Kazusa Nishiyama

Acetyl Tributyl Citrate, the Most Widely Used Phthalate Substitute Plasticizer, Induces Cytochrome P450 3A through Steroid and Xenobiotic Receptor

Steroid and xenobiotic receptor (SXR) is activated by endogenous and exogenous chemicals including steroids, bile acids, and prescription drugs. SXR is highly expressed in the liver and intestine, where it regulates cytochrome P450 3A4 (CYP3A4), which in turn controls xenobiotic and endogenous steroid hormone metabolism. However, it is unclear whether Food and Drug Administration (FDA)-approved plasticizers exert such activity. In the present study, we evaluated the effects of FDA-approved plasticizers on SXR-mediated transcription in vitro by luciferase reporter, SXR-coactivator interaction, quantitative real-time PCR analysis of CYP3A4 expression, CYP3A4 enzyme activity assays, and SXR knockdown. Rats, treated with gavage and intraperitoneal injection of compounds, were examined for CYP3A1 expression in vivo. We found that four of eight FDA-approved plasticizers increased SXR-mediated transcription. In particular, acetyl tributyl citrate (ATBC), an industrial plasticizer widely used in products such as food wrap, vinyl toys, and pharmaceutical excipients, strongly activated human and rat SXR. ATBC increased CYP3A4 messenger RNA (mRNA) levels and enzyme activity in the human intestinal cells but not in human liver cells. Similarly, CYP3A1 mRNA levels were increased in the intestine but not the liver of ATBC-treated rats. These in vitro and in vivo results suggest that ATBC specifically induces CYP3A in the intestine by activating SXR. We suggest that ATBC-containing products be used cautiously because they may alter metabolism of endogenous steroid hormones and prescription drugs.

Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo.

Caenorhabditis elegans galectins LEC-6 and LEC-1 recognize a chemically synthesized Galβ1-4Fuc disaccharide unit which is present in Protostomia glycoconjugates

Galbeta1-4GlcNAc is thought to be a common disaccharide unit preferentially recognized by vertebrate galectins. Eight-amino-acid residues conserved in proteins belonging to the galectin family have been suggested to be responsible for recognition. Meanwhile, we isolated and analyzed endogenous N-glycans of Caenorhabditis elegans that were captured by a C. elegans galectin LEC-6 and demonstrated that the unit of recognition for LEC-6 is a Gal-Fuc disaccharide, though the linkage between these residues was not confirmed. In the present study, we chemically synthesized Galbeta1-4Fuc and Galbeta1-3Fuc labeled with 2-aminopyridine (PA) and demonstrated that LEC-6 interacts with PA-Galbeta1-4Fuc more strongly than PA-Galbeta1-3Fuc by frontal affinity chromatography (FAC). Galbeta1-4Fuc also inhibited hemagglutination caused by LEC-6 more strongly than Galbeta1-3Fuc. FAC analysis using LEC-6 point mutants revealed that some of the conserved amino acid residues which have proven to be important for the recognition of Galbeta1-4GlcNAc are not necessary for the binding to Galbeta1-4Fuc. Another major C. elegans galectin, LEC-1, also showed preferential binding to Galbeta1-4Fuc. These results suggest that Galbeta1-4Fuc is the endogenous unit structure recognized by C. elegans galectins, which implies that C. elegans glycans and galectins may have co-evolved through an alteration in the structures of C. elegans glycans and a subsequent conversion in the sugar-binding mechanism of galectins. Furthermore, since glycans containing the Galbeta1-4Fuc disaccharide unit have been found in organisms belonging to Protostomia, this unit might be a common glyco-epitope recognized by galectins in these organisms.

Mammalian galectins bind Galactoseβ1–4Fucose disaccharide, a unique structural component of protostomial N-type glycoproteins

Galactoseβ1-4Fucose (Galβ1-4Fuc) is a unique disaccharide exclusively found in N-glycans of protostomia, and is recognized by some galectins of Caenorhabditis elegans and Coprinopsis cinerea. In the present study, we investigated whether mammalian galectins also bind such a disaccharide. We examined sugar-binding ability of human galectin-1 (hGal-1) and found that hGal-1 preferentially binds Galβ1-4Fuc compared to Galβ1-4GlcNAc, which is its endogenous recognition unit. We also tested other human and mouse galectins, i.e., hGal-3, and -9 and mGal-1, 2, 3, 4, 8, and 9. All of them also showed substantial affinity to Galβ1-4Fuc disaccharide. Further, we assessed the inhibitory effect of Galβ1-4Fuc, Galβ1-4Glc, and Gal on the interaction between hGal-1 and its model ligand glycan, and found that Galβ1-4Fuc is the most effective. Although the biological significance of galectin-Galβ1-4Fuc interaction is obscure, it might be possible that Galβ1-4Fuc disaccharide is recognized as a non-self-glycan antigen. Furthermore, Galβ1-4Fuc could be a promising seed compound for the synthesis of novel galectin inhibitors.

Caenorhabditis elegans proteins captured by immobilized Galβ1-4Fuc disaccharide units: assignment of 3 annexins

Galβ1-4Fuc is a key structural motif in Caenorhabditis elegans glycans and is responsible for interaction with C. elegans galectins. In animals of the clade Protostomia, this unit seems to have important roles in glycan-protein interactions and corresponds to the Galβ1-4GlcNAc unit in vertebrates. Therefore, we prepared an affinity adsorbent having immobilized Galβ1-4Fuc in order to capture carbohydrate-binding proteins of C. elegans, which interact with this disaccharide unit. Adsorbed C. elegans proteins were eluted with ethylenediaminetetraacetic acid (EDTA) and followed by lactose (Galβ1-4Glc), digested with trypsin, and were then subjected to proteomic analysis using LC-MS/MS. Three annexins, namely NEX-1, -2, and -3, were assigned in the EDTA-eluted fraction. Whereas, galectins, namely LEC-1, -2, -4, -6, -9, -10, and DC2.3a, were assigned in the lactose-eluted fraction. The affinity of annexins for Galβ1-4Fuc was further confirmed by adsorption of recombinant NEX-1, -2, and -3 proteins to the Galβ1-4Fuc column in the presence of Ca(2+). Furthermore, frontal affinity chromatography analysis using an immobilized NEX-1 column showed that NEX-1 has an affinity for Galβ1-4Fuc, but no affinity toward Galβ1-3Fuc and Galβ1-4GlcNAc. We would hypothesize that the recognition of the Galβ1-4Fuc disaccharide unit is involved in some biological processes in C. elegans and other species of the Protostomia clade.

Structural basis of preferential binding of fucose-containing saccharide by the Caenorhabditis elegans galectin LEC-6

Galectins are a group of lectins that can bind carbohydrate chains containing β-galactoside units. LEC-6, a member of galectins of Caenorhabditis elegans, binds fucose-containing saccharides. We solved the crystal structure of LEC-6 in complex with galactose-β1,4-fucose (Galβ1-4Fuc) at 1.5 Å resolution. The overall structure of the protein and the identities of the amino-acid residues binding to the disaccharide are similar to those of other galectins. However, further structural analysis and multiple sequence alignment between LEC-6 and other galectins indicate that a glutamic acid residue (Glu67) is important for the preferential binding between LEC-6 and the fucose moiety of the Galβ1-4Fuc unit. Frontal affinity chromatography analysis indicated that the affinities of E67D and E67A mutants for Galβ1-4Fuc are lower than that of wild-type LEC-6. Furthermore, the affinities of Glu67 mutants for an endogenous oligosaccharide, which contains a Galβ1-4Fuc unit, are drastically reduced relative to that of the wild-type protein. We conclude that the Glu67 in the oligosaccharide-binding site assists the recognition of the fucose moiety by LEC-6.

Purification of galectin-1 mutants using an immobilized Galactoseβ1–4Fucose affinity adsorbent

Galectins are a family of lectins characterized by their carbohydrate recognition domains containing eight conserved amino acid residues, which allows the binding of galectin to β-galactoside sugars such as Galβ1-4GlcNAc. Since galectin-glycan interactions occur extracellularly, recombinant galectins are often used for the functional analysis of these interactions. Although it is relatively easy to purify galectins via affinity to Galβ1-4GlcNAc using affinity adsorbents such as asialofetuin-Sepharose, it could be difficult to do so with mutated galectins, which may have reduced affinity towards their endogenous ligands. However, this is not the case with Caenorhabditis elegans galectin LEC-6; binding to its endogenous recognition unit Galβ1-4Fuc, a unique disaccharide found only in invertebrates, is not necessarily affected by point mutations of the eight well-conserved amino acids. In this study, we constructed mutants of mouse galectin-1 carrying substitutions of each of the eight conserved amino acid residues (H44F, N46D, R48H, V59A, N61D, W68F, E71Q, and R73H) and examined their affinity for Galβ1-4GlcNAc and Galβ1-4Fuc. These mutants, except W68F, had very low affinity for asialofetuin-Sepharose; however, most of them (with the exception of H44F and R48H) could be purified using Galβ1-4Fuc-Sepharose. The affinity of the purified mutant galectins for glycans containing Galβ1-4Fuc or Galβ1-4GlcNAc moieties was quantitatively examined by frontal affinity chromatography, and the results indicated that the mutants retained the affinity only for Galβ1-4Fuc. Given that other mammalian galectins are known to bind Galβ1-4Fuc, our data suggest that immobilized Galβ1-4Fuc ligands could be generally used for easy one-step affinity purification of mutant galectins.