Carbohydrate-binding specificity of calcyclin and its expression in human tissues and leukemic cells.

Binding of biotinylated fetuin in a solid-phase assay served as activity assay for purification of calcyclin, the product of a cell growth-related cDNA with homologies to Ca(2+)-binding proteins. Asialofetuin failed to bind to calcyclin, emphasizing the importance of sialic acids. Binding of fetuin was most effectively reduced by N-glycolylneuraminic acid within a panel of mostly negatively charged sugars. Bovine submaxillary mucin and the ganglioside GM1, but not asialo-GM1, proved more effective than neoglycoproteins, carrying negatively charged carbohydrate moieties. Extension of N-acetyl-neuraminic acid to its lactosyl derivative increased its inhibitory potency. Among charge-free carbohydrate residues, only N-acetylglucosamine, lactose, and mannose, but not fucose, melibiose, or N-acetylgalactosamine affected fetuin binding, substantiating the inherent selectivity. Chemical modification with group-specific reagents revealed that lysine and arginine residues appear to be involved in ligand binding that is optimal in the presence of Ca2+, but not Zn2+ and stable up to 1 m NaCl. Biotinylation of calcyclin by modification of carboxyl groups facilitated performance of solid-phase assays with calcyclin in solution, yielding similar results with (neo)glycoproteins in relation to assays with immobilized calcyclin, thereby excluding an impact of binding to nitrocellulose on calcyclin's specificity. Subcellular fractionation disclosed the presence of fetuin-binding activity in all fractions, the specific activity decreasing from the nuclear to the particulate cytoplasmic fraction and the cytoplasmic supernatant. Affinity-purified antibodies were employed to detect high levels of calcyclin expression in acute lymphoblastic, myelogenous, and monocytic leukemia cell lines, but not in myeloma or lymphoblastoid cells. In comparison, most cells were nearly devoid of an O-acetylsialic acid-specific protein that is more abundant in various tissue types than calcyclin.