Sachiko Nakamura

Comparative analysis of carbohydrate-binding properties of two tandem repeat-type Jacalin-related lectins, Castanea crenata agglutinin and Cycas revoluta leaf lectin

Lectins belonging to the jacalin‐related lectin family are distributed widely in the plant kingdom. Recently, two mannose‐specific lectins having tandem repeat‐type structures were discovered in Castanea crenata (angiosperm) and Cycas revoluta (gymnosperm). The occurrence of such similar molecules in taxonomically less related plants suggests their importance in the plant body. To obtain clues to understand their physiological roles, we performed detailed analysis of their sugar‐binding specificity. For this purpose, we compared the dissociation constants (Kd) of Castanea crenata agglutinin (CCA) and Cycas revoluta leaf lectin (CRLL) by using 102 pyridylaminated and 13 p‐nitrophenyl oligosaccharides with a recently developed automated system for frontal affinity chromatography. As a result, we found that the basic carbohydrate‐binding properties of CCA and CRLL were similar, but differed in their preference for larger N‐linked glycans (e.g. Man7–9 glycans). While the affinity of CCA decreased with an increase in the number of extended α1–2 mannose residues, CRLL could recognize these Man7–9 glycans with much enhanced affinity. Notably, both lectins also preserved considerable affinity for mono‐antennary, complex type N‐linked glycans, though the specificity was much broader for CCA. The information obtained here should be helpful for understanding their functions in vivo as well as for development of useful probes for animal cells. This is the first systematic approach to elucidate the fine specificities of plant lectins by means of high‐throughput, automated frontal affinity chromatography.

The family 42 carbohydrate-binding module of family 54 α-L-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose

Alpha-L-arabinofuranosidase catalyses the hydrolysis of the alpha-1,2-, alpha-1,3-, and alpha-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 alpha-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-alpha-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 10(3) M(-1). These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.

Carbohydrate Specificity of Lectins from Boletopsis leucomelas and Aralia cordate

The carbohydrate specificity of three novel lectins, Boletopsis leucomelas lectin (BLL), Aralia cordate lectin (ACL), and Wasabia japonica lectin (WJL), was examined by frontal affinity chromatography using a panel of fluorescently labeled 47 oligosaccharides. The results indicate that BLL recognizes an agalacto structure of the biantennary chain and its bisecting structure. ACL showed strong affinity for triantennary oligosaccharides, but no affinity for tetraantennary structure. WJL showed no appreciable affinity for any of the 47 glycans examined. These lectins with a unique affinity specificity might be useful for examining alterations in the glycan structures of the glycoconjugates in association with development and various diseases.