Crystal Structure of the Jacalin–T-antigen Complex and a Comparative Study of Lectin–T-antigen Complexes

Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc 1 Man 7-9 GlcNAc 2 present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc 1 Man 7-9 GlcNAc 2 , determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (K b ) and changes in enthalpy of binding (⌬H b°) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19 ؋ 10 4 M ؊1 at 279 K is obtained when glucose occurs in ␣-1,3 linkage to Man␣Me as in Glc␣1-3Man␣Me. The binding constant increases by 25-fold from di-to trisaccharide and doubles from tri-to tetrasaccharide, demonstrating that the entire Glc␣1-3Man␣1-2Man␣1-2Man␣Me structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis á vis legume lectins, which have the same fold as this chaperone, are discussed. Calreticulin (CRT),1 along with calnexin, serves as a molecular chaperone in the endoplasmic reticulum (ER) of eukaryotic cells. Although calreticulin is a soluble, luminal protein, calnexin is a type I membrane protein (1, 2). Segments of these proteins share amino acid identity ranging from 42 to 78% (3). Calreticulin is a highly conserved ubiquitous protein (M r 46,000) and has been implicated in Ca 2ϩ storage and intracellular Ca 2ϩ signaling in the sarcoplasmic and endoplasmic reticula (4, 5). CRT has been divided into three regions: the N-terminal, the C-terminal, and the central P-domain, which consists of short sequence motifs repeated three times in tandem. The N-terminal domain is highly conserved among CRTs from different species and potentially mediates interactions between CRT and the ER folding catalysts, protein disulfide isomerase and ERp57 (6, 7). Recent studies show that the P-domain, previously thought to be involved in oligosaccharide binding, interacts directly with ERp57 (8 -10). The C-domain is characterized by a high content of acidic residues (4, 11), which is consistent with the location of a low affinity (K d ϭ ϳ1-2 mM), high capacity (ϳ25-50 mol) calcium-binding site (12) and contains the ER retrieval sequence.The ER plays an essential role in the folding and maturation of newly synthesized proteins in the secretory pathway. ER quality control operates at various levels; one of the most comm...

Section: Resultsmentioning

confidence: 99%

Interactions of Substrate with Calreticulin, an Endoplasmic Reticulum Chaperone

Kapoor

Srinivas

Kandiah

et al. 2003

“…Comparison with Lectins and Potential Value of GL-BP for Diagnostic Application-To the best of our knowledge, GL-BP is the first example of an SBP having a strong affinity to LNB and GNB; however, a number of lectin structures complexed with these disaccharides have been reported (59,(62)(63)(64)(65)(66)(67). A common feature of the binding site of lectins for GNB or Tantigens is a shallow depression on the surface of the protein, and recognition occurs through hydrogen bonds to hydroxyls and stacking interaction to one side of the sugar.…”

Section: Analysis Of Ligand Specificity By Itc-mentioning

confidence: 99%

Structural and Thermodynamic Analyses of Solute-binding Protein from Bifidobacterium longum Specific for Core 1 Disaccharide and Lacto-N-biose I

Suzuki

Wada

Katayama

et al. 2008

113

Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Gal␤1-3GlcNAc) and galacto-N-biose (GNB; Gal␤1-3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with K d values of 0.087 and 0.010 M, respectively, and the binding process is enthalpydriven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose (Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.Bifidobacteria are Gram-positive anaerobes naturally present in the dominant colonic microbiota and have been considered to be beneficial for human health. As probiotic agents, Bifidobacteria can prevent or alleviate infectious diarrhea through their effects on the immune system and promote the host resistance to colonization by pathogens (1). Carbon source compounds, including oligofructose, inulin, and raffinose, are used as food additives (prebiotics) to selectively promote the growth of Bifidobacteria in the gut (2, 3). Bifidobacteria predominate the intestinal flora of breastfed infants (4, 5), whereas bottle-fed infants do not show rapid colonization of these organisms (6). It has been widely accepted that oligosaccharides other than lactose in human milk (human milk oligosaccharides, HMOs) 4 play a key role in the growth of Bifidobacteria in the gut (7,8). However, it remains unknown what structure, in HMOs, constitutes the bifidus factor responsible for increasing the bifidobacterial population. Human milk is reported to contain more than 100 kinds of oligosaccharides, the building blocks of which are the following three basic core disaccharides: lactose (Gal␤1-4Glc), lacto-N-biose I (LNB; Gal␤1-3GlcNAc), and N-acetyllactosamine (LacNAc; Gal␤1-4GlcNAc). These oligosaccharides are often modified by sialic acid and/or L-fucose (7). Lacto-N-tetraose (LNT; Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc), which is formed by a ␤1-3 linkage between LNB and lactose, and fucosylated derivative (Fuc␣1-2Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc) are the major components of HMOs (9 -11).Recently, we found that Bifidobacterium longum JCM1217 has a unique metabolic pathway specific for LNB and galacto-N-biose (GNB, Gal␤1-3GalNAc), and we presented the hypothesis that the LNB residue in HMOs is the bifidus factor in breastfed infants (12). B...

“…(38), and, more recently, the fungal galectin CGL2 (39). The Tantigen binding lectins have been classified into two groups on the basis of the monosaccharide that occupies the primary binding site (37). According to this classification, ABL belongs to the category that has GalNAc binding at the primary site.…”

Section: Resultsmentioning

confidence: 99%

The Antineoplastic Lectin of the Common Edible Mushroom (Agaricus bisporus) Has Two Binding Sites, Each Specific for a Different Configuration at a Single Epimeric Hydroxyl

Carrizo

Capaldi

Perduca

et al. 2005

The lectin from the common mushroom Agaricus bisporus, the most popular edible species in Western countries, has potent antiproliferative effects on human epithelial cancer cells, without any apparent cytotoxicity. This property confers to it an important therapeutic potential as an antineoplastic agent. The three-dimensional structure of the lectin was determined by x-ray diffraction. The protein is a tetramer with 222 symmetry, and each monomer presents a novel fold with two ␤ sheets connected by a helix-loop-helix motif. Selectivity was studied by examining the binding of four monosaccharides and seven disaccharides in two different crystal forms. The T-antigen disaccharide, Gal␤1-3GalNAc, mediator of the antiproliferative effects of the protein, binds at a shallow depression on the surface of the molecule. The binding of N-acetylgalactosamine overlaps with that moiety of the T antigen, but surprisingly, Nacetylglucosamine, which differs from N-acetylgalactosamine only in the configuration of epimeric hydroxyl 4, binds at a totally different site on the opposite side of the helix-loop-helix motif. The lectin thus has two distinct binding sites per monomer that recognize the different configuration of a single epimeric hydroxyl. The structure of the protein and its two carbohydrate-binding sites are described in detail in this study.