Abstract
A Ca2+-independent sialic acid-specific lectin from two developmental stages of human placenta was similarly purified to apparent homogeneity by DEAE-cellulose chromatography, affinity chromatography on bovine submaxillary mucin, and gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration disclosed a molecular mass of 53 kDa. The specificity of the lectin for O-acetylsialic acids was substantiated by the dependence of hemagglutination on the presence of acetylated sialic acids on the surface of mammalian erythrocytes of various sources, by hapten inhibition in hemagglutination assays with protease-treated rabbit erythrocytes and by hapten inhibition of binding of labeled N-acetylneuraminic acid-bovine serum albumin to the lectin in a solid-phase assay. Bovine and equine submaxillary mucins that contain 9(7,8)-O-acetyl and 4-O-acetylsialic acids were potent inhibitors in contrast to the non-acetylated sialic acids of ovine submaxillary mucin. Absence of inhibitory efficiency of other negatively charged substances like phosphorylated sugars, glucuronic acid, heparin, or oligodeoxynucleotides emphasized the importance of structural features instead of simple ionic interaction. In the presence of acetylation, the pattern of inhibition by gangliosides in the solid-phase assay indicated a preference to alpha-2,8- or alpha-2,6-linked sialic acids in comparison to alpha-2,3-linked moieties. Chemical modification of the lectin by group-specific reagents allowed to emphasize the role of primarily lysine residues, but also, although less pronounced, arginine, tryptophan, and carboxyl groups for ligand binding and/or maintenance of the active conformational state. Application of reagents, specific for histidine or tyrosine residues, failed to affect lectin activity.
Highlights
Were potent inhibitors in contrast to the non-acetylIantesdpite of the ubiquitous nature of sialic acids in mammals, sialic acids of ovine submaxillary mucin
Materials-N-Acetylneuraminic acid (Neu5Ac),’N-glycolylneuraminic acid (NeuBGc), N-acetylneuraminyllactose ((~-2,3:0(-2,6/4:1), fetuin, 9-0-acetyl-N-acetylneuraminic acid (9-OAc-Neu5Ac)-containing bovine submaxillary mucin (BSM, containing 9(7,8)-0-acetylNeu5Ac residues), the a-2,8-linked homopolymer of Neu5Ac, colominic acid, heparin, sperm nuclei and the 9-OAc-Neu5Accontaining gangliosidesGM1, GM3,GD,. and GTlbfrom bovine brain were purchased from Sigma
Purification strategy couldthus be based on the use of BSM as affinity ligand. These tests revealed that the specificity of Ca2+-independent sialic acid-binding protein(s) appeared to be rather restricted.To convenientlyscreen for the presence oflectids) during purification, the ability of the extract to agglutinate protease-treated rabbit erythrocytes served as functional assay to monitor lectin activity
Summary
Neu5Ac-bovineserumalbumin to fixed and paraffin-embedded tissue sections of human placenta at different stages ofdevelopment in PBS without any additionofCaz+ ions [24], preliminaryOuchterlonydoublediffusion experiments were performed to verify this result forextracts and to detect ligands, suitable for affinity purification of the lectin(s). Purification strategy couldthus be based on the use of BSM as affinity ligand. These tests revealed that the specificity of Ca2+-independent sialic acid-binding protein(s) appeared to be rather restricted.To convenientlyscreen for the presence oflectids) during purification, the ability of the extract to agglutinate protease-treated rabbit erythrocytes served as functional assay to monitor lectin activity. This activity was independent of the presence of metal ions (Ca2+,M e , Mn2+).presenceof EDTA (100 mM)had no inhibitory capacity. Following DEAE-cellulose chromatography that removed perturbing pigment while retaining nearly the total activity in the non-binding fractions, BSM-Sepharose 4B yielded a
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