©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium-dependent Conformation of a Mouse Macrophage Calcium-type Lectin
CARBOHYDRATE BINDING ACTIVITY IS STABILIZED BY AN ANTIBODY SPECIFIC FOR A CALCIUM-DEPENDENT EPITOPE (*)

Toshifumi Kimura , Yasuyuki Imai , Tatsuro Irimura (§)

From the (1)Department of Cancer Biology and Molecular Immunology (formerly Division of Chemical Toxicology and Immunochemistry), Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)()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) .

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.


MATERIALS AND METHODS

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 HO (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).

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.


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 10M, 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 (10M, closed circles; 10M, closed squares; or 10M, 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).


RESULTS

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 10M(25) . As shown in Fig. 7, a-d, the ligand binding was negligible at low calcium concentrations (10 and 10M) 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 10M levels of free calcium up to 89 and 68%, respectively, of the level of ligand binding seen at 10M 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 10M calcium levels, respectively. At virtually calcium-free conditions (10M), LOM-11 restored the ligand binding capacity of rML to 68% of the level seen with 10M calcium. In contrast, LOM-14 did not induce ligand binding at 10 and 10M calcium concentrations (Fig. 7, b and d). At a concentration greater than 1 µg/ml, LOM-14 inhibited the ligand binding seen at a 10M 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 10M calcium (Fig. 7e). The binding of mAb LOM-14 to rML was constant throughout the range of calcium concentrations (Fig. 7f).


DISCUSSION

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,()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.

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 10M, whereas almost full activity was observed at 10M. In the present study, we found that pretreatment of rML with mAb LOM-11 preserved the carbohydrate binding activity at free calcium concentrations of 10-10M (Fig. 7, a and c). Moreover, ligand binding of rML was enhanced by LOM-11 at 10-10M 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 10-10M 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.

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.


FOOTNOTES

*
This work was supported by Grants-in-aid from the Ministry of Education, Science, and Culture of Japan (04454591, 05151018, 05152037, 05274101, 05671813, 06282213), Ministry of Health and Welfare, the Japan Health Science Foundation, the Research Association for Biotechnology, Terumo Foundation, Takeda Foundation, and Princess Takamatsu Fund for Cancer Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-3-3812-2111 (ext. 4870); Fax: 81-3-3815-9344; E-mail: irimura@mol.f.u-tokyo.ac.jp.

The abbreviations used are: mAb, monoclonal antibody; ABTS, 2,2`azinobis-(3-ethylbenzthiazoline-6-sulfonate); ELISA, solid phase enzyme-linked immunosorbent assay; FITC-Gal-PLL, FITC-labeled galactosylated poly-L-lysine; MMGL, mouse macrophage galactose/N-acetylgalactosamine-specific calcium-type lectin; rML, recombinant MMGL; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BSA, bovine serum albumin; FITC, fluorescein 5-isothiocyanate; PAGE, polyacrylamide gel electrophoresis.

S. Mizuochi and Y. Imai, unpublished results.


ACKNOWLEDGEMENTS

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.


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