• earthworm Lumbricus terrestris;
  • hemagglutinating activity;
  • NMR titration;
  • R-type lectin;
  • sugar

The R-type lectin EW29, isolated from the earthworm Lumbricus terrestris, consists of two homologous domains (14 500 Da) showing 27% identity with each other. The C-terminal domain (Ch; C-half) of EW29 (EW29Ch) has two sugar-binding sites in subdomains α and γ, and the protein uses these sugar-binding sites for its function as a single-domain-type hemagglutinin. In order to determine the sugar-binding ability and specificity for each of the two sugar-binding sites in EW29Ch, ligand-induced chemical-shift changes in EW29Ch were monitored using 1H–15N HSQC spectra as a function of increasing concentrations of lactose, melibiose, d-galactose, methyl α-d-galactopyranoside and methyl β-d-galactopyranoside. Shift perturbation patterns for well-resolved resonances confirmed that all of these sugars associated independently with the two sugar-binding sites of EW29Ch. NMR titration experiments showed that the sugar-binding site in subdomain α had a slow or intermediate exchange regime on the chemical-shift timescale (Kd = 10−2 to 10−1 mm), whereas that in subdomain γ had a fast exchange regime for these sugars (Kd = 2–6 mm). Thus, our results suggest that the two sugar-binding sites of EW29Ch in the same molecule retain its hemagglutinating activity, but this activity is 10-fold lower than that of the whole protein because EW29Ch has two sugar-binding sites in the same molecule, one of which has a weak binding mode.