N. Sphyris et al., MUTATIONAL ANALYSIS OF THE RICINUS LECTIN B-CHAINS - GALACTOSE-BINDING ABILITY OF THE 2-GAMMA SUBDOMAIN OF RICINUS-COMMUNIS AGGLUTININ B-CHAIN, The Journal of biological chemistry, 270(35), 1995, pp. 20292-20297
Ricin B-chain (RTB) is a galactose-specific lectin that folds into two
globular domains, each of which binds a single galactoside. The two b
inding sites are structurally similar and both contain a conserved tri
peptide kink and an aromatic residue that comprises a sugar-binding pl
atform. Whereas the critical RTB residues implicated in lectin activit
y are conserved in domain 1 of Ricinus communis agglutinin (RCA) B cha
in, 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 p
roduced in Xenopus oocytes in soluble, stable, and core-glycosylated f
orms. Both sites of RCA B chain must be simultaneously modified in ord
er to abolish lectin activity, indicating the presence of two independ
ent, functional binding sites/molecule. Activity associated with the d
omain 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 bin
ding interaction of recombinant RCA B-chain with GalNAc, not observed
with native tetrameric RCA. Finally, the introduction of His at positi
on 248 of RTB severely disrupts but does not abolish GalNAc binding.