Interaction entre l'hémagglutinine de ricin et ses ligands, galactose et lactose. Etude par microcalorimétrie et dialyse à l'équilibre

Zentz, Christian; Frénoy, Jean-Pierre; Bourrillon, R; Tran, d'Anh Tuan

doi:10.1016/s0300-9084(79)80307-7

Cited by 21 publications

(12 citation statements)

References 22 publications

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“…Binding of an individual lectin site (monovalent binding) to a monosaccharide is extremely weak, with K d values typically in the range of 0.1-10 mm [29][30][31]. In the R-type lectin family, the dissociation constants of ricin and RCA120 have been determined mainly by equilibrium dialysis studies and fluorescence polarization studies [32][33][34][35][36][37][38][39][40][41][42]. Ricin has at least two binding sites in its molecule.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2009

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2009

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“…While MOA is specific for Gal␣1,3Gal-containing sugars, ricin binds well with ␤-1,3-or ␤-1,4-linked galactose-terminated sugars (14). Like MOA, ricin shows higher affinity for larger, more complex saccharides than for simple sugars (2,15). The affinity constant for lactose binding to ricin is 10-fold greater than for galactose alone (15).…”

Section: Resultsmentioning

confidence: 97%

“…Like MOA, ricin shows higher affinity for larger, more complex saccharides than for simple sugars (2,15). The affinity constant for lactose binding to ricin is 10-fold greater than for galactose alone (15). Similarly, MOA binds Gal␣1,3Gal with an affinity constant 44-fold greater than that for Me␣-Gal (2).…”

Section: Resultsmentioning

confidence: 99%

Cloning, Expression, and Characterization of the Galα1,3Gal High Affinity Lectin from the Mushroom Marasmius oreades

Kruger¹,

Winter²,

Simonson-Leff³

et al. 2002

Journal of Biological Chemistry

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The purification and unique carbohydrate binding properties, including blood group B-specific agglutination and preferential binding to Gal␣1,3Gal-containing sugar epitopes, of the Marasmius oreades agglutinin (MOA) are reported in an accompanying paper (Winter, H. C., Mostafapour, K., and Goldstein, I. J. (2002) J. Biol. Chem. 277, 14996 -15001). Here we describe the cloning, characterization, and expression of MOA. MOA was digested with trypsin and endoproteinase Asp-N, and the peptide fragments were purified by high performance liquid chromatography. Amino acid sequence data were obtained for eight peptides. Using oligonucleotides deduced from the peptide sequences for a reverse transcriptase-PCR, a 41-base pair cDNA was obtained. The 41-base pair fragment allowed the generation a fulllength cDNA using 5 and 3 rapid amplification of cDNA ends. MOA cDNA encodes a protein of 293 amino acids that contains a ricin domain. These carbohydrate binding domains were first described in subunits of bacterial toxins and are also commonly found in polysaccharidedegrading enzymes. Whereas these proteins are known to display a variety of sugar binding specificities, none to date are known to share MOA's high affinity for Gal␣1,3Gal and Gal␣1,3Gal␤1,4GlcNAc. Recombinantly expressed and purified MOA retains the specificity and affinity observed with the native protein. This study provides the basis for analyzing the underlying cause for the unusual binding specificity of MOA.Specificity varies greatly among carbohydrate-binding proteins. Whereas some lectins broadly recognize all oligosaccharides containing particular terminal sugars, others show increasing affinity for specific di-and trisaccharides. Fewer still show almost no reactivity with a given sugar monomer yet bind strongly and specifically to particular oligosaccharides (1). As described in the accompanying paper (2), the Marasmius oreades agglutinin (MOA) 1 binds to Gal␣1,3Gal-containing sugars and falls into this last category.The Gal␣1,3Gal epitope has received considerable attention, stemming from its presence in the glycoproteins of most mammals and its conspicuous absence in humans, apes, and Old World monkeys (3). This absence is attributable to lack of the specific ␣1,3-galactosyltransferase because of frameshift mutations in its gene (4). The resulting immunogenicity of the Gal␣1,3Gal epitope is a significant barrier to xenotransplantation (5). Despite the importance of Gal␣1,3Gal epitope recognition, MOA is currently the only lectin known to have exclusive specificity for this disaccharide (2).Few proteins have been shown to bind with any specificity to Gal␣1,3Gal. While Clostridium difficile toxin A and antibodies recognizing the ␣-galactosyl epitope both bind well to some Gal␣1,3Gal-containing oligosaccharides (6), the size and species of origin of MOA suggest that it is fundamentally dissimilar to these proteins. On these grounds, the blood group Bspecific Griffonia simplicifolia I-B 4 isolectin is perhaps more appropriate for comparison (7). A recent x...

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“…The R‐type lectins possess common β‐trefoil fold structures, although their sugar‐binding affinities, as well as their ligand specificities, differ significantly. For example, the K d values determined for two sugar‐binding sites of the ricin B‐chain and EW29Ch differ , even though the sugar‐binding properties of the two relevant sugar‐binding sites of these R‐type lectins are quite similar to each other in their crystal structures . To clarify the differences of the sugar‐binding properties between the two sugar‐binding sites of the same molecule (EW29Ch) from the viewpoint of structural biology, we performed high‐resolution NMR structure determination using RDC data, and examined the backbone dynamics by 15 N relaxation experiments.…”

Section: Discussionmentioning

confidence: 99%

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

2012

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The C-terminal domain (Ch; C-half) of the R-type earthworm 29-kDa lectin (EW29), isolated from the earthworm Lumbricus terrestris, has two sugar-binding sites, in subdomains a and c, and the protein uses the two sugar-binding sites for its function as a single domain-type haemagglutinin. Our previous NMR titration experiments showed that the a sugar-binding site is a high-affinity site and the c sugar-binding site is a low-affinity site. However, it remains unclear why the a sugar-binding site of EW29Ch binds to lactose much more strongly because the crystal structure of lactose-bound EW29Ch showed that the interaction between the a sugar-binding site and lactose was almost same as that between the c sugar-binding site and lactose. In the present study, we have determined the NMR structure of EW29Ch in the sugar-free state and performed 15 N relaxation experiments for EW29Ch in both the sugar-free state and the lactosebound states. The conformation of EW29Ch in the sugar-free state was similar to that of EW29Ch in complex with lactose. Conformational changes upon binding of lactose were observed only for the a sugar-binding site. By contrast, the 15 N relaxation experiments revealed a conformational exchange at the a sugar-binding site in the sugar-free state, which was suppressed in the lactose-bound state. The conformational exchange phenomenon observed for the a sugar-binding site was not observed for the c sugar-binding site. Differences in the conformational change and the backbone dynamics between subdomains a and c may be associated with the difference of the sugar-binding modes between the two sugar-binding sites. DatabaseStructural data for the NMR structure of EW29Ch in the sugar-free state have been deposited in the Protein Data Bank database under accession number 2RST.

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Interaction entre l'hémagglutinine de ricin et ses ligands, galactose et lactose. Etude par microcalorimétrie et dialyse à l'équilibre

Cited by 21 publications

References 22 publications

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

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

Cloning, Expression, and Characterization of the Galα1,3Gal High Affinity Lectin from the Mushroom Marasmius oreades

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

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