Isolation, by affinity chromatography and gel filtration in 8 M-urea, of an active subunit from the anti-(blood-group A+N)-specific lectin of Moluccella laevis.
The lectin from Moluccella laevis seeds agglutinates specifically blood-type-A and -N erythrocytes, and both activities are inhibited by micromolar concentrations of N-acetyl-D-galactosamine. The lectin consists of three subunits: a 67 kDa heterodimer, made up of two S-S-linked polypeptides of 28 and 46 kDa, and two non-covalently linked moieties of 26 and 42 kDa, the latter migrating after reduction with an apparent molecular mass of 46 kDa. Here we demonstrate that affinity chromatography of a crude protein fraction from M. laevis seeds on immobilized D-galactose in the presence of 8 M-urea affords a fully active lectin practically devoid of the 42 kDa subunit. We also present data showing that the 26 kDa subunit is devoid of cysteine residues, that the 28 kDa subunit contains two cysteine residues engaged in S-S bonds with the 46 kDa subunit, and that the latter has, in addition, two intramolecular cystine residues. Gel filtration on Sephadex G-150 in 8 M-urea/0.2 M-D-galactose of the lectin, affinity-purified in the presence of urea, afforded a pure 26 kDa subunit which exhibited both anti-A and anti-N activity, as well as high specificity for N-acetyl-D-galactosamine. In addition to demonstrating that the lectin is unusually stable and retains its carbohydrate-binding activity in 8 M-urea, our findings also show that the activity for different blood groups resides in the same subunit.
Alperin
D M
DM
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Latter
H
H
Lis
H
H
Sharon
N
N
D000116:Acetylgalactosamine / Q000494:pharmacology; D001212:Asialoglycoproteins / Q000378:metabolism; D001789:Blood Group Antigens / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes; D006384:Hemagglutination; D006801:Humans; D037102:Lectins / Q000302:isolation & purification* / Q000378:metabolism; D037121:Plant Lectins; D010944:Plants / Q000737:chemistry*; D014508:Urea
D001212:Asialoglycoproteins; D001789:Blood Group Antigens; D037102:Lectins; D037121:Plant Lectins; C026503:asialoglycophorin; D014508:Urea; D000116:Acetylgalactosamine