiner,
Germany) by adding 10 µl of purified MMGL (2.5 ng) dissolved in
buffer C (20 mM MOPS (pH 7.0), 0.15 M NaCl, 20 mM CaCl) containing 5 mM CHAPS to each well
containing 90 µl of buffer C. Adsorption of rML onto ELISA plate
wells was carried out by adding 100 µl of rML solution (2.5
µg/ml in buffer C containing 0.1 mM 2-mercaptoethanol) to
each well. After blocking of the wells using 3% BSA in buffer C
(containing glutathione, 100 µM reduced, 10 µM oxidized) for 2 h at room temperature, hybridoma cell culture
supernatants or purified mAbs diluted in buffer D (20 mM MOPS
(pH 7.0), 0.15 M NaCl, 2 mM CaCl
, 0.1%
Tween 20) containing glutathione and 1% BSA were added. In some
experiments, rML-coated wells (coated and blocked in Dulbecco's
phosphate-buffered saline (containing 0.91 mM CaCl
, 0.49 mM MgCl
) instead of
buffer C) were preincubated with 50 µl of buffer D containing 1%
BSA (buffer D-1% BSA) with or without 20 mM EGTA for 30 min at
room temperature, and then 50 µl of buffer D-1% BSA containing
varying concentrations of purified mAbs were added to each well. After
incubation for 1 h at room temperature, the wells were washed in buffer
D to remove unbound mAbs, and then 100 µl of peroxidase-conjugated
goat anti-rat IgG (H+L) (diluted 1/2000 in buffer D-1% BSA) were
added to detect bound antibody. After 1 h of incubation at room
temperature, the wells were washed in buffer D and then received
substrate solution (100 µl of 1 mM ABTS dissolved in 0.1 M sodium citrate (pH 4.2) mixed with H
O
(1/1000 final dilution of 34% stock solution) just before use).
Absorbance readings were measured at 405 nm in a microplate reader
(MTP-12, Corona Electric, Ibaragi, Japan).
Figure 7:
Preserved ligand binding capacity of rML
in environments with reduced calcium concentrations after binding of
mAb to the calcium-dependent epitope. Panels a-d, the
rML was pre-treated with mAb LOM-11 (a, c) or mAb
LOM-14 (b, d) at concentrations of 10 µg/ml (closed circles), 1 µg/ml (closed squares), 0.1
µg/ml (closed triangles), or without antibodies (open
circles) in buffer D (+glutathione)-1% BSA. After removal of
unbound antibodies, the rML was allowed to interact with FITC-Gal-PLL
at the concentration of 3 µg/ml (a, b) or 0.11
µg/ml (c, d) at three free calcium concentrations (abscissa) by using buffer E (+glutathione)-1% BSA
containing 5 mM EDTA and three levels of calcium chloride:
5.1, 4.96, or 4.57 mM (calcium-EDTA buffers). Free calcium
concentrations in the latter three buffers were calculated to be
10, 10
, and 10
M, respectively. Panels e and f, the rML,
pre-treated with mAb LOM-11 (e) or mAb LOM-14 (f) of
varying concentrations (abscissa), was exposed to an
appropriate calcium-EDTA buffer (10
M, closed circles; 10
M, closed
squares; or 10
M, closed
triangles) or buffer D (+glutathione)-1% BSA (open
circles) in parallel with the ligand binding experiments above.
Levels of bound ligands (panels a-d) or antibodies (panels e and f) were detected colorimetrically as
absorbance readings at 405 nm (shown as OD405, y axis) using an ELISA microplate reader. The values shown represent
means of duplicate determinations, and the errorbars indicate half ranges of determination.
In the mAb competition experiments, rML-coated wells received
50 µl of varying concentrations of mAb (hybridoma culture
supernatant) diluted in buffer D-1% BSA. After a 30-min incubation at
room temperature, 50 µl of biotin-conjugated mAb LOM-8.7 (0.6
µg/ml) or biotin-conjugated mAb LOM-11 (1 µg/ml) in buffer D-1%
BSA were added to each well. The binding of the biotinylated mAbs was
detected by 1 h of incubation at room temperature with 100 µl of
peroxidase-conjugated streptavidin (1/500 dilution in buffer D-1% BSA).
Subclass of mAbs was determined by a kit based on an ELISA method (Rat
MonoAB ID/SP kit) following the manufacturer's instructions.
Flow Cytometric Analysis
RAW264.7 cells
(10 cells) were stained on ice for 30 min in 100 µl of
buffer F (Dulbecco's phosphate-buffered saline containing 0.1%
BSA and 0.1% NaN
) containing hybridoma cell culture
supernatant (1/2 dilution) or purified mAb (10 µg/ml). RAW264.7
cells were subsequently stained with biotin-conjugated monoclonal
anti-rat
and
light chains (1/200 dilution in buffer F),
followed by staining with fluorescein-avidin DCS (1/100 dilution in
buffer F), and then analyzed on a flow cytometer (Cyto ACE-150, Jasco
Co., Tokyo).
Purification of Monoclonal
Antibodies
Culture supernatant was obtained in the form of
GIT serum-free medium supplemented with 200 mML-glutamine, 0.1 mM minimal essential medium
non-essential amino acids solution (Life Technologies, Inc.), 14.5
µg/ml L-cystein solution (NCTC-109, Life Technologies,
Inc.) plus 100 units/ml benzylpenicillin potassium and 100 µg/ml
streptomycin sulfate in a humidified atmosphere containing 5%
CO, 95% air at 37 °C. Antibodies were precipitated from
supernatants by 60% saturation with ammonium sulfate and were dialyzed
against Dulbecco's phosphate-buffered saline and then against 50
mM sodium-phosphate buffer (pH 7.2). The dialysate was applied
to a column of recombinant protein-G Sepharose 4B in the same buffer.
The mAbs were eluted with 200 mM acetic acid and immediately
neutralized with 1 M Tris-HCl buffer (pH 8.5). Aliquots of
mAbs were biotinylated using biotinamidocaproate N-hydroxysuccinimide ester following a standard
procedure(29) . The molar ratio between biotinylation reagent
and mAb was 3:1.
Cell Surface Biotinylation and
Immunoprecipitation
Cell surface proteins were biotinylated
according to a published procedure(30) . Briefly, N-hydroxysuccinimidobiotin (100 µg/ml in MESO)
was added to RAW264.7 cells (10
cells/ml) suspended in 100
mM HEPES (pH 8.0), 0.15 M NaCl. After 40 min of
incubation at room temperature, cells were washed three times with 10
mM HEPES, 0.15 M NaCl, and 1 mM CaCl
(pH 7.2) and extracted with the lysis buffer at a concentration
of 5
10
cells/ml. The lysate (1.8 ml) was subjected
to three cycles of preclearing with 50 µl each of recombinant
protein G-Sepharose 4B beads adsorbed with normal rat serum (0.5 µl
of serum to 250 µl of packed beads). Aliquots (215 µl) of the
extract were incubated for 2 h at 4 °C with 10 µl of
recombinant protein G-Sepharose 4B beads that had been conjugated with
a rat anti-MMGL mAb (1.4 ml of hybridoma culture supernatant), a rabbit
anti-rML antiserum (2.8 µl of serum), or a rat anti-CD45 mAb (1.4
ml of supernatant). The beads were extensively washed with the lysis
buffer, boiled in 20 µl of SDS-PAGE sample buffer (62.5 mM Tris (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol), and analyzed by
SDS-PAGE (10% acrylamide) under nonreducing conditions. Proteins were
transferred to a polyvinylidene difluoride membrane (Immobilon-P,
Millipore Corp., Tokyo). Biotinylated proteins were stained with
peroxidase-conjugated streptavidin (1/1500 in phosphate-buffered saline
containing 0.1% Tween 20) and detected with the ECL system (Amersham
Int'l. Ltd., United Kingdom) according to the
manufacturer's protocol. SDS-PAGE protein reference standards
(phosphorylase b, BSA, aldolase, carbonic anhydrase) were
obtained from Daiichi Pure Chemicals (Tokyo).
Production of Anti-MMGL mAbs That Block
Carbohydrate Binding
Hybridomas were produced by fusion
between Y3-Ag1.2.3 rat myeloma cells and fresh spleen cells from a rat
immunized with native MMGL purified from a mouse macrophage cell line,
RAW264.7. The purity of immunogen is demonstrated in Fig. 1.
Hybridoma culture supernatants were screened for antibody production
against native or recombinant forms of MMGL and for inhibitory activity
against binding of FITC-Gal-PLL (ligand) to rML. Hybridomas of interest
were subjected to cell cloning and subcloning by limiting dilution. Two
blocking mAbs (LOM-4.9 and LOM-4.7) were derived from the same well of
the initial culture, and two others (LOM-8.7 and LOM-8.2) were both
obtained from an additional well of that culture. LOM denotes
``lectin on macrophages.'' Additional mAbs LOM-14 and LOM-11
were established as antibodies with weak blocking activity against
ligand binding. Immunoglobulin heavy chain subclass of mAbs was
determined as IgG2a except for LOM-14, which was IgG2b.
Immunoprecipitation with Anti-MMGL
mAbs
To determine efficiency in binding as well as the
specificity of mAbs, each mAb was tested for its ability to
immunoprecipitate MMGL from crude extract of biotinylated RAW264.7
cells. Each mAb specifically immunoprecipitated an approximately 42-kDa
component (Fig. 2). The 42-kDa component was also seen in the
precipitate produced by a rabbit anti-rML polyclonal antiserum (lane1). A negative control (anti-CD45) mAb did not
immunoprecipitate a 42-kDa component, but it did precipitate a
200-kDa component that corresponded to the CD45 molecule. A lane
of crude lysate (lane9) showed a contiguous smear of
biotinylated proteins even though the amount of lysate applied in this
lane was less than one-tenth of the amount used for immunoprecipitation
in the other lanes. An additional faint band of
80 kDa was seen in
the precipitates with LOM-8.7 and LOM-8.2, whereas a single 42-kDa
component was seen in the precipitates with four other mAbs.
Figure 2:
Immunoprecipitation with anti-MMGL mAbs.
An aliquot of detergent extract of biotinylated RAW264.7 cells (1.1
10
cell equivalents) was immunoprecipitated with
beads coated with rabbit anti-MMGL polyclonal antibody (lane
1), with various rat anti-MMGL mAbs: LOM-4.9 (lane 2),
LOM-4.7 (lane 3), LOM-8.7 (lane 4), LOM-8.2 (lane
5), LOM-11 (lane 6), LOM-14 (lane 7), or with
rat anti-CD45 mAb M1/9.3.4.HL.2 (lane 8). The precipitated
materials and a crude cell lysate (lane 9, 7.5
10
cell equivalents) were electrophoretically separated by
SDS-PAGE (10% gel) under non-reducing conditions, transferred to a
polyvinylidene difluoride membrane, stained with peroxidase-labeled
streptavidin, and detected using the ECL system (Amersham). The
positions and molecular weights of standards are shown on the left. The arrow indicates the position of
MMGL.
Flow Cytometric Analysis
The ability of
mAbs to bind cell surface MMGL was tested by immunofluorescence
staining of RAW264.7 cells. As demonstrated in Fig. 3, each mAb
stained RAW264.7 cells significantly, though the staining intensity was
variable among mAbs. The other sublines LOM-4.9 and LOM-8.2 also
stained (data not shown). Each mAb significantly stained peritoneal
exudate macrophages elicited by OK-432 (data not shown).
Figure 3:
Flow
cytometric analyses of the binding of anti-MMGL mAbs to RAW264.7 cells.
Cells were stained with 10 µg/ml purified LOM-4.7 (a),
LOM-8.7 (b), LOM-11 (c), or LOM-14 (d). Darkcurves represent the immunofluorescence of cells
stained with a mAb, biotinylated anti-rat ( and
chain)
antibody and fluorescein-labeled avidin D. Lightcurves represent background staining with the second and the third
reagents.
Concentration Dependence in the mAb Binding and in
the Inhibition of Ligand Binding to rML
The inhibitory
effects of purified mAbs against the binding of FITC-Gal-PLL (ligand)
to immobilized rML were quantitatively tested with simultaneous
measurement of mAb binding to rML. The ligand binding was inhibited by
EGTA, which was consistent with the characteristics of calcium-type
lectin binding (Fig. 4). With increasing concentration of
LOM-4.9, binding of this mAb to rML and inhibition of ligand binding
were in an inverse relationship (Fig. 4a). A complete
inhibition of ligand binding was achieved at the saturation level of
this mAb. mAbs LOM-4.7 (Fig. 4b) and LOM-8.7 (Fig. 4c) also showed a similar dose response curve. The
inhibitory effect of LOM-8.2 was less prominent, but 80% inhibition was
seen at a concentration of 100 µg/ml (Fig. 4d).
LOM-14, originated from an initial hybridoma cell culture supernatant
with slight inhibitory activity (less than 24% inhibition at 1/4
dilution), displayed significant but less effective inhibition (40%
inhibition at 100 µg/ml) than four other blocking mAbs (Fig. 4f). On the other hand, LOM-11 did not show
significant inhibition despite the fact that a similar dose response
curve was seen in its binding to rML (Fig. 4e).
Figure 4:
Simultaneous measurement of the binding of
anti-MMGL mAbs to rML and their inhibitory effects against the binding
of FITC-Gal-PLL (ligand) to rML. The binding of mAbs (open
circles) and the binding of the ligand (0.5 µg/ml final
concentration) in the presence of mAbs (closed circles) were
measured in response to the concentration of LOM-4.9 (a),
LOM-4.7 (b), LOM-8.7 (c), LOM-8.2 (d),
LOM-11 (e), and LOM-14 (f) as shown on the abscissa. The horizontalbrokenline in each panel represents the ligand binding in the
presence of 7.5 mM EGTA. The indicated values are normalized
as % of control. In each panel, 100% denotes the value for
ligand binding in the absence of mAb and the value for the antibody
binding at 100 µg/ml. Values indicated represent means of duplicate
determinations, and the errorbars indicate half
ranges of determination.
Relationships among Antibody Binding
Sites
To know whether certain epitopes or antibody binding
sites could be responsible for the characteristics of ligand binding
inhibition, we examined competition between these mAbs in binding to
rML. As shown in Fig. 5a, the binding of biotinylated
LOM-8.7 was inhibited not only by (unlabeled) LOM-8.7 itself but also
by LOM-4.7, which produced a dose response pattern identical to that of
LOM-8.7. LOM-4.9 and LOM-8.2 also inhibited the binding of biotinylated
LOM-8.7, exhibiting patterns similar to that for LOM-8.7 (data not
shown). Neither LOM-14 nor LOM-11 inhibited the binding of biotinylated
LOM-8.7. Very similar patterns of inhibition were observed when
biotinylated LOM-4.7 was used for the experiment in place of
biotinylated LOM-8.7 (data not shown). On the other hand, the binding
of biotinylated LOM-11 was inhibited only by LOM-11 itself, and none of
the other mAbs was inhibitory (Fig. 5b). The binding of
biotinylated LOM-11 was markedly enhanced by LOM-4.7 and LOM-8.7, which
both showed very similar dose response patterns, whereas LOM-14 did not
show such an effect. LOM-4.9 and LOM-8.2 also enhanced the binding of
biotinylated LOM-11 in a manner similar to that of LOM-4.7 and LOM-8.7
(data not shown). These results demonstrated 1) that binding sites of
LOM-4.7, LOM-4.9, LOM-8.2, and LOM-8.7 are closely related, 2) that
LOM-11 has a unique binding site, and 3) that the binding site of
LOM-14 is different from those of LOM-11 and LOM-4.7/LOM-8.7.
Figure 5:
Competition between different mAbs in
binding to immobilized rML. Biotin-conjugated mAb LOM-8.7 (a)
or biotin-conjugated LOM-11 (b) was allowed to bind to
immobilized rML in the presence of varying concentrations of unlabeled
LOM-4.7 (open circles), LOM-8.7 (open triangles),
LOM-11 (closed circles), or LOM-14 (closed
triangles). The binding of the biotin-conjugated mAbs was measured
colorimetrically by the use of peroxidase-conjugated streptavidin. The
values presented are normalized as % of control. In each panel, 100%
designates the value in the absence of unlabeled mAbs. All values
represent means of duplicate determinations, and the errorbars indicate half ranges of
determination.
Calcium-dependent Binding of
Antibody
Because MMGL is a calcium-dependent lectin,
calcium might be required not only for direct interaction with ligand
carbohydrates but also for maintenance of conformation. Unlike
lectin-carbohydrate interactions, one can speculate that the presence
of calcium has no effect on the antibody-lectin interaction. However,
if calcium is involved in the maintenance of conformation of MMGL, a
calcium-dependent conformation may be detected by an antibody. We
tested this hypothesis by measuring antibody binding to rML with or
without pretreatment in EGTA (pretreated at 20 mM and final
concentration of 10 mM during incubation with mAbs). LOM-4.9,
LOM-4.7, LOM-8.7, LOM-8.2, and LOM-14 all bound to rML and followed the
same dose response curve regardless of pretreatment with EGTA (Fig. 6, a-d, f). In contrast, the
binding of LOM-11 was inhibited by the pretreatment with EGTA. Thus, in
the presence of EGTA, a 100-fold concentration of LOM-11 was required
to obtain the same level of antibody binding as seen in the absence of
EGTA (Fig. 6e). Therefore, it is likely that the
antibody binding site for LOM-11 represents a calcium-dependent
conformation.
Figure 6:
Demonstration of calcium-dependent epitope
on rML. Immobilized rML was pretreated with 50 µl of either buffer
D-1% BSA (open circles) or with 20 mM EGTA in this
buffer (closed circles) for 30 min at 4 °C. Varying
concentrations (shown as final concentrations) of purified LOM-4.9 (a), LOM-4.7 (b), LOM-8.7 (c), LOM-8.2 (d), LOM-11 (e), and LOM-14 (f) were added
(50 µl each) to the pretreated rML. The binding of mAbs was
measured colorimetrically as absorbance readings at 405 nm (shown as OD405, y axis) using an ELISA microplate reader. The
values represent means of duplicate determinations, and the errorbars indicate half ranges of
determinations.
Binding of LOM-11 Preserves the Ligand Binding
Capacity of rML in Low Calcium Environments
We next
examined the functional significance of the calcium-dependent epitope
detected by LOM-11. rML was treated with the mAb LOM-11 or LOM-14 in
the presence of 2 mM calcium. After removal of unbound
antibody, ligand binding was assessed at various free calcium
concentrations. Our previous results demonstrated that rML exhibited
optimal ligand binding activity at a calcium concentration greater than
10M, whereas rML lost its binding activity
at less than 10
M(25) . As shown in Fig. 7, a-d, the ligand binding was negligible at
low calcium concentrations (10
and 10
M) with either 3 µg/ml (Fig. 7, a and b) or 0.11 µg/ml (Fig. 7, c and d)
of ligand concentration. However, when a 3 µg/ml ligand
concentration was used (Fig. 7a), the preincubation of
rML with 10 µg/ml LOM-11 dramatically increased the ligand binding
capacity at 10
and 10
M levels of free calcium up to 89 and 68%, respectively, of the
level of ligand binding seen at 10
M calcium. Furthermore, when suboptimal concentration of ligands
(0.11 µg/ml) was used (Fig. 7c), 9- and 3-fold
enhancement in ligand binding was seen at 10
and
10
M calcium levels, respectively. At
virtually calcium-free conditions (10
M),
LOM-11 restored the ligand binding capacity of rML to 68% of the level
seen with 10
M calcium. In contrast, LOM-14
did not induce ligand binding at 10
and
10
M calcium concentrations (Fig. 7, b and d). At a concentration greater than 1
µg/ml, LOM-14 inhibited the ligand binding seen at a
10
M calcium level (Fig. 7, b and d). Bound mAbs, which remained on rML after exposure
to an appropriate calcium-EDTA buffer instead of exposure to the
ligands in the buffer, were quantified simultaneously. LOM-11 displayed
significant binding throughout the range of calcium concentrations from
10
to 2 mM, though an approximately 30%
reduction in binding was seen after exposure to the buffer containing
10
M calcium (Fig. 7e). The
binding of mAb LOM-14 to rML was constant throughout the range of
calcium concentrations (Fig. 7f).
(
)indicating 1) that a 42-kDa
component was detected in it by SDS-PAGE/immunoblot analysis using
rabbit polyclonal anti-rML antiserum and 2) that this component was
adsorbed on galactose-Sepharose 4B in the presence of calcium and was
eluted with EDTA.
M, whereas
almost full activity was observed at 10
M.
In the present study, we found that pretreatment of rML with mAb LOM-11
preserved the carbohydrate binding activity at free calcium
concentrations of 10
-10
M (Fig. 7, a and c). Moreover,
ligand binding of rML was enhanced by LOM-11 at
10
-10
M calcium
levels when a suboptimal concentration of ligands was applied (Fig. 7c). These effects were specific to the LOM-11
binding site because pretreatment of rML with mAb LOM-14 did not
produce such effects. These results suggested that the binding of mAb
LOM-11 to rML might provide a physical force to shift the equilibrium
of conformations toward a calcium-dependent conformation in a low
calcium environment. Alternatively, the binding of mAb LOM-11 may have
prevented the release of calcium from a critical site where a ternary
peptide-carbohydrate-calcium complex was formed, possibly due to an
enhanced affinity between calcium and rML. It is noteworthy that the
binding of the mAb LOM-11 to rML was less sensitive than carbohydrate
ligand binding to rML to the reduction in free calcium concentration.
Thus, significant binding of mAb LOM-11 to rML was seen after its
exposure to the buffer with 10
-10
M calcium concentration, whereas its carbohydrate
binding activity was lost (Fig. 7). This might explain why mAb
LOM-11 stabilized the ligand binding activity of rML in low calcium
environments. In contrast to the effect of LOM-11 on the rML
carbohydrate binding activity, LOM-11 did not exert any effect on the
binding of biotinylated LOM-8.7 to rML (Fig. 5a). This
suggested that the LOM-8.7 binding site itself was not under the
influence of mAb LOM-11 binding site, even though the LOM-8.7 site was
in the vicinity of the carbohydrate binding site of rML (Fig. 4).
Interestingly, the mAbs to sites closely associated with the
carbohydrate binding site of rML (LOM-4.9, LOM-4.7, LOM-8.7, and
LOM-8.2) significantly enhanced the binding of biotinylated LOM-11 (Fig. 5b). This result may suggest that the LOM-11
defines a ligand-induced binding on rML site such as those seen in the
case of mAbs against
integrin(32, 33) . These results are consistent
with the promotion of the carbohydrate binding of rML by LOM-11.
Although we focused on the binding sites for LOM-11 in this paper,
effects of the blocking mAbs against LOM-14 binding would be another
interesting point. These issues will be important subjects for further
investigations.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.