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The cloning of full-length cDNA for the first avian tandem-repeat-type galectin which is termed chicken galectin (CG-8) based on its sequence similarity to galectin-8 from mammals is reported. Two proteins with linkers of either 9 (CG-8I) or 28 amino acids (CG-8II) generated by alternative splicing formed monomers with a shape comparable to that of the proto-type proteins CG-1A/B in solution, act as cross-linkers in hemagglutination, and bind cells with a strict dependence on galactose.

Negative cooperativity in the isothermal titration microcalorimetry (ITC) binding data of asialofeutin (ASF), a glycoprotein that possesses nine LacNAc epitopes, to galectin-1, -2, -3, -4, -5, and -7 and truncated, monomer version of galectin-3 and -5 which are members of a family of animal lectins is demonstrated. The results indicate that the galactins bind with fractional, high affinities to multivalent glycoproteins such as ASF, independent of the quaternary structures of the galectins.

Characterization of all members of a gene family established by gene divergence is essential to delineate distinct or overlapping expression profiles and functionalities. Their activity as potent modulators of diverse physiological processes directs interest to galectins (endogenous lectins with β-sandwich fold binding β-galactosides and peptide motifs), warranting their study with the long-term aim of a comprehensive analysis. The comparatively low level of complexity of the galectin network in chicken with five members explains the choice of this organism as model. Previously, the three proto-type chicken galectins CG-1A, CG-1B, and CG-2 as well as the tandem-repeat-type CG-8 had been analyzed. Our study fills the remaining gap to determine gene structure, protein characteristics and expression profile of the fifth protein, that is, chimera-type chicken galectin-3 (CG-3). Its gene has a unique potential to generate variants: mRNA production stems from two promoters, alternative splicing of the form from the second transcription start point (tsp) can generate three mRNAs. The protein with functional phosphorylation sites in the N-terminus generated by transcription from the first tsp (tsp1CG-3) is the predominant CG-3 type present in adult tissues. Binding assays with neoglycoproteins and cultured cells disclose marked similarity to properties of human galectin-3. The expression and localization profiles as well as proximal promoter regions have characteristic features distinct from the other four CGs. This information on CG-3 completes the description of the panel of CGs, hereby setting the stage for detailed comparative analysis of the entire CG family, e.g., in embryogenesis., (Copyright © 2011 Wiley-Liss, Inc.)

Prototype galectins are versatile modulators of cell adhesion and growth via their reactivity to certain carbohydrate and protein ligands. These functions and the galectins' marked developmental regulation explain their attractiveness as models to dissect divergent evolution after gene duplication. Only two members have so far been assumed to constitute this group in chicken, namely the embryonic muscle/liver form {C-16 or CLL-I [16 kDa; chicken lactose lectin, later named CG-16 (chicken galectin-16)]} and the embryonic skin/intestine form (CLL-II or C-14; later named CG-14). In the present study, we report on the cloning and expression of a third prototype CG. It has deceptively similar electrophoretic mobility compared with recombinant C-14, the protein first isolated from embryonic skin, and turned out to be identical with the intestinal protein. Hydrodynamic properties unusual for a homodimeric galectin and characteristic traits in the proximal promoter region set it apart from the two already known CGs. Their structural vicinity to galectin-1 prompts their classification as CG-1A (CG-16)/CG-1B (CG-14), whereas sequence similarity to mammalian galectin-2 gives reason to refer to the intestinal protein as CG-2. The expression profiling by immunohistochemistry with specific antibodies discerned non-overlapping expression patterns for the three CGs in several organs of adult animals. Overall, the results reveal a network of three prototype galectins in chicken.

A library of pentapeptides containing the sequence -Y-X-Y- based on rational design was screened with six different lectins. Sequences were identified that modulate galectin binding to its natural carbohydrate ligand. SPR showed inhibition values 2-3 times stronger than galactose and NMR studies suggested real carbohydrate mimicry.

New and rigid multivalent lactose molecules were prepared. The structures contain lactose-2-aminothiazoline units at the periphery that were formed from a cyclisation of the thiourea sulphur onto the triple bond of the spacer. The lactosides were evaluated as inhibitors against lectin binding in a solid phase inhibition assay. In this assay the glycoprotein asialofetuin was immobilized onto the surface of microtiter plate wells, mimicking cell surface presentation, while mammalian galectins-1, -3 or -5 were in solution. Between the three galectins, the folding pattern and sequence are closely related but the topology of presentation of the carbohydrate recognition domains differs. Strong multivalency effects were observed for the tetravalent lactoside in the inhibition of galectin-3 binding with enhancements of almost 4300-fold compared to lactose. Remarkable selectivity was obtained in the inhibition since relative potencies of the tetravalent lactoside with the proto type galectins-1 and -5 did not exceed a factor of 143 relative to lactose. The binding of the lactosides to galectin-3 was also studied by fluorescence spectroscopy with all components in solution. These studies showed no multivalency effects in the inherent binding affinities.