Key words: galectin-I/human brain/lectin/size exclusion chro- matography ...
single CRD is galectin-1 (GAL1), which is thought to have growth regulatory and
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dothelial cell receptor and DEC-205. Further conclusions about evolution of binding specificities of the CRDs of these receptors must await ligand-binding studies on the endothelial cell receptor and DEC-205. Nomenclature Currently, "mannose receptor family" seems to be the default name for the group of receptors containing multiple C-type CRDs (Wu et al, 1996). While there might be a temptation to consider names like "polylectins," such a designation is probably inappropriate since some, perhaps most, members of the family probably do not bind carbohydrates. A better name for the group must await more information about the functions of each receptor. References Drickamer.K. (1993) Ca2+-dependent carbohydrate-recognition domains in animal proteins. Curr. Opiru Struct. BioL, 3, 393-400. Harrisjvl., Super,M., Rits.M., Chang.G. and Ezekowitz,R.A.B. (1992) Characterization of the murine macrophage mannose receptor demonstration that downregulation of receptor expression mediated by interferon--y occurs at the level of transcription. Blood, 80, 2363-2373. Higashino.K, IshizakiJ., KishinoJ., Ohara,O. and Arita,H. (1994) Structural comparison of phospholipase-A2-binding regions in phospholipase A2 receptors from various mammals. Eur. J. Biochem., 225, 375-382. Higgins.D.G. and Sharp,P.M. (1988) CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene, 73, 237—244. IshizakiJ., HanasakiJC., HigashinojC, KishinoJ., Kikuchi,N., Ohara.O. and Aritaji. (1994) Molecular cloning of pancreatic group I phospholipase A2 receptor. J. BioL Chem., 269, 5897-5904. Jiang.W., SwiggarcLWJ., Heufler.C, Peng.M., MirzaA, SteinmarwR.M. and NussenzweigJvl.C. (1995) The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. Nature, 375, 151-155. Kornblihtt^A.R., Umezawa,K., Vibe-Pedersen.K. and BaralleJ.E. (1985) Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J., 4, 1755-1759. Lambeau.G., AncianJ?., BarhaninJ. and Lazdunski,M. (1994) Cloning and expression of a membrane receptor for secretory phospholipases A2. J. BioL Chem., 269, 1575-1578. Lambeau.G., AncianJ5., Mattei,M.-G. and Lazdunslri,M. (1995) The human 180-kDa receptor for secretory phospholipases A 2 . J. BioL Chem., 270, 8963-8970. Mullin,N.P., Hall^C.T. and Taylor.M.E. (1994) Characterization of ligand binding to a carbohydrate-recognition domain of the macrophage mannose receptor. J. BioL Chem., 269, 28405-28413. Nicolas J.-P-, Lambeau.G. and Lazdunski,M. (1995) Identification of the binding domain for secretory phospholipases A 2 on their M-type 180-kDa membrane receptor. J. BioL Chem., 270, 28869-28873. TaylOT,M.E., ConaryJ.T., Lennarz>i.R., Stahl,P.D. and DrickamerJC. (1990) Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains. J. BioL Chem., 265,12156-12162. Taylor.M.E. and Drickamer.K. (1993) Structural requirements for high affinity binding of complex ligands by the macrophage mannose receptor. /. BioL Chem., 268, 399-404 WuK., YuanJ. and LaskyJ^A. (1996) Characterization of a novel member of the macrophage mannose receptor type C lectin family. J. BioL Chem, 271, 21323-21330.
Is human galectin-1 activity modulated by monomer/dimer equilibrium? Virginie Giudicelli, Didier Lutomski, Matthieu Levi-Strauss1, Dominique Bladier, Raymonde Joubert-Caron and Michel Caron2 Laboratoire de Biochimie et Technologic des Prot6ines, UFR Leonard de Vinci, Universit6 Paris Nord, 74 Rue Marcel Cachin, F-93017 Bobigny Cedex, France and 'INSERM U.I 14, College de France, 11 Place Marcelin-Berthelot, 75005 Paris, France ^ o whom correspondence should be addressed
Key words: galectin-I/human brain/lectin/size exclusion chromatography
The galectins are a family of fi-galactoside-binding mammalian lectins characterized by a highly conserved carbohydraterecognition domain (CRD) showing a characteristic set of highly conserved amino acid residues (Caron et al., 1990; Barondes et al, 1994; Gabius, 1994; Kasai and Hirabayashi, 1996). Both intra- (Avellana-Adalid et al., 1994; Hubert et al., 1995; Wang et al, 1995) and extracellular functions (Li et al, 1992; Fowlis et al, 1995; Ozeki et al, 1995) have been proposed for these lectins. The most common galectin having a single CRD is galectin-1 (GAL1), which is thought to have growth regulatory and immunomodulatory activities (Wells and Malluci, 1991; Lutomski et al., 1995). The principal physiological roles of this protein in human remain unknown. It has been suggested that they differ according to whether GAL1 is predominantly monomeric or dimeric (PeriTlo et al, 1995). The direct demonstration of an equilibrium between monomeric and dimeric forms of GAL1 is restricted to a report showing that recombinant GAL1 from CHO cells occurs in an active monomeric form that can reversibly dimerize (Cho and Cumming, 1995). This work shows clearly that dimerization is dependent on the concentration of galectin. This demonstration is consistent with the observation that, for many oligomers in which there are relatively weak attractive interactions between the subunits, the dissociation into subunits can be accomplished by simple dilution. However, not all subunits in oligomers can be separated in this way. For oligomeric proteins containing identical polypeptide chains, such as GAL1, in most cases the formation of monomers from the oligomer requires dissociating agents of sufficient strength that the tertiary structure of the monomers is disrupted along with the destruction of the quaternary structure. Only rarely are the interchain interactions sufficiently weak and different in kind compared with the intrachain interactions, that the dissociation of oligomers into folded monomers can be achieved (Eisenstein and Schachman, 1989). Therefore, whether or not a monomer-dimer equilibrium occurs for GAL1 in all mammalian species, and serves a functional roles, remains unknown. High resolution size-exclusion chromatography provides an excellent means for separating monomers from dimers according to the difference in their sizes. We used a dextran-based composite gel (Sephacryl) for determining the quaternary structure of recombinant human GAL1 (rGALl) and of purified tissular (human brain) GAL1 (Avellana-Adalid et al., 1990). This matrix is convenient for size exclusion HPLC of soluble proteins (LeMaire et al, 1980); and in experiments with oligomeric plant lectins it has
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amidomethylated rGALl) or on their free amino groups (biotiny lated rGALl), than with unmodified rGALl. Identical elution profiles were also obtained with native GAL1 isolated from the human brain. The position of the peak detected by UV adsorption was confirmed using biotinylated rGALl by determining the amount of biotinylated protein in each fraction. The chromatography profiles obtained by this method were similar to those obtained by UV adsorption and showed no peak which could be interpreted as monomeric GALL Finally, we tested the possibility that the salt concentration might affect the quaternary structure of the lectin though ionic bonds. The behavior of rGALl was not modified by increasing the ionic strength (0.5-1 M NaCl). In summary, it is clear that the concept of an equilibrium
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been reported that results obtained using dextran-based media were more reliable than those obtained using other HPLC media (Young and Jackson, 1984). Typical elution profile obtained for rGALl and the calibration curve for molecular weight determinations are shown in Figure 1. Size exclusion chromatography of human rGALl yielded a fairly symmetrical peak with a calculated molecular weight of about 26,000, consistent with those previously reported for dimeric human placenta GAL1 (Hirabayashi et at, 1987; Niambar et al., 1987). To investigate the possibility of dimer dissociation of human GAL1, we prepared rGALl in buffer at various concentrations. The diluted samples were kept at 4°C for 24 h to promote equilibrium, and the HPLC profile of each sample was examined. The galectin had similar chromatography profile independently of its concentration. Even at low concentration (2mM) of rGALl a dimeric structure was observed (Figure 2). Similar results were obtained with GAL1 derivatives modified either on their free thiol groups (carbox-
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