NMR solution studies of hamster galectin-3 and electron microscopic visualization of surface-adsorbed complexes: evidence for interactions between the N- and C-terminal domains.

Galectin-3, a beta-galactoside binding protein, contains a C-terminal carbohydrate recognition domain (CRD) and an N-terminal domain that includes several repeats of a proline-tyrosine-glycine-rich motif. Earlier work based on a crystal structure of human galectin-3 CRD, and modeling and mutagenesis studies of the closely homologous hamster galectin-3, suggested that N-terminal tail residues immediately preceding the CRD might interfere with the canonical subunit interaction site of dimeric galectin-1 and -2, explaining the monomeric status of galectin-3 in solution. Here we describe high-resolution NMR studies of hamster galectin-3 (residues 1--245) and several of its fragments. The results indicate that the recombinant N-terminal fragment Delta 126--245 (residues 1--125) is an unfolded, extended structure. However, in the intact galectin-3 and fragment Delta 1--93 (residues 94--245), N-terminal domain residues lying between positions 94 and 113 have significantly reduced mobility values compared with those expected for bulk N-terminal tail residues, consistent with an interaction of this segment with the CRD domain. In contrast to the monomeric status of galectin-3 (and fragment Delta 1--93) in solution, electron microscopy of negatively stained and rotary shadowed samples of hamster galectin-3 as well as the CRD fragment Delta 1--103 (residues 104--245) show the presence of a significant proportion (up to 30%) of oligomers. Similar imaging of the N-terminal tail fragment Delta 126--245 reveals the presence of fibrils formed by intermolecular interactions between extended polypeptide subunits. Oligomerization of substratum-adsorbed galectin-3, through N- and C-terminal domain interactions, could be relevant to the positive cooperativity observed in binding of the lectin to immobilized multiglycosylated proteins such as laminin.

Molecular modeling and mutagenesis studies of the N-terminal domains of galectin-3: evidence for participation with the C-terminal carbohydrate recognition domain in oligosaccharide binding.

A model structure (Henrick,K., Bawumia,S., Barboni,E.A.M., Mehul,B. and Hughes, R.C. (1998) Glycobiology:, 8, 45-57) of the carbohydrate recognition domain (CRD, amino acid residues 114-245) of hamster galectin-3 has been extended to include N-terminal domain amino acid residues 91-113 containing one of the nine proline-rich motifs present in full-length hamster galectin-3. The modeling predicts two configurations of the N-terminal tail: in one the tail turns toward the first (SI) and last (S12) beta-strands of the CRD and lies at the apolar dimer interface observed for galectins -1 and -2. In the second folding arrangement the N-terminal tail lies across the carbohydrate-binding pocket of the CRD where it could participate in sugar-binding: in particular tyrosine 102 and adjacent residues may interact with the partly solvent exposed nonreducing N-acetylgalactosamine and fucose substituents of the A-blood group structure GalNAcalpha1,3 [Fucalpha1,2]Galbeta1,4GlcNAc-R. Binding studies using surface plasmon resonance of a recombinant fragment Delta1-93 protein containing residues 94-245 of hamster galectin-3 and a collagenase-derived fragment Delta1-103 containing residues 104-245, as well as alanine mutagenesis of residues 101-105 in Delta1-93 protein, support the prediction that Tyr102 and adjacent residues make significant contributions to oligosaccharide binding.

Kinetic measurements of binding of galectin 3 to a laminin substratum.

Galectin 3, a beta-galactoside binding protein, contains a C-terminal carbohydrate recognition domain (CRD) and an N-terminal segment including multiple repeats of a proline/tyrosine/glycine-rich motif. Previous work has shown that galectin 3 but not the isolated CRD binds to laminin, a multivalent ligand, with positive cooperativety indicating the formation of multiple interactions although the lectin in solution is monomeric. Using surface plasmon resonance, we find that hamster galectin 3 at sub-micromolar concentrations or its isolated CRD at all concentrations binds to a laminin substratum with similar association (k(ass); 10-30,000 M(-1) S(-1)) and dissociation (k(diss); 0.2-0.3 S1(-1)) rates and weak affinity (Ka; 1-3 x 10(5) M(-1)). At higher concentrations of galectin 3 the off rate decreases ten fold leading to increased affinity. Ligation of an N-terminal epitope of galectin 3 with a monoclonal Fab fragment increases association and dissociation rates ten fold. A recombinant protein obtained by deletion of the first 93 N-terminal residues binds to laminin with positive cooperativity and a slowly dissociating fraction (K(diss); 0.002 S(-1)) accumulates on the substratum. The data suggest that homophilic interactions between CRD as well as N terminal domains are implicated in galectin 3 aggregation on the substratum leading to positive binding cooperativity.

Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3.

A model of the carbohydrate recognition domain CRD, residues 111-245, of hamster galectin-3 has been made using homology modeling and dynamics minimization methods. The model is based on the known x-ray structures of bovine galectin-1 and human galectin-2. The oligosaccharides NeuNAc-alpha2,3-Gal-beta1,4-Glc and GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4-Glc, known to be specific high-affinity ligands for galectin-3, as well as lactose recognized by all galectins were docked in the galectin-3 CRD model structure and a minimized binding conformation found in each case. These studies indicate a putative extended carbohydrate-binding subsite in the hamster galectin-3 involving Arg139, Glu230, and Ser232 for NeuNAc-alpha2,3-; Arg139 and Glu160 for fucose-alpha1,2-; and Arg139 and Ile141 for GalNAc-alpha1,3- substituents on the primary galactose. Each of these positions is variable within the whole galectin family. Two of these residues, Arg139 and Ser232, were selected for mutagenesis to probe their importance in this newly identified putative subsite. Residue 139 adopts main-chain dihedral angles characteristic of an isolated bridge structural feature, while residue 232 is the C-terminal residue of beta-strand-11, and is followed immediately by an inverse gamma-turn. A systematic series of mutant proteins have been prepared to represent the residue variation present in the aligned sequences of galectins-1, -2, and -3. Minimized docked models were generated for each mutant in complex with NeuNAc-alpha2,3-Gal-beta1,4-Glc, GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4- Glc, and Gal-beta1,4-Glc. Correlation of the computed protein-carbohydrate interaction energies for each lectin-oligosaccharide pair with the experimentally determined binding affinities for fetuin and asialofetuin or the relative potencies of lactose and sialyllactose in inhibiting binding to asiolofetuin is consistent with the postulated key importance of Arg139 in recognition of the extended sialylated ligand.

Cross-linking of galectin 3, a galactose-binding protein of mammalian cells, by tissue-type transglutaminase.

The 30 kDa beta-galactoside-binding protein of baby hamster kidney (BHK) cells [Mehul et al. (1994), J. Biol. Chem. 269, 18250-18258] homologous to galectin 3, a widely distributed mammalian lectin, has been found to be a substrate for tissue type transglutaminase, as shown by the incorporation in a calcium- and time-dependent manner of 5-(biotinamido) pentylamine in the presence of guinea pig liver transglutaminase. The amino-terminal domain of hamster galectin 3, which is a repetitive sequence rich in glutamine, tyrosine, glycine and proline, is also an excellent substrate. A single lysine residue in the N-terminal domain is an essential requirement for transglutaminase-mediated oligomerization, and two equivalent glutamine residues present in identical sequence repeats within this domain appear to be involved as amine acceptors in cross-linking reactions. Transglutaminase-mediated cross-linking of galectin 3 to itself or to matrix components may be one mechanism for stabilisation of a multivalent binding form of the lectin in cell secretions or in extracellular matrices.

Structure of baby hamster kidney carbohydrate-binding protein CBP30, an S-type animal lectin.

A galactose-binding protein of M(r) = 30,000 previously described in baby hamster kidney cells (Foddy, L., Stamatoglou, S. C., and Hughes, R. C. (1990) J. Cell Sci. 97, 139-148) has been analyzed by the cloning and sequencing of cDNA clones encoding the complete sequence and an amino-terminal fragment. The intact lectin CBP30 contains 245 amino acid residues, including the initiating methionine residue, and is closely homologous to mammalian S-type lectins of similar size characterized in human, rat, and mouse species. The carboxyl-terminal domain contains the carbohydrate binding activity and the amino-terminal domain, which is extremely sensitive to bacterial collagenase, contains a repetitive sequence rich in glycine, tyrosine, and proline. There are 8 repeats in hamster CBP30, as in the human homologue, compared with about 10 in rat and mouse and > 10 in dog homologues. This repeat sequence is also sensitive to the tissue metalloproteinases, gelatinase B and matrilysin, but, unlike the bacterial collagenase, the mammalian enzymes also cause extensive degradation of the carbohydrate binding carboxyl domain. Physical measurements using CD and tryptophan fluorescence spectroscopy indicate that the two domains of CBP30 are structurally, as well as functionally, distinct and independent. Cross-linking studies indicate that the amino-terminal lectin fragment can efficiently self-assemble into oligomeric species, and less efficient but significant aggregation of the intact lectin is also shown. Domain-specific antibodies to hamster CBP30 have been prepared and used to show that only the full-length, undegraded form of CBP30 is present in whole cell lysates.

Affinity of integrin alpha 1 beta 1 from liver sinusoidal membranes for type IV collagen.

Hepatic sinusoidal membranes isolated from adult rats were extracted with detergent and fractionated on a wheat germ agglutinin affinity column. Bound glycoproteins were eluted with N-acetyl glucosamine and chromatographed on a type IV collagen affinity column. Recovery of the bound fraction by EDTA and analysis by SDS-PAGE revealed two glycoproteins with apparent molecular weights of 180,000 and 117,000. These were identified immunologically by Western blotting as the alpha and beta subunits of integrin alpha 1 beta 1.