NMR studies on the interaction of sugars with the C‐terminal domain of an R‐type lectin from the earthworm Lumbricus terrestris

Abstract

Sugar-binding proteins, known as lectins, exist ubiquitously in both animals and plants, but lectins from the annelid phylum have rarely been reported. A 29 kDa lectin (EW29) was isolated from the earthworm Lumbricus terrestris using affinity chromatography on asialofetuin-agarose in the screening of galectin-like proteins. The protein consists of two homologous domains (14 500 Da), i.e. N-and C-terminal domains, which show 27% identity with each other [1]. Both domains of EW29 form a tandem-repeat type structure and contain triple-repeated QXW motifs [2,3]. This short motif has been found in many carbohydraterecognition proteins including the plant lectin ricin B-chain [4]. The 3D structures of R-type lectins have been determined [2,[5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], and these proteins possess common b-trefoil fold structures, although their sugar-binding affinities differ depending on their ligand specificities.Recent biochemical data on EW29 and its truncated mutants showed it to be a single-domain type of 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 a and c, 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 chemicalshift changes in EW29Ch were monitored using )2 to 10 )1 mm), whereas that in subdomain c had a fast exchange regime for these sugars (K d = 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.Abbreviations Ch (C-half), the C-terminal domain; EW29, earthworm 29 kDa lectin; a-Me-Gal, methyl a-D-galactopyranoside; b-Me-Gal, methyl b-D-galactopyranoside; STD, saturation transfer difference.

Directed Evolution of Lectins with Sugar-binding Specificity for 6-Sulfo-galactose

“…A previous x-ray crystallographic analysis revealed that EW29Ch has a ricin-B chain-like ␤-trefoil structure consisting of three homologous (␣, ␤, and ␥) subdomains, with the ␣ and ␥ subdomains possessing the Gal-binding activity (29). Later study by NMR titration experiments demonstrated that the ␣ subdomain predominantly contributes to the Gal-binding activity of EW29Ch (30). Multiple hydroxyl groups attached to the nonreducing terminal Gal, except for the C6 position, have been shown to be directly involved in the hydrogen bonding with the hydrophilic residues Asp-18, Lys-36, and Asn-41 in the ␣ subdomain (supplemental Fig.…”

“…They include 1) adequate size (ϳ14.5 kDa) for stability and error-prone PCR; 2) complete solubility, enabling high productivity in a conventional E. coli expression system; and 3) knowledge of the protein structure on the basis of both x-ray crystallography (29) and NMR analysis (30). A previous x-ray crystallographic analysis revealed that EW29Ch has a ricin-B chain-like ␤-trefoil structure consisting of three homologous (␣, ␤, and ␥) subdomains, with the ␣ and ␥ subdomains possessing the Gal-binding activity (29).…”

Glycosaminoglycans from earthworms (Eisenia andrei)

“…Lectins from Lumbricus terrestris earthworm have also been isolated by affinity chromatography [21], and the C-terminal domain of this lectin was characterized by 1 H-, 13 C-, and 15 N-NMR [22, 23]. Earthworms are also known to have carbohydrate-hydrolyzing enzymes.…”

NMR structure and dynamics of the C‐terminal domain of R‐type lectin from the earthworm Lumbricus terrestris

“…The crystal structures of the complex between EW29Ch and lactose or N ‐acetylgalactosamine [Protein Data Bank (PDB) code: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2ZQN or http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2ZQO] indicate that the overall structure of EW29Ch resembles β‐trefoil fold, and that the protein has two sugar‐binding sites in the subdomains α and γ . Furthermore, our previous NMR titration experiments showed that the α sugar‐binding site of EW29Ch ( K d = 0.01–0.07 m m for lactose) has a much tighter sugar‐binding mode than the γ sugar‐binding site ( K d = 2.66 m m for lactose) . However, the structural basis of the lectin‐sugar interactions of EW29 does not explain why the α sugar‐binding site of EW29Ch binds to lactose much more strongly because the crystal structure of lactose‐bound EW29Ch shows that the interaction between the α sugar‐binding site and lactose is almost same as that between the γ sugar‐binding site and lactose .…”