Mutational analysis of the Ricinus lectin B-chains. Galactose-binding ability of the 2 gamma subdomain of Ricinus communis agglutinin B-chain.
Ricin B-chain (RTB) is a galactose-specific lectin that folds into two globular domains, each of which binds a single galactoside. The two binding sites are structurally similar and both contain a conserved tripeptide kink and an aromatic residue that comprises a sugar-binding platform. Whereas the critical RTB residues implicated in lectin activity are conserved in domain 1 of Ricinus communis agglutinin (RCA) B-chain, the sugar platform aromatic residue Tyr-248 present in domain 2 of RTB is replaced by His in RCA B-chain. In this study, key residues in the vicinity of the binding sites of the Ricinus lectin B-chains were altered by site-directed mutagenesis. The recombinant B-chains were produced in Xenopus oocytes in soluble, stable, and core-glycosylated forms. Both sites of RCA B-chain must be simultaneously modified in order to abolish lectin activity, indicating the presence of two independent, functional binding sites/molecule. Activity associated with the domain 2 site of RCA B-chain is abrogated by the conversion of Trp-258 to Ser. Moreover, the domain 2 site appears responsible for a weak binding interaction recombinant RCA B-chain with GalNAc, not observed with native tetrameric RCA. Finally, the introduction of His at position 248 of RTB severely disrupts but does not abolish GalNAc binding.
Sphyris
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N
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
Lord
J M
JM
Wales
R
R
Roberts
L M
LM
D000116:Acetylgalactosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D005260:Female; D005690:Galactose / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D037121:Plant Lectins; D010947:Plants, Toxic; D017354:Point Mutation; D021382:Protein Sorting Signals / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012276:Ricin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012278:Ricinus / Q000378:metabolism; D014158:Transcription, Genetic; D014981:Xenopus
D037121:Plant Lectins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012276:Ricin; D000116:Acetylgalactosamine; D005690:Galactose