JOURNAL OF MOLECULAR RECOGNITION, VOL. 5, 1-8 (1992)
Lymphoblastoid Cell Adhesion Mediated by a Dimeric and Polymeric Endogenous P-Galactoside-binding Lectin (Galaptin) Hafiz Ahmed, Ashu Sharma, Richard A. DiCiocciot and Howard J. Allen* Departments of Surgical Oncology and tGynecologic Oncology, Roswell Park Cancer Institute, Buffallo, NY 14263, USA
Glutaraldehyde-polymerized human splenic galaptin, a fl-galactoside-binding lectin, was demonstrated to have enhanced hemagglutinating and asialofetuin binding activity relative to native dimeric galaptin when these lectins were present in solution. The polymerized lectin consisted primarily of 2-, 4 and 12-membered species after reductive alkylation. Both forms of galaptin bound, at 4"C, to saturable B lymphoblastoid cell surface receptors. Estimates obtained by Scatchard analyses, with the binding data expressed in terms of 14.5kDa subunit molarity, were 5 X 10' binding sites/cell with affinity constant K, =2.2 X Id M for dimeric galaptin and 17X 10' binding sites/cell with Ka=3.4X 1 6 ~ - for ' polymeric galaptin. Both forms of galaptin adsorbed to polystyrene with high efficiency; however, only plastic-adsorbed polymeric galaptin mediated adhesion of lymphoblastoid cells. Cell adhesion was inhibited by lactose. Plastic-adsorbed polymeric galaptin bound asialofetuin more efficiently than dimeric galaptin. Asialofetuin binding was inhibited 65% and 3040% by lactose for plastic-adsorbed polymeric and dimeric galaptin, respectively. Native fetuin bound to the adsorbed dimeric galaptin in a lactose-insensitive manner. These data indicate that cell surface receptor-galaptin interaction is carbohydrate specific whereas polystyrene-adsorbed galaptin may demonstrate protein-protein interactions with soluble liiands.
INTRODUCTION Galaptin, a thiol-dependent 6-galactoside binding lectin with subunit 14.5 kDa, was found to be present in a variety of human and animal tissues (Allen et al., 1987a; Barondes et al., 1988). This lectin was isolated from mammalian spleens and its physico-chemical and carbohydrate binding characteristics were described (Ahmed ef al., 1990; Allen et al., 1987b; Sharma et al., 1990). Although the molecular biology of human galaptin is being elucidated (Gitt and Barondes, 1991), the function of galaptin is still open to conjecture. Immunohistochemical studies showed that galaptin and similar lectins are present in the basement membrane and stroma of the extracellular matrix for a variety of animal (Barondes et al., 1984; Catt and Harrison, 1985; Kobiler and Barondes, 1977) and human (Allen et al., 1991b; Gabius et al., 1986) tissues. Galaptin is also present in extracellular matrix synthesized in uitro by bovine corneal endothelial cells (Allen et al., 1990a, 1991b). The presence of galaptin in extracellular matrix suggests that one of its functions might be to mediate cell adhesion via interaction with cell surface receptors. Some evidence for galaptinmediated adhesion of ovarian carcinoma cells to extracellular matrix was previously reported (Allen el al., 1990a). A related lectin, metastasis-associated lectin (L-34) (Lotan and Raz, 1988; Raz ef al., 1990) has also been proposed to play a role in tumor cell adhesion via the presence of this lectin at the cell surface. Author to whom correspondence should be addressed. Abbreviations used: ABTS, 2,2'-azinobis (3ethylbenzothiazoline-
sulfonate); BSA, bovine senum albumin; HBSS, Hank's balanced salt solution. O952-34W/92/0 10001-08 $05.00 @ 1992 I by John Wiley & Sons. Ltd.
Leucocytes are known to bear a variety of lactosaminoglycans at the cell surface (Fukuda, 1985) and these oligosaccharides may interact with galaptin. This interaction is of potential significance in view of the possibility that galaptin may have autoimmune suppressive activity (Offner et al., 1990). The presence of buffy coat cell (a population of heterogeneous leucocytes) surface receptors for galaptin was previously noted (Allen et al., 1987a) but these were not analyzed in detail. The trafficking in vivo of leucocyteswould permit these cells to interact with both soluble and matrix-bound forms of galaptin. It was subsequently found that B142 lymphoblastoid cells bound galaptin in a lactose-inhibitable and reversible manner at 4"C, whereas at 37"C, the bound galaptin was internalized (Allen et al., 1990a). During studies to characterize the interaction of galaptin with the lymphoblastoid cell surface, it was observed that polystyrene-adsorbed galaptin failed to mediate the adhesion of lymphoblastoid cells. This was in contrast to the observation that the cells possess galaptin receptors. However, Ricinus communis agglutinin I, another lactosaminoglycan-binding lectin, was found to mediate maximum lymphoblastoid cell adhesion when applied to polystyrene microtiter wells at 0.6Irglwell. Since galaptin appears to be present in basement membranes and along stromal collagen fibrils (Allen et al., 1991b), it was hypothesized that perhaps galaptin had to be present in some type of supramolecular or multimolecular array in order to facilitate cell adhesion. As part of an effort to understand this phenomenon, we describe herein the interaction of galaptin with B lymphoblastoid cell surface receptors and show that a glutaraldehyde-polymerized form of galaptin has enhanced carbohydrate and cell-binding activity. For Received 7 October 1991
Accepted (revbed) 13 Febnrary 1992
2
H. AHMED E T A L .
these studies, glutaraldehyde polymerization was the method chosen for producing multimolecular galaptin since this reagent had been previously used to prepare galaptin-enzyme conjugates that retained carbohydrate-binding activity (Allen et al., 1990a).
EXPERIMENTAL Preparation of galaptin and polygalaptin.. Galaptin was isolated from human spleen by affinity chromatography on lactose-Sepharose 6B (Sharma et al., 1990). It was stored at -20 "C on DEAE-Sephacel following alkylation with iodoacetamide to eliminate the thiol requirement for maintenance of carbohydrate binding activity, as previously described (Allen et al., 1990b). Galaptin was polymerized in the presence of 0.1 M lactose with 0.1% glutaraldehyde according to Allen et al. (1990a) with the following modification. After polymerization at 4 "C for 15 h, the reaction mixture was diluted 20fold with 0 . 0 1 0 ~Tris, pH7.3, and passed through DEAE-Sephacel to adsorb and concentrate galaptin and to remove lactose and excess gutaraldehyde. After washing the column, the polymerized galaptin was eluted with four bed volumes of 1 M NaCU0.008 M PO4, p H 7.3. It was re-purified by affinity chromatography on lactose-Sepharose 6B to remove inactivated lectin. For the data reported here, there was no attempt to prepare different polymeric species of homogeneous molecular size. Preparation of radiolabeled galaptin and polygalaptin.
DEAE-Sephacel (1 mL) containing non-alkylated galaptin (7.9 mg) was washed with 20 bed volumes of 0.010 M Tris, pH 7.3. Galaptin was eluted directly into a ['4C]iodoacetamide (250 pCi) vial with 2 mL of 0.2 M lactose/0.6 M Trid0.2 M NaCl, pH 7.5. The reaction vial was kept in the dark at room temperature for 2 h. Then 37 mg of unlabeled iodoacetamide were dissolved in the reaction mixture to give 0.1 M iodoacetamide and allowed to react for another hour at room temperature. The reaction mixture was diluted 30-fold with cold deionized water and passed through DEAE-Sephacel pre-equilibrated with 0.010 M Tris, pH 7.3, at 4 "C to adsorb the radioactive galaptin. The column was washed with 10mL 0 . 0 1 0 ~Tris, pH 7.3, then with 5 mL 0.008 M PO4, pH 7.3 to remove the alkylation by-products. The radiolabeled galaptin was eluted from DEAE-Sephacel with 2 mL 1 M NaC1/0.008 M PO4, pH7.3, and it was re-purified by chromatography on 10 mL lactose-Sepharose 6B pre-equilibrated with the same buffer. Radiolabeled galaptin was polymerized as described above followed by re-purification by affinity chromatography on lactose-Sepharose 6B. SDS-PAGE. Samples were reduced and alkylated for
electrophoresis as described (Sharma et al., 1990). SDS-PAGE (Laemmli, 1970) and fluorography (Bonner and Laskey, 1974) were carried out as described (Allen et al., 1991a). The percentage of each polymer in radiolabeled polygalaptin was determined by densitometry of fluorographs. Hemagglutination and cytoagglutination assay with splenic galaptin. Hemagglutination assays were performed with
human type 0 and trypsinized glutaraldehyde-fixed
rabbit erythrocytes in microtiter plates as described (Allen et al., 1987b). Cytoagglutination assays were carried out in microtiter plates with neutral red-stained cells (Wang and Cummings, 1987). Mononuclear cells were obtained from fresh peripheral blood by Ficoll-Paque centrifugation (Pharmacia-LKB , Piscataway, NJ, USA). Granulocytes were obtained from the RBC pellet after lysis of RBC in 0.83% NH4Cl (Allen and Johnson, 1976). Lectin binding to asialofetuin. The ELISA of galaptin and polymerized galaptin binding to asialofetuin was essentially as previously described (Ahmed et al., 1990). In brief, two-fold serial dilutions of galaptin (30-0.06 pg/ 100 pL/well) and two-fold serial dilutions of polygalaptin (3.2-0.006 pg/lOO pL/well) were added to microtiter plates precoated with formaldehyde-fixed asialofetuin (2.0 pg/well) and blocked with 0.4% BSA/O.OS0h Tween-20. Plates were then incubated for 1h at 4 "C. The wells were washed and fixed with 2% formaldehyde followed by incubation with anti-galaptin serum (15000). After washing, the wells were incubated with goat anti-rabbit IgG-peroxidase conjugate (0.2 pg/ 100 pL/well). Bound peroxidase was assayed by incubation with 100 pL substrate, 2,2'-azinobis (3ethylbenzothiazolinesulfonate) (ABTS), for 4 min at 37 "C. The reaction was stopped by addition of 100 pL 0.2 M citric acid and the plate was read at 405 nm on a plate reader. Binding inhibition assays with lactose (0.0500.0001 M) as the inhibitor and dimeric galaptin [12 pg in 60 pL 0.010 M P04/0.120 M NaCI/O.Ol% thimerosal, 0.05% Tween-20, pH 7.3 (buffer A)] or polygalaptin (0.12 pg in 60 pL buffer A) were carried out as before (Ahmed et al., 1990). Lectin and two-fold serial dilutions of inhibitor were preincubated for 1 h at 4 "C. Aliquots (50 pL) of the mixtures were added to microtiter plate wells precoated with asialofetuin. After incubation for 1 h at 4 "C, bound galaptin was assayed as above. Concentrations of lactose giving 50% inhibition of galaptin and polygalaptin binding to asialofetuin, I,,, , relative to galaptin and polygalaptin controls, were determined graphically. Binding analysis of cell surface lectin receptors. Cell binding
assays with radiolabeled dimeric and polygalaptin were carried out in tubes containing 2.5 x lo6 B142 cells/ 0.5mL with 0.5% bovine serum albumin (BSA) as previously described for ovarian carcinoma cells (Allen et al., 1990b). The binding reactions were carried out in an ice bath for 90 min. The cells were separated from free lectin by filtration on glass fiber filters presoaked in 1% BSA. The specific activity of [14C]galaptin and ['4C]polygalaptin was 1600 cpm/pg. Controls included tubes with 0.050 M lactose and tubes without cells. This concentration of lactose had been shown to give maximal inhibition of galaptin binding to B142 cells. The data were corrected for non-specific binding (0-5% of binding in the absence of lactose) and plotted according to Scatchard (1949). For these plots, the dimeric and polymeric lectin concentrations were expressed as molarity of carbohydrate binding sites assuming one binding site per 14.5 kDa polypeptide unit (Hirabayashi and Kasai, 1991).
3
BINDING ACTIVITY OF DIMERIC AND POLYMERIC GALAPTIN
Lectin-mediated cell adhesion. This was performed accord-
ing to Landegren (1984). Flat-bottomed ELISA plates (Immulon I, Dynatech Laboratories, Chantilly, VA, USA) were coated with two-fold serial dilutions of galaptin (100 1.1.8-0.4 pg/lOo pL/well) and polygalaptin (10 pg-0.04 pg/100 pL/well) in 0.2 M NaCU0.008 M PO,, pH 7.3, for 17 h at 4°C. The plates were washed three times with cold HBSS (GIBCO, Grand Island, NY, USA), pH7.3, and blocked with 1% BSA in HBSS. A fixed number of B142 lymphoblastoid cells in 100pL, prewashed with HBSS and suspended in 1% BSA/HBSS, were added to each well in duplicate. The plates were centrifuged at 50 x g for 2 min and then were incubated for 90 min at 4 "C. After incubation, the plates were washed three times by immersing in cold Hank's balanced salt solution (HBSS) and flicking off the buffer. Sixty microliters of p-nitrophenyl-N-acetylP-D-glucosaminide solution were added to each well. This substrate was made at 0.00375 M in 0.050 M citrate buffer/0.25(YoTriton X-100, pH 5.0. After 3 h incubation with substrate at 37 "C, the color was developed by adding 2 M NaOH (60 pL/well). The plates were read at 405 nm. For adhesion inhibition assays, wells containing a variable amount of lectin were preincubated with 0.050 M lactose in 1% BSA/HBSS for 1 h at 4 "C. The lactose solutions were aspirated off and to each well was added 1 .0 x lo" cells in 100 pL 0.050 M lactose/l% BSA/HBSS in triplicate. The control wells not treated with lactose contained 1 . 0 lo" ~ cells in 100 pL 1% BSA/HBSS. The percentage of adhesion for each dilution of polygalaptin was determined. Quantitation of adsorbed galaptin (dimeric and polymeric) in ELISA plate. The ELISA plates were coated with two-
fold serial dilution of ',C-labeled dimeric and polymeric galaptin (40 pg-0.08 pg/lOo pL/well in 0.2 M NaCV0.008 M PO,, pH7.3) for 17 h at 4°C. After complete aspiration of galaptin solution, the wells were washed three times with cold HBSS. The wells were separated and were placed directly in Scintiverse cocktail for liquid scintillation counting of radioactivity. Detection of plastic-adsorbed galaptin (dimeric and polymeric) in ELISA plate by asialofetuin and fetuin binding. The
ELISA plates were coated with two-fold serial dilutions of dimeric and polymeric galaptin (10 pg-0.04 pg/ 100pL/well in 0 . 2 ~N a C V 0 . 0 0 8 ~PO,, pH7.3) for 17 h at 4°C. After complete aspiration of galaptin solution, the wells were blocked with 3% BSA/0.010 M PO,/O. 120 M NaCI/O.Ol% thimerosal, pH 7.3, for 1 h at 4°C. The wells were washed three times by immersing in cold buffer and were incubated with fetuin or asialofetuin (1 pg/100 pL/well in 3% BSAIbuffer) at 4 "C for 1 h. After washing six times with cold buffer, the wells were fixed with 2% formaldehyde in buffer (150 pL/ well) for 30 min at 37 "C. After washing the wells with buffer A (Ahmed et al., 1990), rabbit anti-asialofetuin serum (100 pL/well of a 1:5000 dilution in 5% BSAIbuffer A) was added to the wells. After incubation for 1 h at 37°C and washing, the bound antibody was detected with goat anti-rabbit IgG-peroxidase conjugate as described before. Inhibition of fetuin and asialofetuin binding to galaptin was carried out in the presence of 0.1 M lactose. Alternatively, asialofetuin-binding to polystyrene-
Table 1. Minimum galaptin concentration required for agglutination Galaptin (wg/rnL)
1 .o 13.5 13.5 62.0
>loo
Cell type
rabbit RBC" peripheral rnonoclear cells 8142 lyrnphoblastoid cells peripheral granulocytes human type 0 RBCb
Trypsinized and fixed with glutaraldehyde. Native, unfixed RBC or trypsinized and fixed RBC were not agglutinated.
a
adsorbed galaptin and polygalaptin was carried out with glutaraldehyde-coupled asialofetuin-peroxidase conjugates prepared essentially as described for galaptin-fucosidase conjugation (Allen et al., 1990a). The bound peroxidase was assayed with ABTS as described. This approach circumvented the use of antiasialofetuin serum to assay lectin-bound asialofetuin. Rabbit anti-galaptin serum was as described (Sharma et al., 1990). Rabbit anti-asialofetuin serum was obtained by intradermal immunization with 500 pg asialofetuin in Freund's complete adjuvant followed by booster injection of 200 pg in Freund's incomplete adjuvant. Acid-desialylation of fetuin was carried out by hydrolysis of fetuin in 0.1 N H,SO, for 70 min at 80 "C followed by dialysis. Epstein-Barr virusimmortalized B142 lymphoblastoid cells have been previously described (DiCioccio and Brown, 1988). Glutaraldehyde, iodoacetamide and fetuin were products of Sigma Chemical Co., St Louis, MO, USA. [1-I4C] Iodoacetamide, 23 mCi/mmol, was obtained from ICN Radiochemicals, Irvine, CA, USA. Lactose-Sepharose 6B was prepared according to Allen and Johnson (1977). Scintiverse was obtained from Fisher Chemicals, Rochester, NY, USA.
RESULTS Previous experiments showed that heterogeneous buffy coat cells (leucocytes) (Allen et al., 1987a) and B142 lymphoblastoid cells (Allen et al., 1990a) possess receptors for galaptin. It was of interest to gain more quantitative information on these receptors. The results of cytoagglutination assays with purified splenic galaptin are given in Table 1. Mononuclear cells and B142 lymphoblastoid cells were readily agglutinated by galaptin. Granulocytes were poorly agglutinated. As reported previously (Allen et al., 1987b), human galaptin did not agglutinate human RBC. B142 cells were chosen for the studies reported here to investigate the interaction of lymphoid cells with dimeric and polygalaptin. The results for SDS-PAGE of a sample of reduced and alkylated ''C-labeled dimeric and polygalaptin are shown in Fig. 1. The dimeric galaptin showed the presence of a 14.5 kDa subunit, as expected. The polygalaptin was composed of a mixture of different molecular mass species: 14.5 kDa, 4%; 29 kDa, 33%; 43 kDa, 2%; 60 kDa, 22%; 90 kDa, 8 % ; 183 kDa, 23%; and polymer at the gel origin, 9%. For the experiments reported, polygalaptin was used as a mixture of these
H.AHMED E T A L .
4
components. The hemagglutinating activity of dimeric and polygalaptin was compared using rabbit erythrocytes as target cells. Dimeric galaptin had a hemagglutinating specific activity (titer/mg/mL) of 2 x 103whereas polygalaptin had a specific activity of 6 x lo4. The specific activity values varied somewhat among different lectin preparations; however, polygalaptin was always 1 3 0 times better than dimeric galaptin. Hemagglutination was inhibited by 0.1 M lactose. Polygalaptin showed up to a 15-fold enhanced binding on a weight basis, relative to dimeric galaptin, to plastic-adsorbed asialofetuin (Fig. 2). At binding saturation to asialofetuin, this enhancement was 3- to 4fold. Binding was inhibited by lactose but with differing tendency. To demonstrate this, lactose inhibition experiments were carried out with lectin concentrations of 1.2 (based upon the absorption chosen to give ANSnm limit of the plate reader) at zero inhibition. Lactose Z,, for 0.1 pg polygalaptin/well was 1.9 mM whereas lactose for 10 pg dimeric galaptin/well was 0.82 mM. The ratio of lactose Zs0to 0.1 pg polygalaptin was >200 times the corresponding ratio for dimeric galaptin ([so: 10 VB). Galaptin binding to lymphoblastoid cells was previously shown to be reversible at 4 "C and also ta result in endocytosis of the receptors at 37 "C (Allen et al.,
,001
.01
.1
1
10
pg Galaptin/Well
loo 90
-
-
e 80-
[,,
Figure 1. SDS-PAGE and fluorography of 14C-alkylated native and polymerized galaptin. The samples were reduced in 0.6 M Tris10.018 M EDTN0.030 M dithiothreitol, pH 8.4, and alkylated with 0.060 M iodoacetamide under N2 at room temperature. The samples were run on a 7.5% acrylamide slab gel: lane 1, polygalaptin; lane 2, native (dimeric galaptin); lane 3, molecular weight standards.
.1
1
10
100
mM Lactose
Figure2. ELlSA of dimeric (0) and polymeric (0)galaptin binding to asialofetuin. Binding to plastic-adsorbed asialofetuin in microtiter plates (A) and lactose inhibtion of binding (B) was carried out as described in the Experimental section. Bound galaptin was detected with rabbit anti-galaptin serum and goat anti-rabbit IgG-peroxidase complex. For lactose inhibition, dimeric galaptin was present at 12 pg/60 pLlwell and polygalaptin was present at 0.12 pg160 Llwell. These amounts gave b5 nm of 1.2 at 0% inhibition.
1990a). Cell binding assays were carried out in these studies to determine if lymphoblastoid cells possess a finite number of galaptin receptors and to compare the binding of dimeric and polygalaptin. For analysis of the binding data, the molecular mass of the galaptin monomeric subunit, 14.5 kDa, was used for both dimeric galaptin and for polymeric galaptin to calculate carbohydrate binding site molarities. This was based on the apparent presence of one binding site per 14.5 kDa polypeptide (Hirabayashi and Kasai, 1991). The binding of galaptin to B142 cell receptors was a saturable process [Fig. 3(A)]. Binding was inhibited 95-300% by 0.05 M or 0.1 M lactose. Scatchard plots [Fig. 3(B)] were constructed from dimeric and polymeric galaptin saturation binding data. The nature of the Scatchard plot for polygalaptin [Fig. 3(B)] is consistent with positive co-operativity in the binding reaction at low polygalaptin concentration. To estimate affinity constants and receptorskell, the first three data points for polygalaptin were ignored for the computer generation of straight lines. The number of binding sites/cell was 5.3 X lo7 with an affinity constant of 2.2 x 105 M-' for dimeric galaptin. A similar affinity constant, 3.4 x ~O'/M, was obtained for polymeric galaptin. The number of binding sites/cell for polygalaptin was estimated to be 17.4 X lo', about three times greater than for dimeric galaptin. To determine if a multimolecular form of galaptin would show enhanced mediation of cell adhesion, the ability of dimeric and polymeric galaptin to promote B142 cell adhesion was compared. Dimeric galaptin was inactive in promoting cell adhesion up to 100~1.g
BINDING ACTIVITY OF DIMERIC AND POLYMERIC GALAPTIN
galaptin/well [Fig. 4(A)]. Polymeric galaptin promoted cell adhesion over a range of cell concentrations. Maximum adhesion occurred at -0.6 pg polymeric galaptin/well with rapid rise in adhesion to platueau values [Fig. 4[A)]. In the range of maximum polymeric galaptin-mediated adhesion, the adhesion was inhibited up to 80% by 0.05 M lactose [Fig. 4(B)J. Similar inhibition was obtained with 0.1 M lactose. B142 cell adhesion to plastic-adsorbed BSA in the absence of galaptin was 0-5% cell input. Experiments were carried out to determine if dimeric and polymeric galaptin adsorbed to microtiter wells similarly. The two forms of galaptin showed similar adsorption to plastic (Fig. 5 ) . The maximum difference in adsorption at 10 pg galaptin/well was -25%. The ability of the plastic-adsorbed dimeric and polymeric galaptin to bind asialofetuin in a lactoseinhibitable manner was compared to determine if adsorption resulted in inactivation of dimeric galaptin. Both forms of galaptin bound asialofetuin although binding to polymeric galaptin was significantly more efficient [Fig. 6(A)]. In contrast to expectations, binding of the glycoconjugate to the plastic-adsorbed galaptins was only partially inhibited by lactose; -65% for polygalaptin and 30-50% for dimeric galaptin at 1 pg galaptin/well.
5
2.5
d
2.0
3 1.5
1:: nn
B
1.6
1-
4
1.0
0.5
Lymphoblastoid Cell Adhesion Mediated by a Dimeric and Polymeric Endogenous P-Galactoside-binding Lectin (Galaptin) Hafiz Ahmed, Ashu Sharma, Richard A. DiCiocciot and Howard J. Allen* Departments of Surgical Oncology and tGynecologic Oncology, Roswell Park Cancer Institute, Buffallo, NY 14263, USA
Glutaraldehyde-polymerized human splenic galaptin, a fl-galactoside-binding lectin, was demonstrated to have enhanced hemagglutinating and asialofetuin binding activity relative to native dimeric galaptin when these lectins were present in solution. The polymerized lectin consisted primarily of 2-, 4 and 12-membered species after reductive alkylation. Both forms of galaptin bound, at 4"C, to saturable B lymphoblastoid cell surface receptors. Estimates obtained by Scatchard analyses, with the binding data expressed in terms of 14.5kDa subunit molarity, were 5 X 10' binding sites/cell with affinity constant K, =2.2 X Id M for dimeric galaptin and 17X 10' binding sites/cell with Ka=3.4X 1 6 ~ - for ' polymeric galaptin. Both forms of galaptin adsorbed to polystyrene with high efficiency; however, only plastic-adsorbed polymeric galaptin mediated adhesion of lymphoblastoid cells. Cell adhesion was inhibited by lactose. Plastic-adsorbed polymeric galaptin bound asialofetuin more efficiently than dimeric galaptin. Asialofetuin binding was inhibited 65% and 3040% by lactose for plastic-adsorbed polymeric and dimeric galaptin, respectively. Native fetuin bound to the adsorbed dimeric galaptin in a lactose-insensitive manner. These data indicate that cell surface receptor-galaptin interaction is carbohydrate specific whereas polystyrene-adsorbed galaptin may demonstrate protein-protein interactions with soluble liiands.
INTRODUCTION Galaptin, a thiol-dependent 6-galactoside binding lectin with subunit 14.5 kDa, was found to be present in a variety of human and animal tissues (Allen et al., 1987a; Barondes et al., 1988). This lectin was isolated from mammalian spleens and its physico-chemical and carbohydrate binding characteristics were described (Ahmed ef al., 1990; Allen et al., 1987b; Sharma et al., 1990). Although the molecular biology of human galaptin is being elucidated (Gitt and Barondes, 1991), the function of galaptin is still open to conjecture. Immunohistochemical studies showed that galaptin and similar lectins are present in the basement membrane and stroma of the extracellular matrix for a variety of animal (Barondes et al., 1984; Catt and Harrison, 1985; Kobiler and Barondes, 1977) and human (Allen et al., 1991b; Gabius et al., 1986) tissues. Galaptin is also present in extracellular matrix synthesized in uitro by bovine corneal endothelial cells (Allen et al., 1990a, 1991b). The presence of galaptin in extracellular matrix suggests that one of its functions might be to mediate cell adhesion via interaction with cell surface receptors. Some evidence for galaptinmediated adhesion of ovarian carcinoma cells to extracellular matrix was previously reported (Allen el al., 1990a). A related lectin, metastasis-associated lectin (L-34) (Lotan and Raz, 1988; Raz ef al., 1990) has also been proposed to play a role in tumor cell adhesion via the presence of this lectin at the cell surface. Author to whom correspondence should be addressed. Abbreviations used: ABTS, 2,2'-azinobis (3ethylbenzothiazoline-
sulfonate); BSA, bovine senum albumin; HBSS, Hank's balanced salt solution. O952-34W/92/0 10001-08 $05.00 @ 1992 I by John Wiley & Sons. Ltd.
Leucocytes are known to bear a variety of lactosaminoglycans at the cell surface (Fukuda, 1985) and these oligosaccharides may interact with galaptin. This interaction is of potential significance in view of the possibility that galaptin may have autoimmune suppressive activity (Offner et al., 1990). The presence of buffy coat cell (a population of heterogeneous leucocytes) surface receptors for galaptin was previously noted (Allen et al., 1987a) but these were not analyzed in detail. The trafficking in vivo of leucocyteswould permit these cells to interact with both soluble and matrix-bound forms of galaptin. It was subsequently found that B142 lymphoblastoid cells bound galaptin in a lactose-inhibitable and reversible manner at 4"C, whereas at 37"C, the bound galaptin was internalized (Allen et al., 1990a). During studies to characterize the interaction of galaptin with the lymphoblastoid cell surface, it was observed that polystyrene-adsorbed galaptin failed to mediate the adhesion of lymphoblastoid cells. This was in contrast to the observation that the cells possess galaptin receptors. However, Ricinus communis agglutinin I, another lactosaminoglycan-binding lectin, was found to mediate maximum lymphoblastoid cell adhesion when applied to polystyrene microtiter wells at 0.6Irglwell. Since galaptin appears to be present in basement membranes and along stromal collagen fibrils (Allen et al., 1991b), it was hypothesized that perhaps galaptin had to be present in some type of supramolecular or multimolecular array in order to facilitate cell adhesion. As part of an effort to understand this phenomenon, we describe herein the interaction of galaptin with B lymphoblastoid cell surface receptors and show that a glutaraldehyde-polymerized form of galaptin has enhanced carbohydrate and cell-binding activity. For Received 7 October 1991
Accepted (revbed) 13 Febnrary 1992
2
H. AHMED E T A L .
these studies, glutaraldehyde polymerization was the method chosen for producing multimolecular galaptin since this reagent had been previously used to prepare galaptin-enzyme conjugates that retained carbohydrate-binding activity (Allen et al., 1990a).
EXPERIMENTAL Preparation of galaptin and polygalaptin.. Galaptin was isolated from human spleen by affinity chromatography on lactose-Sepharose 6B (Sharma et al., 1990). It was stored at -20 "C on DEAE-Sephacel following alkylation with iodoacetamide to eliminate the thiol requirement for maintenance of carbohydrate binding activity, as previously described (Allen et al., 1990b). Galaptin was polymerized in the presence of 0.1 M lactose with 0.1% glutaraldehyde according to Allen et al. (1990a) with the following modification. After polymerization at 4 "C for 15 h, the reaction mixture was diluted 20fold with 0 . 0 1 0 ~Tris, pH7.3, and passed through DEAE-Sephacel to adsorb and concentrate galaptin and to remove lactose and excess gutaraldehyde. After washing the column, the polymerized galaptin was eluted with four bed volumes of 1 M NaCU0.008 M PO4, p H 7.3. It was re-purified by affinity chromatography on lactose-Sepharose 6B to remove inactivated lectin. For the data reported here, there was no attempt to prepare different polymeric species of homogeneous molecular size. Preparation of radiolabeled galaptin and polygalaptin.
DEAE-Sephacel (1 mL) containing non-alkylated galaptin (7.9 mg) was washed with 20 bed volumes of 0.010 M Tris, pH 7.3. Galaptin was eluted directly into a ['4C]iodoacetamide (250 pCi) vial with 2 mL of 0.2 M lactose/0.6 M Trid0.2 M NaCl, pH 7.5. The reaction vial was kept in the dark at room temperature for 2 h. Then 37 mg of unlabeled iodoacetamide were dissolved in the reaction mixture to give 0.1 M iodoacetamide and allowed to react for another hour at room temperature. The reaction mixture was diluted 30-fold with cold deionized water and passed through DEAE-Sephacel pre-equilibrated with 0.010 M Tris, pH 7.3, at 4 "C to adsorb the radioactive galaptin. The column was washed with 10mL 0 . 0 1 0 ~Tris, pH 7.3, then with 5 mL 0.008 M PO4, pH 7.3 to remove the alkylation by-products. The radiolabeled galaptin was eluted from DEAE-Sephacel with 2 mL 1 M NaC1/0.008 M PO4, pH7.3, and it was re-purified by chromatography on 10 mL lactose-Sepharose 6B pre-equilibrated with the same buffer. Radiolabeled galaptin was polymerized as described above followed by re-purification by affinity chromatography on lactose-Sepharose 6B. SDS-PAGE. Samples were reduced and alkylated for
electrophoresis as described (Sharma et al., 1990). SDS-PAGE (Laemmli, 1970) and fluorography (Bonner and Laskey, 1974) were carried out as described (Allen et al., 1991a). The percentage of each polymer in radiolabeled polygalaptin was determined by densitometry of fluorographs. Hemagglutination and cytoagglutination assay with splenic galaptin. Hemagglutination assays were performed with
human type 0 and trypsinized glutaraldehyde-fixed
rabbit erythrocytes in microtiter plates as described (Allen et al., 1987b). Cytoagglutination assays were carried out in microtiter plates with neutral red-stained cells (Wang and Cummings, 1987). Mononuclear cells were obtained from fresh peripheral blood by Ficoll-Paque centrifugation (Pharmacia-LKB , Piscataway, NJ, USA). Granulocytes were obtained from the RBC pellet after lysis of RBC in 0.83% NH4Cl (Allen and Johnson, 1976). Lectin binding to asialofetuin. The ELISA of galaptin and polymerized galaptin binding to asialofetuin was essentially as previously described (Ahmed et al., 1990). In brief, two-fold serial dilutions of galaptin (30-0.06 pg/ 100 pL/well) and two-fold serial dilutions of polygalaptin (3.2-0.006 pg/lOO pL/well) were added to microtiter plates precoated with formaldehyde-fixed asialofetuin (2.0 pg/well) and blocked with 0.4% BSA/O.OS0h Tween-20. Plates were then incubated for 1h at 4 "C. The wells were washed and fixed with 2% formaldehyde followed by incubation with anti-galaptin serum (15000). After washing, the wells were incubated with goat anti-rabbit IgG-peroxidase conjugate (0.2 pg/ 100 pL/well). Bound peroxidase was assayed by incubation with 100 pL substrate, 2,2'-azinobis (3ethylbenzothiazolinesulfonate) (ABTS), for 4 min at 37 "C. The reaction was stopped by addition of 100 pL 0.2 M citric acid and the plate was read at 405 nm on a plate reader. Binding inhibition assays with lactose (0.0500.0001 M) as the inhibitor and dimeric galaptin [12 pg in 60 pL 0.010 M P04/0.120 M NaCI/O.Ol% thimerosal, 0.05% Tween-20, pH 7.3 (buffer A)] or polygalaptin (0.12 pg in 60 pL buffer A) were carried out as before (Ahmed et al., 1990). Lectin and two-fold serial dilutions of inhibitor were preincubated for 1 h at 4 "C. Aliquots (50 pL) of the mixtures were added to microtiter plate wells precoated with asialofetuin. After incubation for 1 h at 4 "C, bound galaptin was assayed as above. Concentrations of lactose giving 50% inhibition of galaptin and polygalaptin binding to asialofetuin, I,,, , relative to galaptin and polygalaptin controls, were determined graphically. Binding analysis of cell surface lectin receptors. Cell binding
assays with radiolabeled dimeric and polygalaptin were carried out in tubes containing 2.5 x lo6 B142 cells/ 0.5mL with 0.5% bovine serum albumin (BSA) as previously described for ovarian carcinoma cells (Allen et al., 1990b). The binding reactions were carried out in an ice bath for 90 min. The cells were separated from free lectin by filtration on glass fiber filters presoaked in 1% BSA. The specific activity of [14C]galaptin and ['4C]polygalaptin was 1600 cpm/pg. Controls included tubes with 0.050 M lactose and tubes without cells. This concentration of lactose had been shown to give maximal inhibition of galaptin binding to B142 cells. The data were corrected for non-specific binding (0-5% of binding in the absence of lactose) and plotted according to Scatchard (1949). For these plots, the dimeric and polymeric lectin concentrations were expressed as molarity of carbohydrate binding sites assuming one binding site per 14.5 kDa polypeptide unit (Hirabayashi and Kasai, 1991).
3
BINDING ACTIVITY OF DIMERIC AND POLYMERIC GALAPTIN
Lectin-mediated cell adhesion. This was performed accord-
ing to Landegren (1984). Flat-bottomed ELISA plates (Immulon I, Dynatech Laboratories, Chantilly, VA, USA) were coated with two-fold serial dilutions of galaptin (100 1.1.8-0.4 pg/lOo pL/well) and polygalaptin (10 pg-0.04 pg/100 pL/well) in 0.2 M NaCU0.008 M PO,, pH 7.3, for 17 h at 4°C. The plates were washed three times with cold HBSS (GIBCO, Grand Island, NY, USA), pH7.3, and blocked with 1% BSA in HBSS. A fixed number of B142 lymphoblastoid cells in 100pL, prewashed with HBSS and suspended in 1% BSA/HBSS, were added to each well in duplicate. The plates were centrifuged at 50 x g for 2 min and then were incubated for 90 min at 4 "C. After incubation, the plates were washed three times by immersing in cold Hank's balanced salt solution (HBSS) and flicking off the buffer. Sixty microliters of p-nitrophenyl-N-acetylP-D-glucosaminide solution were added to each well. This substrate was made at 0.00375 M in 0.050 M citrate buffer/0.25(YoTriton X-100, pH 5.0. After 3 h incubation with substrate at 37 "C, the color was developed by adding 2 M NaOH (60 pL/well). The plates were read at 405 nm. For adhesion inhibition assays, wells containing a variable amount of lectin were preincubated with 0.050 M lactose in 1% BSA/HBSS for 1 h at 4 "C. The lactose solutions were aspirated off and to each well was added 1 .0 x lo" cells in 100 pL 0.050 M lactose/l% BSA/HBSS in triplicate. The control wells not treated with lactose contained 1 . 0 lo" ~ cells in 100 pL 1% BSA/HBSS. The percentage of adhesion for each dilution of polygalaptin was determined. Quantitation of adsorbed galaptin (dimeric and polymeric) in ELISA plate. The ELISA plates were coated with two-
fold serial dilution of ',C-labeled dimeric and polymeric galaptin (40 pg-0.08 pg/lOo pL/well in 0.2 M NaCV0.008 M PO,, pH7.3) for 17 h at 4°C. After complete aspiration of galaptin solution, the wells were washed three times with cold HBSS. The wells were separated and were placed directly in Scintiverse cocktail for liquid scintillation counting of radioactivity. Detection of plastic-adsorbed galaptin (dimeric and polymeric) in ELISA plate by asialofetuin and fetuin binding. The
ELISA plates were coated with two-fold serial dilutions of dimeric and polymeric galaptin (10 pg-0.04 pg/ 100pL/well in 0 . 2 ~N a C V 0 . 0 0 8 ~PO,, pH7.3) for 17 h at 4°C. After complete aspiration of galaptin solution, the wells were blocked with 3% BSA/0.010 M PO,/O. 120 M NaCI/O.Ol% thimerosal, pH 7.3, for 1 h at 4°C. The wells were washed three times by immersing in cold buffer and were incubated with fetuin or asialofetuin (1 pg/100 pL/well in 3% BSAIbuffer) at 4 "C for 1 h. After washing six times with cold buffer, the wells were fixed with 2% formaldehyde in buffer (150 pL/ well) for 30 min at 37 "C. After washing the wells with buffer A (Ahmed et al., 1990), rabbit anti-asialofetuin serum (100 pL/well of a 1:5000 dilution in 5% BSAIbuffer A) was added to the wells. After incubation for 1 h at 37°C and washing, the bound antibody was detected with goat anti-rabbit IgG-peroxidase conjugate as described before. Inhibition of fetuin and asialofetuin binding to galaptin was carried out in the presence of 0.1 M lactose. Alternatively, asialofetuin-binding to polystyrene-
Table 1. Minimum galaptin concentration required for agglutination Galaptin (wg/rnL)
1 .o 13.5 13.5 62.0
>loo
Cell type
rabbit RBC" peripheral rnonoclear cells 8142 lyrnphoblastoid cells peripheral granulocytes human type 0 RBCb
Trypsinized and fixed with glutaraldehyde. Native, unfixed RBC or trypsinized and fixed RBC were not agglutinated.
a
adsorbed galaptin and polygalaptin was carried out with glutaraldehyde-coupled asialofetuin-peroxidase conjugates prepared essentially as described for galaptin-fucosidase conjugation (Allen et al., 1990a). The bound peroxidase was assayed with ABTS as described. This approach circumvented the use of antiasialofetuin serum to assay lectin-bound asialofetuin. Rabbit anti-galaptin serum was as described (Sharma et al., 1990). Rabbit anti-asialofetuin serum was obtained by intradermal immunization with 500 pg asialofetuin in Freund's complete adjuvant followed by booster injection of 200 pg in Freund's incomplete adjuvant. Acid-desialylation of fetuin was carried out by hydrolysis of fetuin in 0.1 N H,SO, for 70 min at 80 "C followed by dialysis. Epstein-Barr virusimmortalized B142 lymphoblastoid cells have been previously described (DiCioccio and Brown, 1988). Glutaraldehyde, iodoacetamide and fetuin were products of Sigma Chemical Co., St Louis, MO, USA. [1-I4C] Iodoacetamide, 23 mCi/mmol, was obtained from ICN Radiochemicals, Irvine, CA, USA. Lactose-Sepharose 6B was prepared according to Allen and Johnson (1977). Scintiverse was obtained from Fisher Chemicals, Rochester, NY, USA.
RESULTS Previous experiments showed that heterogeneous buffy coat cells (leucocytes) (Allen et al., 1987a) and B142 lymphoblastoid cells (Allen et al., 1990a) possess receptors for galaptin. It was of interest to gain more quantitative information on these receptors. The results of cytoagglutination assays with purified splenic galaptin are given in Table 1. Mononuclear cells and B142 lymphoblastoid cells were readily agglutinated by galaptin. Granulocytes were poorly agglutinated. As reported previously (Allen et al., 1987b), human galaptin did not agglutinate human RBC. B142 cells were chosen for the studies reported here to investigate the interaction of lymphoid cells with dimeric and polygalaptin. The results for SDS-PAGE of a sample of reduced and alkylated ''C-labeled dimeric and polygalaptin are shown in Fig. 1. The dimeric galaptin showed the presence of a 14.5 kDa subunit, as expected. The polygalaptin was composed of a mixture of different molecular mass species: 14.5 kDa, 4%; 29 kDa, 33%; 43 kDa, 2%; 60 kDa, 22%; 90 kDa, 8 % ; 183 kDa, 23%; and polymer at the gel origin, 9%. For the experiments reported, polygalaptin was used as a mixture of these
H.AHMED E T A L .
4
components. The hemagglutinating activity of dimeric and polygalaptin was compared using rabbit erythrocytes as target cells. Dimeric galaptin had a hemagglutinating specific activity (titer/mg/mL) of 2 x 103whereas polygalaptin had a specific activity of 6 x lo4. The specific activity values varied somewhat among different lectin preparations; however, polygalaptin was always 1 3 0 times better than dimeric galaptin. Hemagglutination was inhibited by 0.1 M lactose. Polygalaptin showed up to a 15-fold enhanced binding on a weight basis, relative to dimeric galaptin, to plastic-adsorbed asialofetuin (Fig. 2). At binding saturation to asialofetuin, this enhancement was 3- to 4fold. Binding was inhibited by lactose but with differing tendency. To demonstrate this, lactose inhibition experiments were carried out with lectin concentrations of 1.2 (based upon the absorption chosen to give ANSnm limit of the plate reader) at zero inhibition. Lactose Z,, for 0.1 pg polygalaptin/well was 1.9 mM whereas lactose for 10 pg dimeric galaptin/well was 0.82 mM. The ratio of lactose Zs0to 0.1 pg polygalaptin was >200 times the corresponding ratio for dimeric galaptin ([so: 10 VB). Galaptin binding to lymphoblastoid cells was previously shown to be reversible at 4 "C and also ta result in endocytosis of the receptors at 37 "C (Allen et al.,
,001
.01
.1
1
10
pg Galaptin/Well
loo 90
-
-
e 80-
[,,
Figure 1. SDS-PAGE and fluorography of 14C-alkylated native and polymerized galaptin. The samples were reduced in 0.6 M Tris10.018 M EDTN0.030 M dithiothreitol, pH 8.4, and alkylated with 0.060 M iodoacetamide under N2 at room temperature. The samples were run on a 7.5% acrylamide slab gel: lane 1, polygalaptin; lane 2, native (dimeric galaptin); lane 3, molecular weight standards.
.1
1
10
100
mM Lactose
Figure2. ELlSA of dimeric (0) and polymeric (0)galaptin binding to asialofetuin. Binding to plastic-adsorbed asialofetuin in microtiter plates (A) and lactose inhibtion of binding (B) was carried out as described in the Experimental section. Bound galaptin was detected with rabbit anti-galaptin serum and goat anti-rabbit IgG-peroxidase complex. For lactose inhibition, dimeric galaptin was present at 12 pg/60 pLlwell and polygalaptin was present at 0.12 pg160 Llwell. These amounts gave b5 nm of 1.2 at 0% inhibition.
1990a). Cell binding assays were carried out in these studies to determine if lymphoblastoid cells possess a finite number of galaptin receptors and to compare the binding of dimeric and polygalaptin. For analysis of the binding data, the molecular mass of the galaptin monomeric subunit, 14.5 kDa, was used for both dimeric galaptin and for polymeric galaptin to calculate carbohydrate binding site molarities. This was based on the apparent presence of one binding site per 14.5 kDa polypeptide (Hirabayashi and Kasai, 1991). The binding of galaptin to B142 cell receptors was a saturable process [Fig. 3(A)]. Binding was inhibited 95-300% by 0.05 M or 0.1 M lactose. Scatchard plots [Fig. 3(B)] were constructed from dimeric and polymeric galaptin saturation binding data. The nature of the Scatchard plot for polygalaptin [Fig. 3(B)] is consistent with positive co-operativity in the binding reaction at low polygalaptin concentration. To estimate affinity constants and receptorskell, the first three data points for polygalaptin were ignored for the computer generation of straight lines. The number of binding sites/cell was 5.3 X lo7 with an affinity constant of 2.2 x 105 M-' for dimeric galaptin. A similar affinity constant, 3.4 x ~O'/M, was obtained for polymeric galaptin. The number of binding sites/cell for polygalaptin was estimated to be 17.4 X lo', about three times greater than for dimeric galaptin. To determine if a multimolecular form of galaptin would show enhanced mediation of cell adhesion, the ability of dimeric and polymeric galaptin to promote B142 cell adhesion was compared. Dimeric galaptin was inactive in promoting cell adhesion up to 100~1.g
BINDING ACTIVITY OF DIMERIC AND POLYMERIC GALAPTIN
galaptin/well [Fig. 4(A)]. Polymeric galaptin promoted cell adhesion over a range of cell concentrations. Maximum adhesion occurred at -0.6 pg polymeric galaptin/well with rapid rise in adhesion to platueau values [Fig. 4[A)]. In the range of maximum polymeric galaptin-mediated adhesion, the adhesion was inhibited up to 80% by 0.05 M lactose [Fig. 4(B)J. Similar inhibition was obtained with 0.1 M lactose. B142 cell adhesion to plastic-adsorbed BSA in the absence of galaptin was 0-5% cell input. Experiments were carried out to determine if dimeric and polymeric galaptin adsorbed to microtiter wells similarly. The two forms of galaptin showed similar adsorption to plastic (Fig. 5 ) . The maximum difference in adsorption at 10 pg galaptin/well was -25%. The ability of the plastic-adsorbed dimeric and polymeric galaptin to bind asialofetuin in a lactoseinhibitable manner was compared to determine if adsorption resulted in inactivation of dimeric galaptin. Both forms of galaptin bound asialofetuin although binding to polymeric galaptin was significantly more efficient [Fig. 6(A)]. In contrast to expectations, binding of the glycoconjugate to the plastic-adsorbed galaptins was only partially inhibited by lactose; -65% for polygalaptin and 30-50% for dimeric galaptin at 1 pg galaptin/well.
5
2.5
d
2.0
3 1.5
1:: nn
B
1.6
1-
4
1.0
0.5
