A novel galactosyl-binding lectin from the plasma of the blood clam, Anadara granosa (L) and a study of its combining site.
The marine blood clam species Anadara granosa (L) belong to arcidae, a family with some extraordinary haematological features. The plasma of this species exhibited strong haemagglutinating activities, from which a galactosyl binding lectin, Anadarin P, was purified in a single step affinity chromatography using Sepharose 4B-asialofetuin as an affinity matrix. The purified lectin, eluted with lactose, was found to be homogeneous by alkaline polyacrylamide disc gels, gel-filtration and isoelectric focusing. Native M(r) of the lectin was 130,000 having a PI value of 6.82 and was composed of two subunits of M(r) 17,000 and M(r) 16,000 which were noncovalently bound. The lectin was remarkably thermostable; the agglutinating titre remained unchanged over a wide range of pH (from 5 to 10) but increased with neuraminidase treated rabbit erythrocytes. Anadarin P combining site has been proposed to be small pocket-like structure which recognised only C-3 and C-4 hydroxyl groups of D-galactose. Presence of bulky groups at C-2 and C-6 exert strong steric hindrance as L-arabinose, 2-deoxy-D-galactose and D-xylose are better inhibitors than D-galactose. The lectin fails to differentiate methyl substituted galactosides as both alpha- and beta- methyl galactosides are equally active; but in case of substituted phenyl glycosides, the lectin shows different affinity towards alpha and beta anomers. The avidity of the lectin to bind the aromatic aglycons of galactosides suggests the presence of a hydrophobic region in the combining site. Interactions with some disaccharides indicate the presence of an extended area near the monosaccharide binding site.
Dam
T K
TK
Department of Marine Science, University Colleges of Science, Technology and Agriculture, Calcutta, India.
Sarkar
M
M
Ghosal
J
J
Choudhury
A
A
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D049872:Bivalvia / Q000378:metabolism*; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D004912:Erythrocytes / Q000378:metabolism; D005690:Galactose / Q000378:metabolism*; D006385:Hemagglutination Inhibition Tests; D006386:Hemagglutination Tests; D006863:Hydrogen-Ion Concentration; D037102:Lectins / Q000302:isolation & purification / Q000378:metabolism*; D009439:Neuraminidase; D013696:Temperature; D014357:Trypsin
D037102:Lectins; D009439:Neuraminidase; D014357:Trypsin; D005690:Galactose