Abstract
A sialic acid-binding lectin with high specificity for 9-O-acetyl- and 4-O-acetylsialic acids was purified from the hemolymph of the California coastal crab, Cancer antennarius, by affinity chromatography using bovine submaxillary mucin coupled to agarose. The binding specificity of the crab lectin distinguishes it from other known sialic acid-specific lectins from Limulus polyphemus and Limax flavus which show a broader range of specificity for sialic acids. The purified lectin is homogenous on sodium dodecyl sulfate-polyacrylamide electropherograms with a subunit molecular weight of about 36 kDa. The specificity of the lectin for O-acetylsialic acids appears to account for the fact that it agglutinates mouse, rat, rabbit, and horse erythrocytes, which contain O-acetylsialic acids on cell surface glycoconjugates, but not human monkey, sheep, goat, and chicken erythrocytes which contain only NeuAc or N-glycolylneuraminic acid (NeuGc). This conclusion was supported by the potent inhibition of hemagglutination by bovine and equine submaxillary mucins which contain 9(7,8)-O-acetyl- and 4-O-acetylsialic acids, respectively, and also by free 9-O-acetyl-N-acetylneuraminic acid (9-O-Ac-NeuAc) and 4-O-Ac-NeuAc relative to NeuAc and NeuGc. Further support for the role of O-Ac-sialic acids in hemagglutination of erythrocytes was obtained by enzymatic modification of human erythrocytes. Sialidase-treated erythrocytes were resialylated with purified sialyltransferases and various CMP-sialic acid donor substrates to contain NeuAc or NeuGc or 9-O-Ac-NeuAc in the Sia alpha 2,3Gal or Sia alpha 2,6Gal linkages. Cells resialylated to contain NeuAc or NeuGc were not agglutinated, but cells resialylated to contain 9-O-Ac-NeuAc were agglutinated with high titer, comparable to that of mice or horse erythrocytes.
Highlights
A sialic acid-binding lectin with high specificity for been widely used in purification of polysaccharides and gly
The results indicate that the crab lectin utilizes sialic acids asa primary binding determinantand exhibits high affinity for 9-0-acetyl-NeuAc and 4-0-acetylNeuAc relative to NeuAc and NeuGc
Purification ofC. antennurius Lectin-Basedon preliminary hemagglutination inhibition results demonstratinghigh affinity of the C. antennurius lectin for bovine submaxillary mucin, purification was attempted by affinity chromatography using BSM-agarose
Summary
Materials-Affi-Gel 15and polypropylene Econo columns were purchased from Bio-Rad. Bovine submaxillary mucin, fetuin, transferrin, thyroglobulin, acid glycoprotein (bovine fraction IV), human al-acid glycoprotein, C. perfringens neuraminidase type X, molecular weight standards, N-glycolyl-, and N-acetylneuraminic acids were purchased from Sigma. (Beckman T 65 rotor) to sediment the major portion of hemocyanin [32] This sera is referred to as "clarified sera." Preparation of BSM-Agarose Affinity Columns-Affi-Gel 15 (50 ml) was washed as described by the manufacturer and added to 100 ml of cold HEPES containing 200 mg of BSM. 25 pl of a solution of the purified lectin, giving an HA titer of 4 with horse erythrocytes, was added. The lectin-inhibitor mixture was incubated for 1 h at room temperature,and 25 ~l of a 1.5% suspension of horse erythrocytes was added. De-0-acetylation of sialic acids was performed following the procedures of Sarris andPalade [34] and Schauer [18].A solution of 750 p1 of BSM (5 mg/ml) was added to 250 pI of0.4 or 0.04 N of. For determining the molecular weight of unreduced lectin, samples were heated for 3 min a t 100 "C in sample buffer without the reducing agent
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