Functional Domain of Marine Invertebrate Lectin

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The amino acid sequences of multiple galactose-binding lectins isolated from the coelomic fluid of the acorn barnacle, Megabalanus rosa have been determined. 140K lectin was composed of identical subunits (Mr 22K) which were cross-linked by interchain disulfide bonds. The subunit was a glycoprotein on 173 amino acids. 64K lectin was composed of four identical subunits (Mr 16K) of 138 amino acids. The subunits were crossーlinked by disulfide bonds to form dimers at the C-terminal segments. They had a domain which was homologous with the carbohydrate-recognition domain of the calcium-dependent (C-type) animal lectins. The denaturation by urea and other agents of the lectins was followed by high-performance size exclusion chromatography and agglutinating activity against rabbit erythrocytes. The subunit structure of 64K lectin was highly stable either in 6M urea and 50% isopropanol, sustainig the activity, whereas 140K lectin easily denatured.A photoactivatable D-galactosamine derivative was prepared for the photoaffinity labeling of the lectins to localize carbohydrate binding sites. The lectins were also subjected to various chemical modification in order to ascertain the amino acid residues responsible for its carbohydrate-binding property. Modification of lysine and tyrosine did not affect their activities. However, modification of tryptophan, arginine and histidine led to complete losses of the activities.Upon treatment with cyanogen bromide, 64K lectin yielded two fragments, a small one including interchain disulfide bonds and a large one corresponding to the carbohydrate-recognition domain, by gel filtration. The isolated domain was demonstrated to retain the carbohydrate binding activity by affinity chromatography on a HCI treated Sepharose 4B column.The above results indicate that marine invertebrate lectins can be used as a functional domain to construct various kinds of multifunctional proteins.