Abstract
The detailed characterization of a galectin from the toad (Bufo arenarum Hensel) ovary in its primary structure, carbohydrate specificity, and overall biochemical properties has provided novel information pertaining to structural and evolutionary aspects of the galectin family. The lectin consists of identical single-chain polypeptide subunits composed of 134 amino acids (calculated mass, 14,797 daltons), and its N-terminal residue, alanine, is N-acetylated. When compared to the sequences of known galectins, the B. arenarum galectin exhibited the highest identity (48% for the whole molecule and 77% for the carbohydrate recognition domain (CRD)) with the bovine spleen galectin-1, but surprisingly less identity (38% for the whole molecule and 47% for the CRD) with a galectin from Xenopus laevis skin (Marschal, P., Herrmann, J., Leffler, H., Barondes, S. H., and Cooper, D. N. W. (1992) J. Biol. Chem. 267, 12942-12949). Unlike the X. laevis galectin, the binding activity of the B. arenarum galectin for N-acetyllactosamine, the human blood group A tetrasaccharide and Galbeta1,3GalNAc relative to lactose, was in agreement with that observed for the galectin-1 subgroup and those galectins having "conserved" (type I) CRDs (Ahmed, H., and Vasta, G. R. (1994) Glycobiology 4, 545-549). Moreover, the toad galectin shares three of the six cysteine residues that are conserved in all mammalian galectins-1, but not in the galectins from X. laevis, fish, and invertebrates described so far. Based on the homologies of the B. arenarum galectin with the bovine spleen galectin-1 and X. laevis skin galectin, it should be concluded that within the galectin family the correlation between conservation of primary structure and phylogenetic distances among the source species may not be a direct one as proposed elsewhere (Hirabayashi, J., and Kasai, K. (1993) Glycobiology 3, 297-304). Furthermore, galectins with conserved (type I) CRDs, represented by the B. arenarum ovary galectin, and those with "variable" (type II) CRDs, represented by the X. laevis 16-kDa galectin, clearly constitute distinct subgroups in the extant amphibian taxa and may have diverged early in the evolution of chordate lineages.
Highlights
Galectins (Barondes et al, 1994a), comprise S-type -galactosyl binding lectins, present in both homeotherm and poykilotherm vertebrates and invertebrates, that require a reducing environment but do not require divalent cations for their binding activity (Hirabayashi and Kasai, 1993; Barondes et al, 1994b)
Among the amino acid residues that are substantially conserved among various galectins, His44, Asn46, Arg48, Asn61, Trp68, Glu71, and Arg73 (residue numbers are those of bovine spleen (Liao et al, 1994)) are recognized as critical for sugar binding (Lobsanov et al, 1993; Bourne et al, 1994; Liao et al, 1994) whereas Ser29, Phe30, Asn33, His44, Asn46, Arg48, Asn61, and Arg111 interact with each other to provide the architecture of the CRD1 (Liao et al, 1994)
The concentration-dependent monomer-dimer equilibrium observed for the purified toad ovary galectin is noteworthy and similar to that reported for the galectin-1 from CHO cells (Cho and Cummings, 1995)
Summary
Reagents—The protein assay reagent was from Bio-Rad. Ampholine PAGplate and gel permeation chromatography molecular weight standards and ribonuclease A were purchased from Pharmacia Biotech Inc. Stability in a Nonreducing Environment—The purified toad ovary galectin (100 g) was absorbed on 1 ml each lactosyl-Sepharose and asialofetuin-Sepharose, and each matrix was thoroughly washed with aerated PBS (20 ml/1 ml matrix) and stored at room temperature. The preincubation of the galectin-HRP conjugate (24 ng in 60 l of azide-free PBS-Tween 20 buffer) for binding inhibition assays was carried out by mixing equal volumes conjugate and the test ligand at varying concentrations. Under the elution conditions used, PTCCys(CM) was eluted between PTC-Asp and PTC-Glu. Acyl N 3 O Shift on Blocked N-terminal Fragments, Ac-(1–13) and Ac-(1–21)—The purified peptides (150 –200 pmol) were dried in polypropylene test tubes, dissolved in dry trifluoroacetic acid containing 0.001% dithiothreitol (50 l, sequencer reagent R3), and sealed under argon. These conditions provided the best signal to noise ratio for the ions of interest
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
