Laminin Binds, Specifically to Sulfated Glycolipids (Sulfatides/Fibronectin/Agglutination/Gangliosides) DAVID D
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Proc. Natl. Acad. Sci. USA Vol. 82, pp. 1306-1310, March 1985 Biochemistry Laminin binds, specifically to sulfated glycolipids (sulfatides/fibronectin/agglutination/gangliosides) DAVID D. ROBERTS*, C. NAGESWARA RAOt, JOHN L. MAGNANI*, STEVEN L. SPITALNIK*, LANCE A. LIOTTAt, AND VICTOR GINSBURG* *Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, and tLaboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205 Communicated by Phillips W. Robbins, October 15, 1984 ABSTRACT Previous studies of the agglutination of and with high affinity. In contrast, fibronectin, another cell erythrocytes by the basement membrane glycoprotein laminin attachment protein that agglutinates erythrocytes (20), binds have suggested that laminin binds to gangliosides [Kennedy, with low affinity to all anionic lipids tested. D. W., Rohrbach, D. H., Martin, G. R., Momoi, T. & Ya- mada, K. M. (1983) J. CeU. Physiol. 114, 257-262]. Based on the following evidence, however, we find that laminin binds MATERIALS AND METHODS specifically to sulfatides, not gangliosides. Monogalactosyl sul- Materials. Laminin was purified from 0.5 M NaCl extracts fatides, purified from sheep erythrocytes with a yield of 4.3 of mouse Engelbreth Holm Swarm tumor by DEAE-cellu- mg/kg of packed cells, bound laminin with high affinity as did lose chromatography (2) and 4.0 M NaCl precipitation. Pro- authentic bovine brain sulfatide (galactosylceramide-I3-sul_ tease-derived fragments of laminin were purified and exam- fate). The binding activity of these lipids and of total erythro- ined by rotary shadowing electron microscopy as described cyte lipids was stable to alkali and neuraminidase treatment (6, 21). Antibodies to laminin were raised in New Zealand but labile to dilute acid under conditions that destroy sulfa- White adult rabbits and purified by affinity chromatography. tides but not gangliosides. Of various glycolipid and phospho- These antibodies did not crossreact with type IV collagen or lipid standards tested, only sulfatides bound laminin with high heparan sulfate proteoglycan of the tumor matrix. Human affinity. Sulfatide binding and agglutinating activities of pro- plasma fibronectin was prepared by gelatin affinity chroma- teolytic fragments of laminin indicated that the globular end tography (22). Goat anti-human fibronectin was from Cappel regions of the 200-kDa subunits are required for both activi- Laboratories (Cochranville, PA). Protein A, DEAE-Sepha- ties. Thus, monogalactosylsulfatides, and possibly other more rose CL-6B, and Sephadex G-25 were from Pharmacia. Bio- complex sulfated glycolipids, are probably involved in the ag- Sil HA (minus 325 mesh) was from Bio-Rad. Lipid standards glutination oferythrocytes. These results also suggest a physio- were obtained from Supelco (Bellefonte, PA) and Sigma. logical function of sulfatides in cell adhesion. The agglutina- Sheep blood was from the National Institutes of Health Ani- tion of erythrocytes by fibronectin is also inhibited by ganglio- mal Farm (Poolesville, MD). Protein A and fibronectin were sides [Yamada, K. M., Kennedy, D. W., Grotendorst, G. R. labeled with Na125I (ICN) by the Iodo-Gen method (23) to & Momoi, T. (1981) J. Cell. Physiol. 109, 343-351]. Fibronec- specific activities of40-60 and 18 ,Ci/,g, respectively (1 Ci tin, however, did not bind to sulfatides with high affinity but = 37 GBq). Laminin was iodinated using immobilized lacto- rather bound with low affinity to all anionic lipids tested, in- peroxidase (Enzymobeads, Bio-Rad). cluding phospholipids, gangliosides, and sulfatides. Sheep Erythrocyte Glycolipids. Glycolipids were isolated from 2 liters of sheep blood. Ghosts were prepared by hypo- Laminin is a major basement membrane glycoprotein that tonic lysis of washed erythrocytes (24) and were extracted may participate in the attachment of cells to the basement twice with chloroform/methanol/water, 4:8:3 (vol/vol) (25). membrane network through binding to heparan sulfate, type The combined extracts were evaporated to dryness and IV collagen, entactin, and the cell surface (1-5). Specific do- treated with 100 ml of 0.2 M NaOH in methanol for 1 hr at mains of the laminin molecule are involved in its interaction 37°C. The reaction mixture was neutralized with an equal with cells and matrix components (1, 6, 7). In addition to cell volume of 0.2 M HCl in methanol, evaporated to 1/4th vol, attachment (2, 8, 9), laminin is also involved in related phe- dialyzed extensively against distilled water, and lyophilized. nomena including cell growth and differentiation (10), mor- Neutral and acidic lipids were separated on a 3.5 x 10 cm phogenesis (11), cell migration (12), neurite outgrowth (13), column of DEAE-Sepharose, bicarbonate form, equilibrated and cancer metastases (4). A protein receptor, which binds with chloroform/methanol/water, 30:60:8 (vol/vol) (26). Af- laminin with a Kd of 2 x 10-9 M, has been purified from the ter elution of neutral lipids with the same solvent (600 ml), plasma membrane of some laminin-dependent cell lines (14- acidic lipids were eluted with 600 ml each of0.01 M, 0.02 M, 16). Monoclonal antibodies to the receptor blocked laminin 0.05 M, 0.1 M, and 0.5 M NH4HCO3 in methanol. Active binding to these cells (17). lipids in the 0.01 M NH4HCO3 fraction were further purified Laminin agglutinates aldehyde-fixed sheep, human, and by silicic acid chromatography on a 0.8 x 92 cm column of rabbit erythrocytes (18, 19). Inhibition studies ofhemaggluti- Bio-Sil HA. Glycolipids were applied to the column in chlor- nation suggested that gangliosides and some anionic phos- oform/methanol, 96:4 (vol/vol) and eluted with a linear gra- pholipids might also be receptors for laminin (19). We have dient (2 liters) from chloroform/methanol, 9:1 (vol/vol) to tested this hypothesis by measuring binding of laminin to chloroform/methanol/water, 47:47:6 (vol/vol). Purity of the erythrocyte membrane lipids, gangliosides, and other glyco- isolated glycolipids was determined by TLC on silica gel lipids. We report here that sulfated glycolipids, but not gan- high performance plates (Merck) developed with chloro- gliosides or anionic phospholipids, bind laminin specifically form/methanol/0.25% KCl, 5:4:1 (vol/vol), chloroform/ methanol, 4:1 (vol/vol), or 1-propanol/15 M ammonia/ The publication costs of this article were defrayed in part by page charge water, 60:9.5:11.5 (vol/vol). Glycolipids were visualized payment. This article must therefore be hereby marked "advertisement" with orcinol/H2SO4. Other lipid components were visual- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ized using 0.25% ninhydrin in ethanol or by charring. 1306 Downloaded by guest on September 25, 2021 Biochemistry: Roberts et aL Proc. Natl. Acad Sci USA 82 (1985) 1307 Analytical Procedures. Purified erythrocyte sulfatides were quantified by the dye binding assay of Kean (27) using 12 bovine brain sulfatide (galactosylceramide-I3-sulfate, Su- pelco) as a standard. Sugar composition was determined by GLC analysis of alditol acetates (28) on a column packed were desulfated with SP 2340 (Supelco). Isolated glycolipids m using 0.05 M HCl in dry methanol (29) at room temperature 0 for 16 hr. Glycolipids were treated with Clostridium perfrin- x 8 gens neuraminidase (0.1 unit/ml; Sigma, type VI) in 50 mM E sodium acetate/0.15 M NaCl/9 mM CaCl2, pH 5.5. C) Laminin Binding to Lipids. A solid-phase RIA for detec- Q tion of laminin binding to membrane lipid was developed 0 based on previously described methods using monoclonal .0 antibodies (30, 31) for detection of glycolipid antigens. Lam- 4 inin binding to lipids separated on thin layer chromatograms was detected by immunostaining based on a method using monoclonal antibodies (31) for the detection of glycolipid antigens. RESULTS 0.1 1 As reported by Kennedy et al. (19) laminin agglutinates glu- Lipid, ug taraldehyde-fixed sheep erythrocytes and trypsinized fixed human erythrocytes (Table 1). Treatment of the cells with FIG. 1. Binding of laminin to sheep erythrocyte lipids. Lipids in neuraminidase had no effect on sheep erythrocyte agglutina- 25 A.1 of methanol were dried onto the wells ofround-bottomed poly- tion by laminin but increased the agglutinability of human vinylchloride microtiter plates (Dynatech, Alexandria, VA). The erythrocytes, suggesting that neuraminidase-sensitive sialyl wells were then filled with 50 mM Tris-HCl/110 mM NaCl/5 mM EDTA CaCl2/0.02% NaN3/1% bovine serum albumin, pH 8.0 (Tris/albu- residues are not involved in binding laminin. partially min buffer). After 30 min, the wells were emptied and 25 p1 of lam- inhibited agglutination at 2 mM, consistent with the results inin (10 pg/pl) in Tris/albumin buffer was added. The plate was of Ozawa et al. (18) who also reported that Ca2+ is required covered, incubated 2 hr at room temperature, and then washed for agglutination. Kennedy et al. (19) reported no inhibition twice with 0.1 M sodium phosphate/0.15 M NaCl, pH 7.4 (Pj/NaCl). by EDTA. To each well was added 25 Al of a 1:1000 dilution of rabbit anti- A total lipid extract of sheep erythrocytes was tested for laminin in Tris/albumin buffer. The plate was covered, incubated 1 laminin binding by solid phase RIA. Initially, 125I-labeled hr, and washed twice with Pi/NaCl, and 25 1A of "2I-labeled protein laminin was used but it was less active by a factor of at least A ('100,000 cpm) was added. After 1 hr of incubation the wells 10 as an agglutinin than unlabeled laminin and did not bind to were again washed; then, they were cut from the plate, and bound or not There- "I was determined. Control experiments were conducted in which fixed erythrocytes isolated lipids (data shown).