BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS Pages 1674-1680
Vol. 186, No. 3, 1992 August 14, 1992
DICHOTOMY IN THE LAMININ-BINDING PROPERTIES OF SOLUBLE AND MEMBRANE-BOUND HUMAN GALACTOSIDE-BINDING PROTEIN Josiah Ochieng, Mark Gerold and Avraham Raz Metastasis Program, Michigan Cancer Foundation, 110 East Warren Avenue, Detroit, MI 48201 Received July 2, 1992
SUMMARY: Recent studies indicate that galactoside-binding proteins may bind the poly-Nacetyllactosamine sequences of laminin. We questioned whether human galactoside-binding protein (hL-31) binds to laminin and whether cells that express hL-31 on their surface use it as a laminin receptor to promote cellular attachment. The data show that both lectin and cells bind to immobilized laminin. The binding of soluble lectin to laminin is inhibited by lactose, while cell adhesion to it is not. The results indicate that laminin may be a ligand for soluble galactoside-binding proteins. ~ 1992A c a d e m i c P ..... inc.
Laminin is the major non-collagenous polypeptide of basement membranes (1), and is a glycoprotein containing N-linked oligosaccharides (2) which serves as a ligand for several cell surface integrins that bind to different domains of the protein, modulating various normal and transformed cell phenotypes. These include cell adhesion and migration, growth and differentiation, invasion and metastasis (1,3). The biological role of the poly-Nacetyllactosamine sequences of laminin, although implicated to play a role in cellular recognition and adhesion (4), is less characterized compared to the various functional properties ascribed for its amino acid sequence. Recently, on macrophages, a major nonintegrin laminin binding protein was identified and found to be the Mac-2 cell surface antigen (5). Following molecular cloning and sequencing the Mac-2 antigen was found to be a carbohydrate-binding protein homologous to the low affinity IgE-receptor (6); the 35 kDa carbohydrate-binding protein (7); the rat, mouse and human 29 kDa galactoside-binding lectin (8), and to the murine and human tumor associated 34 kDa and 31 kDa galactosidebinding lectins, respectively (9-10).
Abbreviations: SDS, Sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; hL31, human galactoside binding protein; rhL-31, recombinant human galactoside binding protein; EHS, Englebreth-Holm Swarm. 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1674
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Two other carbohydrate-binding proteins with the same sugar specificity were ,=viously shown to bind laminin; the soluble 14 kDa lactose binding lectin (L-14) (11,12) and 67 kDa elastin/laminin-binding protein (13). Since the normal function of the lectin is not fully understood and its natural ligand remains elusive, we questioned its possible ction as a non-integrin laminin receptor on human breast epithelial cells. We have recently ,ned the human 31 kDa galactoside-binding lectin (hL-31) (10) and expressed it in E. coli ~). The availability of the recombinant hL-31 (rhL-31) made it possible to directly test its ~ding properties to laminin. Our studies demonstrate that soluble lectin binds to "nobilized laminin and that the binding is inhibited by lactose, while cell binding to laminin is )endent upon divalent ions but not lactose-dependent.
MATERIALS AND METHODS =terials EHS Laminin and 12~l-goat anti-rabbit IgG were obtained from Sigma, St. Louis, MO., ~tinylated and enzyme label reagents from Biogenex, San Ramon, CA; lactose was from dak, Rochester, NY, FITC-goat anti-rabbit from Zymed, San Francisco, CA; and romogen-substrate reagents from Hyclone, Logan, UT. The recombinant lectin (rhL-31), ,s isolated and purified as described (t4). Antibodies to purified hL-31 were raised in rabbit Bethyl laboratories, Montgomery, IX. Ceils: Normal human breast epithelial cell line ;M-153, was kindly provided by Dr. H. Soule of the Michigan Cancer Foundation. The cells ,re maintained in DMEM/F12 with 5% chelexed equine serum and containing 0.04 mM Ca +* is as described (15). tirect immunofluorescence and Imrnunoblotting SCM-153 cells were plated on glass coverslips until 70-80% confluent and processed indirect immunofluorescence as described (16), using anti-hL-31. Cells similarly grown on tstic were lysed with phosphate lysis buffer and separated by SDS-PAGE (20/~g/lane). ~e samples from the slab gel were electroblotted to immobilon-P. The membranes were lenched in 5% non-fat dried milk in PBS for 4 hrs and then incubated with the primary ~tibody for 1 hr (23*C) in the same quench solution. The membrane was washed five times 3 min each) with the quench solution containing 0.1% tween, and subsequently incubated th the secondary antibody (iodinated goat anti-rabbit IgG) for 1 hr (23*C) and washed as ~ove.
=,11attachment to laminin coated wells Laminin coated wells (2/~g/well) were prepared as described (12). To assess the :achment of SCM-153 cells to these wells in the presence or absence of divalent ions (1.4 VI Ca *÷, 0.8 mM Mg ÷*) and the effect of lactose (0.1M) on cell attachment, 5 x l 0 ' cells were ]ted and allowed to attach for 2 hrs at 37*C after which the non-adherent cells were tshed off with PBS. The adherent cells were fixed with methanol, and stained with toluidine Je and photographed. The cells were also plated in increasing number in PBS with and thout the above mentioned divalent ions, allowed to attach as above, adherent cells fixed th methanol, stained with methylene blue which was released by the addition of HCI;ohol and the optical density (650 nm) determined by a plate reader as described (17). 1675
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Lectin binding to laminin The rhL-31 in PBS was divided into two portions. One portion was incubated with lactose (0.1M) in PBS and the other with PBS alone for 30 min at 4°C. The lectin samples were then added to the laminin coated plates at concentrations ranging from 20-100/~g/well and incubated for at least one hour at 37°C after which the wells were drained, washed with PBS (x3) and incubated with rabbit anti-rhL-31 (1:200) for 30 min at 37°C. The wells were washed with PBS (x3) containing 0.05% tween and then incubated with biotinylated secondary antibodies for 5 min at 37°C. After washing with PBS (x3), the peroxidase label was added and incubated for a further 5 min at 37°C. The wells were once more washed (x6) and the chromogen-substrate reagent added and the color developed for at least 15 min. The O.D. at 405 nm was determined for each well. rhL-31 and SCM-153 cell binding to electroblotted laminin Three laminin samples (10/~g each) were separated on 8% SDS-PAGE gel, electroblotted onto immobilon-P overnight and quenched in 5% dried non-fat milk in PBS for 4 hrs. The membrane was then cut into three equal strips with each strip representing a laminin lane. Two strips were incubated overnight at 4 C with 400 ~g of rhL-31 plus or minus 0.1 M lactose. The strips were washed with quench solution containing 0.05% tween, incubated with anti-rhL-31 and bound hL-31 detected with 12~l-secondary antibody as described above. The other strip was incubated overnight with SCM-153 cells in serum-free medium at 37°C, washed with PBS (x3) and stained with toluidine blue for 30 min at 37°C, briefly washed with methanol and the bound cells photographed. RESULTS AND DISCUSSION The location and spatial distribution of hL-31 on the cell surface of SCM-153 human breast epithelial cells is depicted in Figure 1. Staining of viable cells was in the form of microclusters distributed randomly at the cell circumference, similar to the staining pattern observed with other cells (16). Western analysis confirmed that the anti-lectin antibodies recognized only the hL-31 out of total cell protein extract (Fig. 1, inset). Next, we sought to determine the binding properties of the SCM-153 cells to laminin. Cells were plated onto laminin-coated substrates in the presence (Fig. 2A) or absence of (Fig. 2B) divalent cations. From the results it is obvious that the binding of the human breast epithelial cells to laminin is highly dependent on the presence of the divalent cations (Figs. 2A, B and D).
The addition
of lactose to the adhesion media had no obvious effect on the adhesive properties of the cells to the immobilized laminin (Fig. 2C). Furthermore, anti-lectin antibodies also failed to inhibit the binding of the SCM-153 cells to laminin (data not shown). These results suggest that at least on the human breast epithelial cells the galactoside-lectin is not functioning as a non-integrin laminin receptor and that adhesion to laminin is most probably integrin-mediated Furthermore, recently it was shown that EDTA but not lectin-specific antibodies abolishes the binding of baby hamster kidney cells to the immobilized laminin (18) and thiodigalactose had no effect on myoblast adhesion to laminin (12). These results posed two possibilities concerning the function of galactoside-lectin as laminin receptor; a) the human hL-31 is not a laminin-binding protein or b) it is not functioning as a cell surface non-integrin laminin binding 1676
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
Figure 1. Indirect immunofluorescence labeling of viable SCM-153 cells with anti-hL-31 antibodies (x600). Insert is the Western blot of the SCM-153 cell lysate probed with anti-hL31 showing the expression of 31 kDa hL-31. protein while being able to recognize the carbohydrate side chains of laminin. To address these questions we performed the following experiments. Laminin was immobilized on the tissue culture well micro-titer plates (2/~g/well) then lectin in suspension was added and the amount bound in each well was determined (Fig. 3), implying that laminin can serve as a ligand for soluble lectin. Binding of the lectin to laminin was saturable and completely blocked by lactose (Fig. 3), and is independent of divalent ons. Similar results were obtained with the low molecular weight calf heart agglutinin (9). Secondly, purified laminin was separated on SDS-PAGE under reducing conditions and then electroblotted onto Immobilon-P. The laminin migrated as two bands, the A chain ( - 4 0 0 KDa) and the B chains ( - 200 kDa) (Fig. 4a). The B1 and B2 chain are not well separated under the experimental conditions. Laminin blots were overlaid with rhL-31 in the absence (Fig. 4b) or presence of 0.1M lactose (Fig. 4c). After washing, the blots were incubated with 1677
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
D 0.4 0.3 O
0.2
O0.1 o( o
20
40
60
80
10c
C e l l N u m b e r ( x l O "3) Figure 2. Binding of SCM-153 cells to laminin coated wells. The cells (5 x 104) were plated on laminin coated wells (2/~g/well) and bound cells in the presence (A), absence of divalent ions (B) or presence of divalent ions and 0.1 M lactose (C). Cells were photographed after washing (x3) with PBS (x200). The binding of the cells to laminin coated wells in a dosedependent manner in the presence (-0-0-) or absence (-0-0-) of divalent ions was also determined (D).
anti-lectin antibodies followed by '2'l-goat anti-rabbit IgG and autoradiography. From Fig. 4b it is clear the rhL-31 exhibited a higher affinity to the A chain and the binding is significantly reduced by lactose (Fig. 4c). Likewise, SCM-153 cells show a greater preference binding to the A chain of laminin (Fig. 4d and e) and this binding is not lactose-sensitive. The results reported here suggest that laminin may serve as a ligand for the soluble hL-31 as previously shown for the low molecular weight (14 kD) counterpart lectins (11,12). However, unlike the Mac-2 antigen, the major cell surface non-integrin laminin binding protein of macrophages,
1678
V o l . 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
h
J ....
C
~J~
0
I
6 Q
0
@ 0
20 40 60 80 I00 hL-31 (,ug/well)
Figure 3. Binding of rhL-31 to laminin coated wells. Binding of rhL-31 in a dosedependent manner to laminin coated wells (2 #g/well) in the absence (-0-0-) or presence (-el-) of 0.1 M lactose was assayed as described in Materials and Methods. Each determination is an average (+ S.E.) of two experiments done in triplicate. Figure 4. Binding of hL-31 and SCM-153 cells to the subunits of laminin. Laminin (10 ~g/lane) was separated into A and B subunits on 8% SDS-PAGE gel which was stained with coomassie blue (a) or transferred to immobilon-P which was cut into strips (each strip representing a laminin lane). The strips were incubated with hL-31 without (b) or with (c) lactose and incubated with antibodies to hL-31 as described in Materials and Methods. Another strip was incubated with SCM-153 cells for 24 hrs and cells bound to the A subunit (d) and B subunits (e) photographed (x] 30). The hL-31 of the human breast epithelial SCM-153 cells most probably does not function as a cell surface laminin receptor. This dichotomy in the functional properties and biological significance of the soluble and the membrane-bound lectins is now under investigation. Acknowledgments:
This work was supported in part by grant CTR-2902 from the Council
for Tobacco Research (A.R.) by R01-CA46120 from NIH (A.R.) and R01-CA51714-0A2S1 from NIH (J.O.). A Raz is the recipient of the Paul Zuckerman support Foundation for Cancer Research. REFERENCES .
2. 3. 4. 5. ,
7. 8. .
10. 11.
Martin, G.K. and Timple, R. (1987) Ann. Rev. Cell Biol. 3, 57-85. Knibbs, R.N., Ferini, F. and Goldstein, I.J. (1989) Biochemistry 28, 6379-6392. Hynes, R.O. (1992) Cell 69, 11-25. Runyan, R.B., Versalovic, J. and Shur, B.D. (1988) J. Cell Biol. 107:1863-1871. Woo, H.J., Shaw, L.M., Messier, J.M. and Mercurio, A.M. (1990) J. Biol. Chem. 265, 7097-7099. Alaberandt K., Orida, N.K. and Liu, F.T. (1987) Proc. Natl. Acad. Sci. 84, 6859-6863. Jia, S. and Wang, J.L. (1988) J. Biol. Chem. 263, 6009-6011. Oda, Y., Leffler, H., Sakakura, Y., Kasai, K-I. and Barondes, S.H. (1991) Gene 99, 279283. Raz, A., Pazerini, G., and Carmi, P. (1989) Cancer Res. 49, 3489-3493. Raz, A., Carmi, P., Raz, T., Hogan, V., Mohammed, A. and Wolman, S.R. (1991) Cancer Res. 51, 2173-2178. Zhou, Q. and Cummings, R.D. (1990) Arch. Biochem. Biophys. 281, 27-35. 1679
Vol. 186, No. 3, 1 9 9 2 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Cooper, D.N.W., Massa, S.M. and Barondes, S.H. (1991) J. Cell Biol. 115, 1437-1448 Mecham, R.P., Hinek, A., Griffin, G.L., Senior, R.M. and Liotta, L.A. (1989) J. Biol. Chem. 264, 16652-16657. Ochieng, J., Platt, D., Tait, L., Hogan, V., Raz, T., Carmi, P. and Raz, A. (1992) (Submitted). Ochieng, J., Tait, L. and Russo, J. (1990) In Vitro 26, 318-324. Raz, A., Meromsky, L., Carmi, P., Karakash, R., Lotan, D. and Lotan, R. (1984) EMBO J. 3, 2979-2983. Oliver, M.H., Harrison, N.K., Bishop, J.E., Cole, P.J. and Laurent, G.J. (1989) J. Cell Sci. 92, 513-518. Sato, S. and Hughes, R.C. (1992) J. Biol. Chem. 267, 6983-6990.
1680
Vol. 186, No. 3, 1992 August 14, 1992
DICHOTOMY IN THE LAMININ-BINDING PROPERTIES OF SOLUBLE AND MEMBRANE-BOUND HUMAN GALACTOSIDE-BINDING PROTEIN Josiah Ochieng, Mark Gerold and Avraham Raz Metastasis Program, Michigan Cancer Foundation, 110 East Warren Avenue, Detroit, MI 48201 Received July 2, 1992
SUMMARY: Recent studies indicate that galactoside-binding proteins may bind the poly-Nacetyllactosamine sequences of laminin. We questioned whether human galactoside-binding protein (hL-31) binds to laminin and whether cells that express hL-31 on their surface use it as a laminin receptor to promote cellular attachment. The data show that both lectin and cells bind to immobilized laminin. The binding of soluble lectin to laminin is inhibited by lactose, while cell adhesion to it is not. The results indicate that laminin may be a ligand for soluble galactoside-binding proteins. ~ 1992A c a d e m i c P ..... inc.
Laminin is the major non-collagenous polypeptide of basement membranes (1), and is a glycoprotein containing N-linked oligosaccharides (2) which serves as a ligand for several cell surface integrins that bind to different domains of the protein, modulating various normal and transformed cell phenotypes. These include cell adhesion and migration, growth and differentiation, invasion and metastasis (1,3). The biological role of the poly-Nacetyllactosamine sequences of laminin, although implicated to play a role in cellular recognition and adhesion (4), is less characterized compared to the various functional properties ascribed for its amino acid sequence. Recently, on macrophages, a major nonintegrin laminin binding protein was identified and found to be the Mac-2 cell surface antigen (5). Following molecular cloning and sequencing the Mac-2 antigen was found to be a carbohydrate-binding protein homologous to the low affinity IgE-receptor (6); the 35 kDa carbohydrate-binding protein (7); the rat, mouse and human 29 kDa galactoside-binding lectin (8), and to the murine and human tumor associated 34 kDa and 31 kDa galactosidebinding lectins, respectively (9-10).
Abbreviations: SDS, Sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; hL31, human galactoside binding protein; rhL-31, recombinant human galactoside binding protein; EHS, Englebreth-Holm Swarm. 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1674
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Two other carbohydrate-binding proteins with the same sugar specificity were ,=viously shown to bind laminin; the soluble 14 kDa lactose binding lectin (L-14) (11,12) and 67 kDa elastin/laminin-binding protein (13). Since the normal function of the lectin is not fully understood and its natural ligand remains elusive, we questioned its possible ction as a non-integrin laminin receptor on human breast epithelial cells. We have recently ,ned the human 31 kDa galactoside-binding lectin (hL-31) (10) and expressed it in E. coli ~). The availability of the recombinant hL-31 (rhL-31) made it possible to directly test its ~ding properties to laminin. Our studies demonstrate that soluble lectin binds to "nobilized laminin and that the binding is inhibited by lactose, while cell binding to laminin is )endent upon divalent ions but not lactose-dependent.
MATERIALS AND METHODS =terials EHS Laminin and 12~l-goat anti-rabbit IgG were obtained from Sigma, St. Louis, MO., ~tinylated and enzyme label reagents from Biogenex, San Ramon, CA; lactose was from dak, Rochester, NY, FITC-goat anti-rabbit from Zymed, San Francisco, CA; and romogen-substrate reagents from Hyclone, Logan, UT. The recombinant lectin (rhL-31), ,s isolated and purified as described (t4). Antibodies to purified hL-31 were raised in rabbit Bethyl laboratories, Montgomery, IX. Ceils: Normal human breast epithelial cell line ;M-153, was kindly provided by Dr. H. Soule of the Michigan Cancer Foundation. The cells ,re maintained in DMEM/F12 with 5% chelexed equine serum and containing 0.04 mM Ca +* is as described (15). tirect immunofluorescence and Imrnunoblotting SCM-153 cells were plated on glass coverslips until 70-80% confluent and processed indirect immunofluorescence as described (16), using anti-hL-31. Cells similarly grown on tstic were lysed with phosphate lysis buffer and separated by SDS-PAGE (20/~g/lane). ~e samples from the slab gel were electroblotted to immobilon-P. The membranes were lenched in 5% non-fat dried milk in PBS for 4 hrs and then incubated with the primary ~tibody for 1 hr (23*C) in the same quench solution. The membrane was washed five times 3 min each) with the quench solution containing 0.1% tween, and subsequently incubated th the secondary antibody (iodinated goat anti-rabbit IgG) for 1 hr (23*C) and washed as ~ove.
=,11attachment to laminin coated wells Laminin coated wells (2/~g/well) were prepared as described (12). To assess the :achment of SCM-153 cells to these wells in the presence or absence of divalent ions (1.4 VI Ca *÷, 0.8 mM Mg ÷*) and the effect of lactose (0.1M) on cell attachment, 5 x l 0 ' cells were ]ted and allowed to attach for 2 hrs at 37*C after which the non-adherent cells were tshed off with PBS. The adherent cells were fixed with methanol, and stained with toluidine Je and photographed. The cells were also plated in increasing number in PBS with and thout the above mentioned divalent ions, allowed to attach as above, adherent cells fixed th methanol, stained with methylene blue which was released by the addition of HCI;ohol and the optical density (650 nm) determined by a plate reader as described (17). 1675
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Lectin binding to laminin The rhL-31 in PBS was divided into two portions. One portion was incubated with lactose (0.1M) in PBS and the other with PBS alone for 30 min at 4°C. The lectin samples were then added to the laminin coated plates at concentrations ranging from 20-100/~g/well and incubated for at least one hour at 37°C after which the wells were drained, washed with PBS (x3) and incubated with rabbit anti-rhL-31 (1:200) for 30 min at 37°C. The wells were washed with PBS (x3) containing 0.05% tween and then incubated with biotinylated secondary antibodies for 5 min at 37°C. After washing with PBS (x3), the peroxidase label was added and incubated for a further 5 min at 37°C. The wells were once more washed (x6) and the chromogen-substrate reagent added and the color developed for at least 15 min. The O.D. at 405 nm was determined for each well. rhL-31 and SCM-153 cell binding to electroblotted laminin Three laminin samples (10/~g each) were separated on 8% SDS-PAGE gel, electroblotted onto immobilon-P overnight and quenched in 5% dried non-fat milk in PBS for 4 hrs. The membrane was then cut into three equal strips with each strip representing a laminin lane. Two strips were incubated overnight at 4 C with 400 ~g of rhL-31 plus or minus 0.1 M lactose. The strips were washed with quench solution containing 0.05% tween, incubated with anti-rhL-31 and bound hL-31 detected with 12~l-secondary antibody as described above. The other strip was incubated overnight with SCM-153 cells in serum-free medium at 37°C, washed with PBS (x3) and stained with toluidine blue for 30 min at 37°C, briefly washed with methanol and the bound cells photographed. RESULTS AND DISCUSSION The location and spatial distribution of hL-31 on the cell surface of SCM-153 human breast epithelial cells is depicted in Figure 1. Staining of viable cells was in the form of microclusters distributed randomly at the cell circumference, similar to the staining pattern observed with other cells (16). Western analysis confirmed that the anti-lectin antibodies recognized only the hL-31 out of total cell protein extract (Fig. 1, inset). Next, we sought to determine the binding properties of the SCM-153 cells to laminin. Cells were plated onto laminin-coated substrates in the presence (Fig. 2A) or absence of (Fig. 2B) divalent cations. From the results it is obvious that the binding of the human breast epithelial cells to laminin is highly dependent on the presence of the divalent cations (Figs. 2A, B and D).
The addition
of lactose to the adhesion media had no obvious effect on the adhesive properties of the cells to the immobilized laminin (Fig. 2C). Furthermore, anti-lectin antibodies also failed to inhibit the binding of the SCM-153 cells to laminin (data not shown). These results suggest that at least on the human breast epithelial cells the galactoside-lectin is not functioning as a non-integrin laminin receptor and that adhesion to laminin is most probably integrin-mediated Furthermore, recently it was shown that EDTA but not lectin-specific antibodies abolishes the binding of baby hamster kidney cells to the immobilized laminin (18) and thiodigalactose had no effect on myoblast adhesion to laminin (12). These results posed two possibilities concerning the function of galactoside-lectin as laminin receptor; a) the human hL-31 is not a laminin-binding protein or b) it is not functioning as a cell surface non-integrin laminin binding 1676
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
Figure 1. Indirect immunofluorescence labeling of viable SCM-153 cells with anti-hL-31 antibodies (x600). Insert is the Western blot of the SCM-153 cell lysate probed with anti-hL31 showing the expression of 31 kDa hL-31. protein while being able to recognize the carbohydrate side chains of laminin. To address these questions we performed the following experiments. Laminin was immobilized on the tissue culture well micro-titer plates (2/~g/well) then lectin in suspension was added and the amount bound in each well was determined (Fig. 3), implying that laminin can serve as a ligand for soluble lectin. Binding of the lectin to laminin was saturable and completely blocked by lactose (Fig. 3), and is independent of divalent ons. Similar results were obtained with the low molecular weight calf heart agglutinin (9). Secondly, purified laminin was separated on SDS-PAGE under reducing conditions and then electroblotted onto Immobilon-P. The laminin migrated as two bands, the A chain ( - 4 0 0 KDa) and the B chains ( - 200 kDa) (Fig. 4a). The B1 and B2 chain are not well separated under the experimental conditions. Laminin blots were overlaid with rhL-31 in the absence (Fig. 4b) or presence of 0.1M lactose (Fig. 4c). After washing, the blots were incubated with 1677
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
D 0.4 0.3 O
0.2
O0.1 o( o
20
40
60
80
10c
C e l l N u m b e r ( x l O "3) Figure 2. Binding of SCM-153 cells to laminin coated wells. The cells (5 x 104) were plated on laminin coated wells (2/~g/well) and bound cells in the presence (A), absence of divalent ions (B) or presence of divalent ions and 0.1 M lactose (C). Cells were photographed after washing (x3) with PBS (x200). The binding of the cells to laminin coated wells in a dosedependent manner in the presence (-0-0-) or absence (-0-0-) of divalent ions was also determined (D).
anti-lectin antibodies followed by '2'l-goat anti-rabbit IgG and autoradiography. From Fig. 4b it is clear the rhL-31 exhibited a higher affinity to the A chain and the binding is significantly reduced by lactose (Fig. 4c). Likewise, SCM-153 cells show a greater preference binding to the A chain of laminin (Fig. 4d and e) and this binding is not lactose-sensitive. The results reported here suggest that laminin may serve as a ligand for the soluble hL-31 as previously shown for the low molecular weight (14 kD) counterpart lectins (11,12). However, unlike the Mac-2 antigen, the major cell surface non-integrin laminin binding protein of macrophages,
1678
V o l . 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
h
J ....
C
~J~
0
I
6 Q
0
@ 0
20 40 60 80 I00 hL-31 (,ug/well)
Figure 3. Binding of rhL-31 to laminin coated wells. Binding of rhL-31 in a dosedependent manner to laminin coated wells (2 #g/well) in the absence (-0-0-) or presence (-el-) of 0.1 M lactose was assayed as described in Materials and Methods. Each determination is an average (+ S.E.) of two experiments done in triplicate. Figure 4. Binding of hL-31 and SCM-153 cells to the subunits of laminin. Laminin (10 ~g/lane) was separated into A and B subunits on 8% SDS-PAGE gel which was stained with coomassie blue (a) or transferred to immobilon-P which was cut into strips (each strip representing a laminin lane). The strips were incubated with hL-31 without (b) or with (c) lactose and incubated with antibodies to hL-31 as described in Materials and Methods. Another strip was incubated with SCM-153 cells for 24 hrs and cells bound to the A subunit (d) and B subunits (e) photographed (x] 30). The hL-31 of the human breast epithelial SCM-153 cells most probably does not function as a cell surface laminin receptor. This dichotomy in the functional properties and biological significance of the soluble and the membrane-bound lectins is now under investigation. Acknowledgments:
This work was supported in part by grant CTR-2902 from the Council
for Tobacco Research (A.R.) by R01-CA46120 from NIH (A.R.) and R01-CA51714-0A2S1 from NIH (J.O.). A Raz is the recipient of the Paul Zuckerman support Foundation for Cancer Research. REFERENCES .
2. 3. 4. 5. ,
7. 8. .
10. 11.
Martin, G.K. and Timple, R. (1987) Ann. Rev. Cell Biol. 3, 57-85. Knibbs, R.N., Ferini, F. and Goldstein, I.J. (1989) Biochemistry 28, 6379-6392. Hynes, R.O. (1992) Cell 69, 11-25. Runyan, R.B., Versalovic, J. and Shur, B.D. (1988) J. Cell Biol. 107:1863-1871. Woo, H.J., Shaw, L.M., Messier, J.M. and Mercurio, A.M. (1990) J. Biol. Chem. 265, 7097-7099. Alaberandt K., Orida, N.K. and Liu, F.T. (1987) Proc. Natl. Acad. Sci. 84, 6859-6863. Jia, S. and Wang, J.L. (1988) J. Biol. Chem. 263, 6009-6011. Oda, Y., Leffler, H., Sakakura, Y., Kasai, K-I. and Barondes, S.H. (1991) Gene 99, 279283. Raz, A., Pazerini, G., and Carmi, P. (1989) Cancer Res. 49, 3489-3493. Raz, A., Carmi, P., Raz, T., Hogan, V., Mohammed, A. and Wolman, S.R. (1991) Cancer Res. 51, 2173-2178. Zhou, Q. and Cummings, R.D. (1990) Arch. Biochem. Biophys. 281, 27-35. 1679
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BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
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