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• W2016107310 abstract "The lecticans are a group of chondroitin sulfate proteoglycans characterized by the presence of C-type lectin domains. Despite the suggestion that their lectin domains interact with carbohydrate ligands, the identity of such ligands has not been elucidated. We previously showed that brevican, a nervous system-specific lectican, binds the surface of B28 glial cells (Yamada, H., Fredette, B., Shitara, K., Hagihara, K., Miura, R., Ranscht, B., Stallcup, W. B., and Yamaguchi, Y. (1997) J. Neurosci. 17, 7784–7795). In this paper, we demonstrate that two classes of sulfated glycolipids, sulfatides and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs), act as cell surface receptors for brevican. The lectin domain of brevican binds sulfatides and SGGLs in a calcium-dependent manner as expected of a C-type lectin domain. Intact, full-length brevican also binds both sulfatides and SGGLs. The lectin domain immobilized as a substrate supports adhesion of cells expressing SGGLs or sulfatides, which was inhibited by monoclonal antibodies against these glycolipids or by treatment of the substrate with SGGLs or sulfatides. Our findings demonstrate that the interaction between the lectin domains of lecticans and sulfated glycolipids comprises a novel cell substrate recognition system, and suggest that lecticans in extracellular matrices serve as substrate for adhesion and migration of cells expressing these glycolipids in vivo. The lecticans are a group of chondroitin sulfate proteoglycans characterized by the presence of C-type lectin domains. Despite the suggestion that their lectin domains interact with carbohydrate ligands, the identity of such ligands has not been elucidated. We previously showed that brevican, a nervous system-specific lectican, binds the surface of B28 glial cells (Yamada, H., Fredette, B., Shitara, K., Hagihara, K., Miura, R., Ranscht, B., Stallcup, W. B., and Yamaguchi, Y. (1997) J. Neurosci. 17, 7784–7795). In this paper, we demonstrate that two classes of sulfated glycolipids, sulfatides and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs), act as cell surface receptors for brevican. The lectin domain of brevican binds sulfatides and SGGLs in a calcium-dependent manner as expected of a C-type lectin domain. Intact, full-length brevican also binds both sulfatides and SGGLs. The lectin domain immobilized as a substrate supports adhesion of cells expressing SGGLs or sulfatides, which was inhibited by monoclonal antibodies against these glycolipids or by treatment of the substrate with SGGLs or sulfatides. Our findings demonstrate that the interaction between the lectin domains of lecticans and sulfated glycolipids comprises a novel cell substrate recognition system, and suggest that lecticans in extracellular matrices serve as substrate for adhesion and migration of cells expressing these glycolipids in vivo. The lecticans are a family of chondroitin sulfate proteoglycans (CSPGs) 1The abbreviations used are: CSPG, chondroitin sulfate proteoglycan; PLD, proteoglycan lectin domain; CRP, complement regulatory protein; ECM, extracellular matrix; SGGL, sulfoglucuronylglycolipid; rCLD, recombinant C-type lectin domain; FNIII, fibronectin type III; EGF, epidermal growth factor; CHO, Chinese hamster ovary; TBS, Tris-buffered saline; MDCK, Madin-Darby canine kidney; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; CMF, calcium- and magnesium-free; HBSS, Hank's balanced salt solution. characterized by the presence of a C-type lectin domain in their core proteins (1Ruoslahti E. Glycobiology. 1996; 6: 489-492Crossref PubMed Scopus (375) Google Scholar, 2Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1349) Google Scholar). The C-terminal globular domains of lecticans, or “proteoglycan lectin domain” (PLD), consist of one or two epidermal growth factor (EGF)-like domains, a C-type lectin domain, and a complement regulatory protein (CRP) domain. This arrangement of domains is similar to that of selectins, suggesting that lecticans are also involved in the recognition of carbohydrate ligands. Lecticans are the most abundantly expressed family of proteoglycans in the nervous system. The lectican family includes aggrecan (3Doege K. Sasaki M. Horigan E. Hassel J.R. Yamada Y. J. Biol. Chem. 1987; 262: 17757-17767Abstract Full Text PDF PubMed Google Scholar), versican (4Zimmermann D.R. Ruoslahti E. EMBO J. 1989; 8: 2975-2981Crossref PubMed Scopus (502) Google Scholar), neurocan (5Rauch U. Karthikeyan L. Maurel P. Margolis R.U. Margolis R.K. J. Biol. Chem. 1992; 267: 19536-19547Abstract Full Text PDF PubMed Google Scholar), and brevican (6Yamada H. Watanabe K. Shimonaka M. Yamaguchi Y. J. Biol. Chem. 1994; 269: 10119-10126Abstract Full Text PDF PubMed Google Scholar), all of which are expressed in the nervous system at certain stages of development (1Ruoslahti E. Glycobiology. 1996; 6: 489-492Crossref PubMed Scopus (375) Google Scholar, 7Yamaguchi Y. Perspect. Dev. Neurobiol. 1996; 3: 307-317PubMed Google Scholar). Although aggrecan and versican were initially characterized as connective tissue proteoglycans, their expression in the nervous system has been demonstrated in a number of reports (8Bignami A. Perides G. Rahemtulla F. J. Neurosci. Res. 1993; 34: 97-106Crossref PubMed Scopus (97) Google Scholar, 9Bode-Lesniewska B. Dours-Zimmermann M.T. Odermatt B.F. Briner J. Heitz P.U. Zimmermann D.R. J. Histochem. Cytochem. 1996; 44: 303-312Crossref PubMed Scopus (163) Google Scholar, 10Li H. Domowicz M. Hennig A. Schwartz N.B. Brain Res. Mol. Brain Res. 1996; 36: 309-321Crossref PubMed Scopus (28) Google Scholar, 11Schwartz N.B. Domowicz M. Krueger Jr., R.C. Li H. Mangoura K. Perspect. Dev. Neurobiol. 1996; 3: 291-306PubMed Google Scholar, 12Milev P. Maurel P. Chiba A. Mevissen M. Popp S. Yamaguchi Y. Margolis R.K. Margolis R.U. Biochem. Biophys. Res. Commun. 1998; 247: 207-212Crossref PubMed Scopus (189) Google Scholar). Brevican and neurocan are specifically expressed in the nervous system (6Yamada H. Watanabe K. Shimonaka M. Yamaguchi Y. J. Biol. Chem. 1994; 269: 10119-10126Abstract Full Text PDF PubMed Google Scholar, 13Rauch U. Gao P. Janetzko A. Flaccus A. Hilgenberg L. Tekotte H. Margolis R.K. Margolis R.U. J. Biol. Chem. 1991; 266: 14785-14801Abstract Full Text PDF PubMed Google Scholar, 14Oohira A. Matsui F. Watanabe E. Kushima Y. Maeda N. Neuroscience. 1994; 60: 145-157Crossref PubMed Scopus (163) Google Scholar, 15Seidenbecher C.I. Richter K. Rauch U. Fässler R. Garner C.C. Gundelfinger E.D. J. Biol. Chem. 1995; 270: 27206-27212Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Structural similarities with selectins and the abundant expression in the nervous system suggest that lecticans play major roles in carbohydrate recognition in the nervous system. The identity of the ligand to PLDs has been a focus of our interest. We previously showed that the PLD of versican binds tenascin-R, an extracellular matrix (ECM) protein predominantly expressed in the nervous system (16Aspberg A. Binkert C. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10590-10594Crossref PubMed Scopus (124) Google Scholar). More recently, we demonstrated that the PLDs of all lecticans bind tenascin-R, and that brevican and tenascin-R indeed form a complex in adult rat brain extracts (17Aspberg A. Miura R. Bourdoulous S. Shimonaka M. Heinegård D. Schachner M. Ruoslahti E. Yamaguchi Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10116-10121Crossref PubMed Scopus (241) Google Scholar). These results suggest that the lectican-tenascin-R interactions, especially the brevican-tenascin-R interaction, are relevant to the assembly of the adult brain ECM. However, these interactions are not carbohydrate-protein interactions expected of C-type lectin domains; they are protein-protein interactions between the PLDs and fibronectin type III domains (FNIII) 3–5 of tenascin-R (17Aspberg A. Miura R. Bourdoulous S. Shimonaka M. Heinegård D. Schachner M. Ruoslahti E. Yamaguchi Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10116-10121Crossref PubMed Scopus (241) Google Scholar). Regarding carbohydrate interactions by PLDs, several studies have demonstrated that PLDs can bind simple sugars and heparin/heparan sulfate in vitro (18Halberg D.F. Proulx G. Doege K. Yamada Y. Drickamer K. J. Biol. Chem. 1988; 263: 9486-9490Abstract Full Text PDF PubMed Google Scholar, 19Saleque S. Ruiz N. Drickamer K. Glycobiology. 1993; 3: 185-190Crossref PubMed Scopus (28) Google Scholar, 20Ujita M. Shinomura T. Ito K. Kitagawa Y. Kimata K. J. Biol. Chem. 1994; 269: 27603-27609Abstract Full Text PDF PubMed Google Scholar). However, these studies failed to identify the nature of any physiological carbohydrate ligands for PLDs. Thus the ability of PLDs to behave as C-type lectins in vivo and the identity of physiological carbohydrate ligands for PLDs are issues that have not been addressed. We have previously shown that purified brevican binds to the surface of primary astrocytes as well as of several immortalized rat neural cell lines (21Yamada H. Fredette B. Shitara K. Hagihara K. Miura R. Ranscht B. Stallcup W.B. Yamaguchi Y. J. Neurosci. 1997; 17: 7784-7795Crossref PubMed Google Scholar). Binding studies with B28 glial cells demonstrated that the binding is mediated by the C-terminal 80-kDa fragment of the brevican core protein which includes the PLD (21Yamada H. Fredette B. Shitara K. Hagihara K. Miura R. Ranscht B. Stallcup W.B. Yamaguchi Y. J. Neurosci. 1997; 17: 7784-7795Crossref PubMed Google Scholar). It was initially suspected that tenascin-R deposited to the surface of these cells may act as the “receptor” for brevican. However, we have found that these cells do not have any tenascin-R on their surface nor did they secrete any tenascin-R into culture supernatants. Furthermore, a number of assays failed to identify any cell surface protein that specifically binds brevican PLD. Since it has been reported that the lectin domains of P- and L-selectins bind sulfated glycolipids, such as sulfatides (22Aruffo A. Kolanus W. Walz G. Fredman P. Seed B. Cell. 1991; 67: 35-44Abstract Full Text PDF PubMed Scopus (264) Google Scholar) and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs) (23Needham L.K. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1339-1363Crossref Scopus (181) Google Scholar), we examined the possibility that the cell surface brevican receptor is a glycolipid rather than a glycoprotein. In this paper, we report that the PLDs of lecticans bind these sulfated glycolipids. We also show that the interaction between brevican and sulfated glycolipids supports adhesion of cells expressing these glycolipids on their surfaces. These observations suggest that the PLD-sulfated glycolipid interactions are a novel cell substrate recognition system. Mixed bovine brain gangliosides, purified bovine brain sulfatides, and galactosylceramides were purchased from Sigma. Mixed neutral glycosphingolipids were obtained from Matreya (Pleasant Gap, PA). The HNK-1 monoclonal antibody was purchased from Becton Dickinson (Bedford, MA). Mouse monoclonal anti-tenascin-R antibody (clone 596) (24Pesheva P. Spiess E. Schachner M. J. Cell Biol. 1989; 109: 1765-1778Crossref PubMed Scopus (233) Google Scholar) and monoclonal anti-sulfatide antibody were gifts from Drs. Melitta Schachner (University of Hamburg, Hamburg, Germany) and Yoshio Hirabayashi (RIKEN, Wako, Japan), respectively. The Fc fusion protein of L-selectin (L-selectin Ig chimera) and a HeLa cell line transfected with cDNAs for HNK-1 sulfotransferase, glucuronyltransferase, and N-CAM were obtained from Dr. Minoru Fukuda (Burnham Institute, La Jolla, CA). A chimeric protein of brevican PLD fused with Fc region of human IgG (brevican PLD chimera) and biotinylated recombinant lectin domains (rCLDs) of all four lecticans were prepared as described previously (17Aspberg A. Miura R. Bourdoulous S. Shimonaka M. Heinegård D. Schachner M. Ruoslahti E. Yamaguchi Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10116-10121Crossref PubMed Scopus (241) Google Scholar). Brevican PLD chimera consists of a short segment of the central domain, a EGF domain, a lectin domain, and a CRP domain of rat brevican. rCLDs consist only of the lectin domains. For preparation of recombinant brevican with no fusion partner, CHO cell were stably transfected with an expression vector pcDNA-rBV, which has a full-length rat brevican cDNA (3.0-kilobase pairEcoRI-EcoRI fragment) (25Yamada H. Watanabe K. Shimonaka M. Yamasaki M. Yamaguchi Y. Biochem. Biophys. Res. Commun. 1995; 216: 957-963Crossref PubMed Scopus (46) Google Scholar) inserted into a uniqueEcoRI site of pcDNAIAmp (Invitrogen, San Diego, CA). For the preparation of radiolabeled probe for TLC-ligand binding assay, cultures of a cloned CHO transfectant were metabolically labeled with 100 μCi/ml Tran35S-Label (ICN, Costa Mesa, CA).35S-Labeled or unlabeled brevican was purified from culture supernatants by affinity chromatography on the FNIII3–5 fragment of tenascin-R as follows. The glutathione S-transferase fusion protein of the FNIII3–5 fragment (17Aspberg A. Miura R. Bourdoulous S. Shimonaka M. Heinegård D. Schachner M. Ruoslahti E. Yamaguchi Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10116-10121Crossref PubMed Scopus (241) Google Scholar) was bound to glutathione-agarose and covalently coupled with dimethylpimelimidate according to Gersten and Marchalonis (26Gersten D.M. Marchalonis J.J. J. Immunol. Methods. 1978; 24: 305-309Crossref PubMed Scopus (108) Google Scholar). Culture supernatants of CHO transfectants were incubated overnight at 4 °C with the FNIII3–5 affinity resin preequilibrated with TBS containing 5 mm CaCl2. After extensive washing, the labeled brevican was eluted from the affinity resin with 20 mm EDTA. For flow cytometry, cells were dissociated with trypsin-EDTA (Irvine Scientific, Irvine, CA), suspended in 10% fetal calf serum in Dulbecco's modified Eagle's medium, and incubated in the medium for 2 h at 37 °C. Cell were then washed three times with PBS containing 0.1% sodium azide, and incubated with PBS containing 1% BSA and 0.1% sodium azide for 20 min on ice. Cells were then incubated with brevican PLD chimera or primary antibodies for 30 min on ice. After washing three times with PBS containing 0.1% sodium azide, the cells were incubated with fluorescein-conjugated goat antibodies to human IgG (Sigma) or to mouse IgG+IgM (Biosource) for 20 min on ice, washed again, and resuspended in 0.5–1.0 ml of PBS containing 0.1% sodium azide. The cells were examined on a FACSort (Becton Dickinson, Oxford, CA). Cell surface biotinylation was performed with sulfo-NHS-biotin (Pierce) according to the manufacture's instruction. Biotinylated molecules were collected by streptavidin-agarose. Ligand overlay and immunoblotting assays were performed as described previously (17Aspberg A. Miura R. Bourdoulous S. Shimonaka M. Heinegård D. Schachner M. Ruoslahti E. Yamaguchi Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10116-10121Crossref PubMed Scopus (241) Google Scholar). For preparation of total glycolipid fraction, tissues or cells were extracted by sonication with chloroform/methanol (2:1) and then with a chloroform/methanol/water (1:2:0.8). After removal of the remaining precipitates by centrifugation, the supernatant was dried under N2 stream. The resulting residue was dissolved in chloroform/methanol (2:1), and then 1/10 volume of 2 m KOH in methanol was added. The solution was incubated at 37 °C for 2 h to degrade phospholipids. The supernatant was collected by centrifugation, neutralized with 1/20 volume of acetic acid, and dried under N2 stream. The residue was suspended in water, dialyzed against water, evaporated to dryness, and then dissolved in chloroform/methanol/water (60:35:8). Acidic glycolipids were purified as described previously (27Miura Y. Yamagata T. Biochem. Biophys. Res. Commun. 1997; 241: 698-703Crossref PubMed Scopus (36) Google Scholar). HNK-1-reactive SGGLs were purified from dog sciatic nerve endoneurium as described previously (28Needham L.K. Schnaar R.L. J. Cell Biol. 1993; 121: 397-408Crossref PubMed Scopus (38) Google Scholar). This sample contains 41% SO3-GlcU-nLc-4-Cer and 59% SO3-GlcU-nLc-6-Cer, both of which are recognized by anti-HNK-1 antibody. These assays were performed according to Taki et al. (29Taki T. Ogura K. Rokukawa C. Hara T. Kawakita M. Endo T. Kobata A. Handa S. Cancer Res. 1991; 51: 1701-1707PubMed Google Scholar). Briefly, glycolipid samples were developed twice on high performance thin layer chromatography plates (Baker Inc., Phillipsburg, NJ) in chloroform/methanol/0.2% aqueous CaCl2 (60:35:8). After drying, the plates were dipped for 30 s in 0.4% polyisobutylmethacrylate (2.5% polyisobutylmethacrylate in chloroform was diluted with hexane to the final concentration of 0.4%), and air-dried. The dried plates were then incubated with various probes (antibodies, brevican PLD chimera, rCLDs, or 35S-labeled native brevican) in TBS containing 1–3% BSA at 4 °C overnight. After washing with TBS, the plates were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies or HRP-conjugated avidin for 1.5 h, followed by the visualization of reactive bands with chemiluminescence. In the case of 35S-labeled brevican, the dried plates were directly exposed to Kodak BioMax film for 3 days. For preparation of substrates, solutions of brevican PLD chimera or human IgG (100 μg/ml) in calcium- and magnesium-free Hank's balanced salt solution (CMF/HBSS) were applied on nitrocellulose-coated plastic (21Yamada H. Fredette B. Shitara K. Hagihara K. Miura R. Ranscht B. Stallcup W.B. Yamaguchi Y. J. Neurosci. 1997; 17: 7784-7795Crossref PubMed Google Scholar), and incubated at 37 °C for 2 h. After washing three times with CMF/HBSS, uncoated surfaces were blocked by incubating with CMF/HBSS containing 2% heat-inactivated BSA (HBSS/BSA) for 2 h at 37 °C. After washing three times with CMF/HBSS, cells suspended in Opti-MEM (Life Technologies, Inc.) containing 0.1% heat-inactivated BSA were plated at a density of 5 × 105 (B28 cells), or 1 × 106 (MDCK cells) per ml, and incubated for 1 h at 37 °C. After gentle washing, attached cells were fixed with 4% paraformaldehyde in PBS and counted under microscope at 200× magnification. For perturbation with antibodies, cells were preincubated with 100 μg/ml HNK-1 or anti-sulfatide monoclonal antibodies diluted in CMF/HBSS/BSA for 30 min on ice prior to the plating of the cells. For perturbation by glycolipids, the substrate of brevican PLD chimera was preincubated with SGGLs, sulfatide, or galactosylceramide at concentration of 5 μg/ml in CMF/HBSS for 1 h at 37 °C before plating of the cells. We have shown that the PLD-containing C-terminal 80-kDa fragment of brevican core protein binds to primary astrocytes and B28 cells, which is an immortalized glial cell line. Binding studies with cell monolayers ruled out hyaluronan, heparan sulfate, and chondroitin sulfate as cell surface “receptors” for the brevican C-terminal fragment (21Yamada H. Fredette B. Shitara K. Hagihara K. Miura R. Ranscht B. Stallcup W.B. Yamaguchi Y. J. Neurosci. 1997; 17: 7784-7795Crossref PubMed Google Scholar). To facilitate the identification of the putative brevican receptor, a fusion protein of brevican PLD and Fc region of human IgG (brevican PLD chimera) and a biotin-labeled recombinant lectin domain of brevican (brevican rCLD) were produced. We first examined the binding of brevican PLD chimera to a series of immortalized neural cell lines derived from BDIX rats (30Stallcup W.B. Cohn M. Exp. Cell Res. 1976; 98: 285-297Crossref PubMed Scopus (63) Google Scholar) in flow cytometric assay. Among 16 cell lines tested, brevican PLD chimera bound to B28 cells (Fig.1 A) and four other glial cell lines, namely B9, B15, B49, and B92. The chimera showed no binding to other cell lines with fibroblastic or neuronal phenotypes, including B23 (Fig. 1 B), B19, B27, B35, B50, B65, B82, B103, B104, B108, or B111 cells (data not shown). These results suggest that the cell surface binding of the 80-kDa fragment is mediated by the PLD of the brevican core protein, and that the brevican receptor is expressed in various neural cell lines. The lectin domain of brevican binds tenascin-R by a protein-protein interaction (17Aspberg A. Miura R. Bourdoulous S. Shimonaka M. Heinegård D. Schachner M. Ruoslahti E. Yamaguchi Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10116-10121Crossref PubMed Scopus (241) Google Scholar). Although tenascin-R is a secreted protein, it is possible that tenascin-R is present on the surface of B28 cells through interaction with cell surface tenascin-R binding proteins (e.g. contactin) or by nonspecific aggregation, thereby acting as an apparent brevican receptor. However, as described previously (21Yamada H. Fredette B. Shitara K. Hagihara K. Miura R. Ranscht B. Stallcup W.B. Yamaguchi Y. J. Neurosci. 1997; 17: 7784-7795Crossref PubMed Google Scholar), no tenascin-R was detected on the surface of B28 cells either by flow cytometric assay or by immunocytochemistry (data not shown). Furthermore, the ligand overlay assay with brevican PLD chimera, which we used to identify tenascin-R as a protein ligand to lectican PLDs in adult rat brain extracts (17Aspberg A. Miura R. Bourdoulous S. Shimonaka M. Heinegård D. Schachner M. Ruoslahti E. Yamaguchi Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10116-10121Crossref PubMed Scopus (241) Google Scholar), did not detect tenascin-R in surface-biotinylated proteins from B28 cells (Fig.1 C). Having ruled out tenascin-R as the putative receptor for brevican PLD, we next examined the possibility that cell surface glycoproteins carrying specific carbohydrates would be the receptor for brevican PLD, as is the case with selectins. However, the ligand overlay experiment demonstrated that B28 cells lacks not only tenascin-R but also any cell surface proteins that specifically interact with brevican PLD chimera (Fig. 1 C), suggesting that there are no glycoprotein ligands for the brevican PLD in B28 cells. We further searched for cell surface brevican-binding proteins in B28 cells by immunoprecipitation of surface-labeled materials and affinity chromatography on a column bearing the 80-kDa brevican fragment that includes PLD. None of these experiments could identify glycoproteins that would specifically bind brevican PLD (data not shown). It has been reported that sulfated cell surface glycolipids, sulfatides and HNK-1-reactive SGGLs, bind to the lectin domains of P- and L-selectin (22Aruffo A. Kolanus W. Walz G. Fredman P. Seed B. Cell. 1991; 67: 35-44Abstract Full Text PDF PubMed Scopus (264) Google Scholar, 23Needham L.K. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1339-1363Crossref Scopus (181) Google Scholar). Therefore, we investigated the possibility that glycolipids would act as cell surface receptors for brevican PLD. To test this, we prepared glycolipids from the adult rat cerebellum and probed with brevican PLD chimera in the TLC-ligand overlay assay. As shown in Fig.2 A, brevican PLD chimera reacted with a single band (lane 2) among a number of glycolipid species extracted from the cerebellum (lane 1). The reactive band migrated at the same position as purified sulfatides (compare panel A,lane 2 with panel B,lane 1), suggesting that the band represents sulfatides. To further identify this reactive band, standard glycolipids were examined by TLC-ligand overlay assay (Fig. 2,B–F) The brevican PLD chimera bound to purified sulfatides (C, lane 1), but not to any of the neutral glycosphingolipids (lane 2) or gangliosides (lane 3). Binding to sulfatides was abolished in the presence of EDTA (D, lane 1), as expected of a C-type lectin interaction. The brevican PLD chimera did not bind galactosylceramide, an unsulfated precursor of sulfatides (indicated by asterisk in B,lane 2), suggesting that a sulfate group is necessary for binding. L-selectin Ig chimera also bound to sulfatides, but not to other glycolipids (E), consistent with previous reports (23Needham L.K. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1339-1363Crossref Scopus (181) Google Scholar). Human IgG did not bind any glycolipids (F). The brevican PLD chimera contains not only the C-type lectin domain but also EGF and CRP domains flanking the lectin domain (17Aspberg A. Miura R. Bourdoulous S. Shimonaka M. Heinegård D. Schachner M. Ruoslahti E. Yamaguchi Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10116-10121Crossref PubMed Scopus (241) Google Scholar). To examine the location of the sulfatide binding site, the binding of biotinylated recombinant protein consisting of only the lectin domain (brevican rCLD) was examined. Like the PLD chimera, brevican rCLD specifically bound to sulfatides (Fig. 3 E,lane 1), but not to neutral glycolipids, including galactosylceramide, or to gangliosides (Fig. 3 E,lane 2). Moreover, rCLDs of aggrecan, neurocan, and versican also showed specific binding to sulfatide (Fig. 3,B–D). All of these interactions were completely suppressed in the presence of EDTA (data not shown). These results show that the C-type lectin domain of all four lecticans bind sulfatides in a divalent cation-dependent manner. Considering that L- and P-selectins bind another class of sulfated glycolipid, HNK-1-reactive SGGLs (23Needham L.K. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1339-1363Crossref Scopus (181) Google Scholar), we tested the binding of PLDs to the HNK-1-reactive SGGLs. A sample of dog sciatic nerve-derived SGGLs consisting of roughly equal amounts of SO3-GlcU-nLc-4-Cer and SO3-GlcU-nLc-6-Cer was tested for binding to brevican PLD chimera (Fig.4). The brevican PLD chimera bound to both SO3-GlcU-nLc-4-Cer and SO3-GlcU-nLc-6-Cer (lane 3). The binding was EDTA-sensitive (lane 4). Consistent with previous reports,L-selectin Ig chimera bound SGGLs (lane 5), but human IgG did not (lane 6). These results demonstrate that PLD binds HNK-1-reactive SGGLs. HNK-1-reactive carbohydrates are present on several glycoproteins as well as on glycolipids (31Jungalwala F.B. Neurochem. Res. 1994; 19: 945-957Crossref PubMed Scopus (111) Google Scholar, 32Schachner M. Martini R. Trends Neurosci. 1995; 18: 183-191Abstract Full Text PDF PubMed Scopus (181) Google Scholar). Thus, there remained a possibility that glycoproteins carrying HNK-1-reactive glycans could bind PLDs and act as PLD receptors. To address this question, we performed two experiments. First, we prepared extracts containing large amounts of HNK-1-positive glycoproteins from E19 mouse brain. These brain extracts indeed contained several protein bands intensely reactive with the HNK-1 antibody in immunoblotting (Fig.5 A, lane 1). We probed this sample with brevican PLD chimera in ligand overlay assay. Brevican PLD chimera, while it efficiently bound to tenascin-R in adult brain extracts (see Fig. 1 C,lane 4), did not bind any of these HNK-1-positive glycoproteins (Fig. 5 A, lane 2). Second, we examined by flow cytometry the binding of brevican PLD chimera to a HeLa-derived cell line that expresses HNK-1 carbohydrates only on glycoproteins, not on glycolipids. This cell line has been transfected with cDNAs for N-CAM, a glucuronyltransferase (33Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar), and the HNK-1 sulfotransferase (34Ong E. Yeh J.C. Ding Y. Hindsgaul O. Fukuda M. J. Biol. Chem. 1998; 273: 5190-5195Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The glucuronyltransferase acts only on glycoprotein glycans and not on glycolipids (33Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar). Because the parental HeLa cells express no HNK-1-reactive SGGLs, all of the HNK-1 carbohydrates expressed by these cells are contained on glycoproteins, mainly on N-CAM. 2M. Fukuda, personal communication. Flow cytometric analysis confirmed that these cells express high levels of cell surface HNK-1 carbohydrates (Fig. 5 B, a). Despite this, these cells do not bind brevican PLD chimera at all (Fig.5 B, b). In contrast, B28 cells, which express HNK-1-reactive SGGL (see below), show strong binding of brevican PLD chimera (see Fig. 1). Although these results do not entirely rule out the possibility that HNK-1 carbohydrates carried by glycoproteins could be recognized by PLD under some conditions, they demonstrate that HNK-1 carbohydrates attached to glycoproteins are not recognized by PLDs as efficiently as HNK-1-reactive SGGLs. We next examined whether intact, full-length brevican, not just the recombinant PLD fragment, binds these sulfated glycolipids. To test this, we isolated 35S-labeled brevican from culture supernatants of CHO cells transfected with full-length rat brevican cDNA, and used it as a probe in the TLC-ligand overlay assay. As shown in Fig.6, native brevican bound to both sulfatides and SGGLs (lane 1). No binding to neutral glycosphingolipids or gangliosides was found (lane 2). The glycolipid binding by brevican was inhibited with EDTA (lane 3), as was the case with the PLD chimera and the rCLDs. These results demonstrate that not only recombinant PLD fragment but also intact brevican binds sulfated glycolipids. To determine if the cell surface binding of brevican observed with the B28 cells (see Fig.1 A) (21Yamada H. Fredette B. Shitara K. Hagihara K. Miura R. Ranscht B. Stallcup W.B. Yamaguchi Y. J. Neurosci. 1997; 17: 7784-7795Crossref PubMed Google Scholar) is indeed mediated by sulfated glycolipids, we examined whether these cells contain sulfatides and/or SGGLs. Analysis of an acidic glycolipid fraction" @default.
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