Isolation and characterization of galactose-specific carbohydrate-binding protein from Guerin tumor cells.
Carbohydrate-binding protein with specificity towards galactose was isolated from Guerin tumor cells. This protein had molecular weight of 51 kDa in dissociating and reducing conditions. It was phosphorylated, but not glycosylated, having two isoforms with pIs corresponding to 7.3 and 7.9. We found predominantly cytoplasmic and nuclear, but not plasma membrane, localization of the isolated protein. Oxidative conditions and presence of the ligand are required for the protein to oligomerize. Probing of the carbohydrate-binding domain with sugar derivatives showed that hydroxyl groups at C3, C4 and C6 positions of galactose, as well as at C3 and C6 positions of the glucose part of NAcLactosamine are involved in ligand binding. Tyrosine, tryptophan and histidine amino acids were found to participate in binding of the galactose ligand. N-linked multivalent macromolecular ligands, containing up to four antennae, bound to the isolated protein with positive cooperativity. Affinity for NAcLactosamine, as measured by its I(50) value, was 7918-times higher than that for galactose. Binding of galactose to the combining site was enthalpically driven, dH=-32.16 (kJ mol(-1)), with K(d) in the micromolar range, 32.25 x 10(4) mol(-1).
Stoyloff
J
J
Institute of Experimental Pathology and Parasitology, Bulgarian Acad. of Sciences, Sofia, Bulgaria. jstoyloff@gmail.com
Ivanov
S
S
D000818:Animals; D002135:Calcium-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D045744:Cell Line, Tumor / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004195:Disease Models, Animal; D005690:Galactose / Q000302:isolation & purification*; D015972:Gene Expression Regulation, Neoplastic; D006735:Horseradish Peroxidase / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D033902:Periplasmic Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D011134:Polysaccharides; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar
D002135:Calcium-Binding Proteins; D037102:Lectins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011134:Polysaccharides; C023550:galactose-binding protein; C037199:poly-N-acetyllactosamine; D007770:L-Lactate Dehydrogenase; D006735:Horseradish Peroxidase; D005690:Galactose