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The C-Type Lectin Domains of Lecticans, a Family Of Proc. Natl. Acad. Sci. USA Vol. 94, pp. 10116–10121, September 1997 Biochemistry The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein– protein interactions independent of carbohydrate moiety ANDERS ASPBERG*†‡,RYU MIURA*‡,SANDRINE BOURDOULOUS*, MOTOYUKI SHIMONAKA*, DICK HEINEGÅRD†, MELITTA SCHACHNER§,ERKKI RUOSLAHTI*¶, AND YU YAMAGUCHI*¶ *The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037; †Department of Cell and Molecular Biology, Lund University, P.O. Box 94, S-221 00 Lund, Sweden; and §Zentrum fu¨r Molekulare Neurobiologie, University of Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany Communicated by George E. Palade, University of California School of Medicine, La Jolla, CA, July 17, 1997 (received for review May 16, 1997) ABSTRACT The lecticans are a family of chondroitin pected of a carbohydrate–protein interaction mediated by a sulfate proteoglycans including aggrecan, versican, neurocan, C-type lectin domain (11). This observation suggested that and brevican. The C-terminal globular domains of lecticans other members of the lectican family, notably brevican and are structurally related to selectins, consisting of a C-type neurocan, which are specifically expressed in the nervous lectin domain flanked by epidermal growth factor and com- system (4, 13), may also interact with tenascin-R. In this paper, plement regulatory protein domains. The C-type lectin do- we report that all lecticans do bind tenascin-R, but surprisingly main of versican has been shown to bind tenascin-R, an the interaction is mediated by the fibronectin type III (FNIII) extracellular matrix protein specifically expressed in the domains of the protein backbone of tenascin-R independent of nervous system, and the interaction was presumed to be any carbohydrates. Results of surface plasmon resonance mediated by a carbohydrate–protein interaction. In this pa- analysis and coprecipitation experiments suggest that brevican per, we show that the C-type lectin domain of brevican, is a physiological tenascin-R ligand in the adult brain. another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is EXPERIMENTAL PROCEDURES mediated by a protein–protein interaction through the fi- bronectin type III domains 3–5 of tenascin-R, independent of Recombinant Lectin Domains. The NotI–SalI fragment of any carbohydrates or sulfated amino acids. The lectin domains the versican lectin domain construct (11) and a synthetic of versican and other lecticans also bind the same domain of fragment encoding a hexahistidine (his)-tag flanked by SalI tenascin-R by protein–protein interactions. Surface plasmon and XbaI sites were ligated into pcDNA3. For the construction resonance analysis revealed that brevican lectin has at least a of his-tagged expression vector for other lecticans, the frag- 10-fold higher affinity than the other lectican lectins. Tena- ment encoding the lectin domain of versican was excised, scin-R is coprecipitated with brevican from adult rat brain leaving the his-tag in the vector. cDNAs for the lectin domains extracts, suggesting that tenascin-R and brevican form com- of other lecticans were amplified from rat cartilage (for plexes in vivo. These results demonstrate that the C-type lectin aggrecan) or rat brain (for brevican and neurocan) cDNA by domain can interact with fibronectin type III domains PCR based on published sequence (2, 4, 14) and ligated into through protein–protein interactions, and suggest that brevi- this vector. For production of his-tagged recombinant lectin can is a physiological tenascin-R ligand in the adult brain. domains of aggrecan and neurocan, human 293 cells were cotransfected with respective expression vectors and pSV2- The lecticans are a family of chondroitin sulfate proteoglycans pac, and spent media were collected from stable transfectants. (reviewed in ref. 1), encompassing aggrecan, versican, neuro- For lectin domains of versican and brevican, for which stable can, and brevican (2–5). The core proteins of these proteo- 293 cell transfectants producing high levels of recombinant glycans share structural domains consisting of a central gly- proteins were not isolated, the expression cassette for each cosaminoglycan carrying region and N-terminal and C- lectin domain was subcloned into pCEP4 (Invitrogen), and terminal globular domains. The C-terminal globular domain of transfected into 293–Epstein–Barr virus-encoded nuclear an- lecticans consists of one or two epidermal growth factor (EGF) tigen cells (Invitrogen). The recombinant lectin domains with domains, a C-type lectin domain, and a complement regulatory their C-terminal his-tag were purified from conditioned media protein domain. These are the same structural elements as on a Ni-NTA (nickel-nitrotriacetic acid) agarose (Qiagen, seen in selectins, which mediate leukocyte–endothelium ad- Chatsworth, CA) column. These recombinant proteins are hesion through various carbohydrate ligands (6, 7). The struc- referred to as brevican rCLD (recombinant C-type lectin tural similarities between selectins and the C-terminal globular domain), versican rCLD, etc. in this paper. For the surface domains of lecticans suggested that the lecticans also bind plasmon resonance analysis, recombinant rCLDs were further carbohydrate ligands. Earlier studies have shown that the purified on a Mono Q column. Biotinylation was performed as C-type lectin domain of aggrecan and versican bind simple described (11). Brevican–Ig Chimera. A cDNA fragment (0.9 kb) consisting carbohydrates as well as heparinyheparan sulfate (8–11). Previous studies (11) have demonstrated that recombinant of the EGF, lectin, and complement regulatory protein do- versican lectin domain binds tenascin-R, a member of tenascin mains (ELC) of rat brevican (corresponding to nucleotides 1791–2669; ref. 14) was amplified by PCR and fused in-frame family glycoproteins expressed specifically in the nervous to another PCR fragment (0.2 kb) of brevican containing its system (12). This interaction is calcium-dependent, as ex- signal peptide (nucleotides 1–182). The resultant 1.1-kb frag- The publication costs of this article were defrayed in part by page charge Abbreviations: FNIII, fibronectin type III; GST, glutathione S- payment. This article must therefore be hereby marked ‘‘advertisement’’ in transferase; rCLD, recombinant C-type lectin domain; EGF, epider- accordance with 18 U.S.C. §1734 solely to indicate this fact. mal growth factor. © 1997 by The National Academy of Sciences 0027-8424y97y9410116-6$2.00y0 ‡A.A. and R.M. contributed equally to this paper. PNAS is available online at http:yywww.pnas.org. ¶To whom reprint requests should be addressed. 10116 Downloaded by guest on September 30, 2021 Biochemistry: Aspberg et al. Proc. Natl. Acad. Sci. USA 94 (1997) 10117 ment was inserted into the BamHI site of pcDNAIyFc (15), containing 10% fetal calf serum. Tunicamycin and chlorate which contains human genomic DNA encoding the Fc region treatments were performed according to Yasugi et al. (17) and of IgG1. The plasmid was cotransfected with pSV2-dhfr into Pouyani et al. (18), respectively. Effectiveness of tunicamycin CHOyDG44 cells by the calcium phosphate method. The treatment was monitored by the reduction of the apparent chimera was purified from serum-free culture supernatants of molecular weight of tenascin-R in SDSyPAGE. Inhibition of these cells by affinity chromatography on protein A- sulfation was monitored by anti-HNK-1 immunoblotting of Sepharose. tenascin-R secreted by B9 cells (the HNK-1 epitope includes Ligand Blotting, Immunoblotting, and Immunoprecipita- sulfated glucuronic acid). The chlorate treatment almost com- tion Assays. Preparation of soluble and membrane fractions of pletely abolished the reactivity of anti-HNK-1 antibody with adult rat brain and immunoblotting were performed as de- tenascin-R, confirming the effectiveness of the treatment. scribed (5). Ligand overlay assays with Ig chimeras and bio- Glutathione S-Transferase (GST) Fusion Proteins. GST tinylated recombinant lectin domains, as well as precipitation fusion proteins consisting of various domains of rat tenascin-R, of tenascin-R with Ig chimera were performed according to except for FN3–4, FN4–5, FN3, FN4, and FN5 fusion proteins, Aspberg et al. (11). For immunoprecipitation with anti- have been described (19). Constructs for FN3–4, FN4–5, FN3, brevican antibodies, soluble brain extracts were incubated with FN4, and FN5 fusion proteins were similarly prepared in purified RB18 mouse monoclonal antibodies (28) and the pGEX-KG by using the following primers: FN3 forward, immune complex was precipitated with protein G-Sepharose. 59-CGAATTCCTGTCATTGACGGGC-39; FN3 backward, Precipitated materials were resolved in SDSyPAGE and an- 59-CGTGTCGACTATGTTGTAAACTGGGTGC-39; FN4 alyzed by the ligand overlay assay with brevican ELC chimera forward, 59-CGAATTCCTGAAATTGATGCTCCC-39; FN4 as described above. Enzymatic deglycosylation was performed backward, 59-CGTGTCGACTAAGTCCTGGCATTCAT-39; as described previously (11). FN5 forward, 59-CGAATTCCTGAGCTTGACAGTCCC-39; Tunicamycin and Chlorate Treatment. B9 cells (16) were FN5 backward, 59-CGTGTCGACTATGTGAAGGCATC- maintained in Dulbecco’s modified minimal essential medium CAC-39. For FN3–4 and FN4–5 fusion proteins, primer com- FIG. 1. Brevican ELC chimera binds tenascin-R. (A) Brevican ELC chimera binds to 160y180 kDa bands in ligand blotting. Soluble
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