title: Galactosyl-binding lectins from the tunicate Didemnum candidum. Carbohydrate specificity and characterization of the combining site.
abstract: The plasma of the ascidian Didemnum candidum possesses lectin activity directed toward galactosyl moieties. We report the characterization of the affinity chromatography-purified galactosyl-binding lectins from the plasma of this protochordate species in terms of their hemagglutination patterns, temperature stability, saccharide specificities, divalent cation requirements, and the comparison of the properties of their combining sites to those of other characterized lectins. The major galactosyl-specific lectin, termed DCL-I, has an apparent mass of 14,500 daltons and a minor lectin (DCL-II) has an apparent subunit mass of 15,500 daltons. The two molecules differed somewhat in their hemagglutination profiles with untreated and enzyme-treated erythrocytes: a 10-fold increase in DCL-II concentration is required to obtain agglutination titers comparable to those of DCL-I. Although both DCL-I and DCL-II will agglutinate neuraminidase-treated erythrocytes from all vertebrate species tested and most Pronase-treated erythrocytes, DCL-I will agglutinate some untreated erythrocytes which are not agglutinated by DCL-II. Both lectins required divalent cations, were inactivated by temperatures above 70 degrees C, and both exhibited optimal agglutinating activity over a wide range of pH (from 5 to 11). The DCL-I molecule was characterized for its saccharide specificity by binding and inhibition assays using characterized sugars and glycoproteins. Galactose and oligosaccharides bearing nonreducing terminal galactose were the best inhibitors. The inhibition analysis indicated that the DCL-I combining site is small, interacts only with hydroxyls on carbons 2, 3, and 4 of galactose, and exhibits moderate steric hindrance for voluminous groups on carbon 6 and the alpha-anomeric linkage. The data suggest that the combining site would be smaller than the peanut lectin combining site for galactose since DCL-I does not interact with the subterminal monosaccharide hydroxyls for C4 and C6 as does peanut agglutinin. To our knowledge, this is the first isolation and detailed characterization of a lectin from a protochordate species.
mesh_terms: D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000494:pharmacology; D002413:Cations, Divalent; D004912:Erythrocytes / Q000187:drug effects; D005690:Galactose / Q000494:pharmacology; D037161:Galectins; D006386:Hemagglutination Tests; D006388:Hemagglutinins / Q000302:isolation & purification; D006358:Hot Temperature; D006801:Humans; D009439:Neuraminidase / Q000494:pharmacology; D011134:Polysaccharides / Q000494:pharmacology; D011402:Pronase / Q000494:pharmacology; D014561:Urochordata / Q000032:analysis*
chemical_list: D002241:Carbohydrates; D002413:Cations, Divalent; D037161:Galectins; D006388:Hemagglutinins; D011134:Polysaccharides; D009439:Neuraminidase; D011402:Pronase; D005690:Galactose