Galectin-1 from Bovine Spleen: Biochemical Characterization, Carbohydrate Specificity and Tissue-Specific Isoform Profiles

Abstract

Selected biochemical properties, including the charge heterodispersity profile and carbohydrate specificity, of bovine galectin-1 were determined in detail. The lectin was purified through an improved purification protocol that yielded 35-40 mg/kg of wet tissue with a specific activity of 1.7-2 x 10(4) mg-1.ml. The galectin is a homodimer of approximately 14.5 kDa subunits with E(280)mg/ml of 0.65 ml.mg-1.cm-1. When stored in the presence of its carbohydrate ligand, the lectin's binding activity remained stable in a non-reducing environment even at room temperature. The optimal pH for binding to the ligand was 6.5-8.0. The overall carbohydrate specificity of the bovine galectin-1 isolated from spleen is similar to that of the galectin isolated from heart and to other mammalian galectins that exhibit "conserved" (Type I) carbohydrate recognition domains (CRDs) [Ahmed, H. and Vasta, G.R. (1994) Glycobiology 4, 545-549], but differs from those from Xenopus laevis and rat intestine domain I. The fluorescence of 4-methylumbelliferyl alpha-D-galactopyranoside was quenched on binding to bovine spleen galectin-1. Scatchard plots of data obtained at 5, 15, and 30 degrees C showed that the galectin has two sugar exothermic binding sites with association constants of 3.4 x 10(5), 1.0 x 10(5), and 0.3 x 10(5), respectively. Chemical modification studies indicated that histidine, tryptophan, carboxylic acid, and arginine, but not lysine or tyrosine, are involved in the binding to the carbohydrate ligand. On isoelectric focusing, the spleen galectin-1 appeared as six isoforms ranging from pI4.56-4.88 with main components at pI 4.63 (34.0%), 4.73 (42.6%), and 4.88 (16.6%). The galectin-1 isolated from heart yielded a quali- and quantitatively different profile with four isoforms ranging from pI 4.53-4.73, those with pIs of 4.56, 4.63, and 4.73 being common to the spleen homolog. Edman degradation of selected peptides purified from the spleen galectin-1 digest revealed amino acid sequences identical to those obtained for the heart galectin-1. This suggests that although point mutations in the subunit primary structure may not be the likely source of isolectins, as observed for X. laevis, tissue-specific co- or post-translational modifications may be the possible cause of the differences in the galectin isoform profile between bovine spleen and heart.

The Primary Structure and Carbohydrate Specificity of a β-Galactosyl-binding Lectin from Toad (Bufo arenarum Hensel) Ovary Reveal Closer Similarities to the Mammalian Galectin-1 than to the Galectin from the Clawed Frog Xenopus laevis

“…Like other galectins examined with regard to their thermal stability, the toad ovary galectin was irreversibly inactivated by exposure to high temperatures during a relatively short period of time. Therefore, in this and all the aforementioned biochemical aspects examined, the toad ovary lectin closely resembles those galectins isolated from mammalian sources, such as that from bovine spleen (Ahmed et al, 1996), possibly reflecting a conservation of biological roles of galectins from such phylogenetically distant taxa.…”

“…Galectin Purification and Homogeneity-The improved protocol optimized for a mammalian galectin-1 (Ahmed et al, 1996) was equally satisfactory for purification of the toad ovary galectin. The use of DEAE-Sepharose prior to affinity chromatography allowed the partial isolation of the galectin and the removal of lactose present in the extraction buffer.…”

“…Purification of Toad Ovary Galectin-The galectin was purified through an improved protocol as reported elsewhere (Ahmed et al, 1996). Briefly, toad ovaries (500 g) were homogenized with cold (4°C) phosphate-buffered saline (diluted 1:10), 0.01 M 2-mercaptoethanol, 0.1 M lactose (PBS(1:10)/ME/lactose) containing 0.1 mM phenylmethylsulfonyl fluoride (2 ml buffer/g wet tissue).…”

“…Comparison to Other Galectins-The amino acid sequence of the toad ovary 14.8-kDa lactose-binding lectin was compared to those of galectins from bovine (Abbott et al, 1989;Ahmed et al, 1996) human (Hirabayashi et al, 1989), rat (Clerch et al, 1988), mouse (Wells and Malluci, 1991), chicken (Hirabayashi et al, 1987;Sakakura et al, 1990), frogs (Ozeki et al, 1991;Marschal et al, 1992), teleost fishes (Paroutaud et al, 1987;Muramoto and Kamiya, 1992), nematode (Hirabayashi et al, 1992, and a sponge (Pfeifer et al, 1993) (Fig. 7).…”

Isolation and Characterization of a Novel Eosinophil-specific Galectin Released into the Lungs in Response to Allergen Challenge

“…It contains six of the seven residues reported to be important for sugar recognition and binding by galectin-1 (14,15), and the amino acid substitution that does occur is a conservative change. Additionally, the arginine residues known to be important for sugar binding, hemagglutination, and eosinophil chemotactic activity of ecalectin/galectin-9 are also conserved in galectin-14 (Arg 65 and Arg 239 of ecalectin (23)).…”