Excited state dynamics of protonated dopamine: hydration and conformation effects.

Electronic and vibrational spectroscopy in a cryogenic ion trap has been applied to protonated dopamine water clusters and assigned with the help of quantum chemistry calculations performed in the ground and electronic excited states. A dramatic hydration effect is observed when dopamine is solvated by three water molecules. The broad electronic spectra recorded for the bare and small water clusters containing protonated dopamine turn to sharp, well-resolved vibronic transitions in the 1-3 complex. This reflects the change induced by hydration in the photodynamics of protonated dopamine which is initially controlled by an excited state proton transfer (ESPT) reaction from the ammonium group toward the catechol ring. Interestingly, conformer selectivity is revealed in the 1-3 complex which shows two low lying energy conformers for which the ESPT reaction is prevented or not depending on the H-bond network formed between the dopamine and water molecules.

Hydration-controlled excited-state relaxation in protonated dopamine studied by cryogenic ion spectroscopy.

Ultraviolet (UV) and infrared (IR) spectra of protonated dopamine (DAH+) and its hydrated clusters DAH+(H2O)1-3 are measured by cryogenic ion spectroscopy. DAH+ monomer and hydrated clusters with up to two water molecules show a broad UV spectrum, while it turns to a sharp, well-resolved one for DAH+-(H2O)3. Excited state calculations of DAH+(H2O)3 reproduce these spectral features. The conformer-selected IR spectrum of DAH+(H2O)3 is measured by IR dip spectroscopy, and its structure is assigned with the help of quantum chemical calculations. The excited state lifetime of DAH+ is much shorter than 20 ps, the cross correlation of the ps lasers, revealing a fast relaxation dynamics. The minimal energy path along the NH → π proton transfer coordinate exhibits a low energy barrier in the monomer, while this path is blocked by the high energy barrier in DAH+(H2O)3. It is concluded that the excited state proton transfer in DAH+ is inhibited by water-insertion.

Galactoseß1-4fucose: A unique disaccharide unit found in N-glycans of invertebrates including nematodes.

Galactoseß1-4fucose (Galß1-4Fuc), a unique disaccharide unit found only on the N-glycans of Protostomia, has been intensively studied, particularly in Nematoda. Galß1-4Fuc attached to the 6-OH of the innermost GlcNAc of N-glycans has been identified as an endogenous target recognized by Caenorhabditis elegans galectin LEC-6 and might function as an endogenous ligand for other galectins as well. Interactions between galectins and N-glycans might be subject to fine-tuning through modifications of the penultimate GlcNAc and the Galß1-4Fuc unit. Similar fine-tuning is also observable in vertebrate galectins, although their major recognition unit is a Galß1-4GlcNAc. In Protostomia, it can be postulated that glycan-binding proteins and their ligands have coevolved; however, epitopes such as Galß1-4Fuc were then hijacked as targets by other organisms. Fungal (Coprinopsis cinerea) galectin 2, CGL2, binds the Galß1-4Fuc on C. elegans glycans to exert its nematotoxicity. Some human and mouse galectins bind to synthesized Galß1-4Fuc; as some parasitic nematodes express this motif, its recognition by mammalian galectins could hypothetically be involved in host defense, similar to fungal CGL2. In this review, we discuss the Galß1-4Fuc unit in Protostomia as a possible equivalent for the Galß1-4GlcNAc unit in vertebrates and a potential non-self glycomarker useful for pathogen recognition.

Preparation of a polyclonal antibody that recognizes a unique galactoseß1-4fucose disaccharide epitope.

Galactoseß1-4fucose (Galß1-4Fuc) is a unique disaccharide unit that has been found only in the N-glycans of protostomia. We demonstrated that this unit has a role as an endogenous ligand for Caenorhabditis elegans galectins. This unit is also recognized by fungal and mammalian galectins possibly as a non-self glycomarker. In order to clarify its biological function, we made a polyclonal antibody using (Galß1-4Fuc)n-BSA as the antigen, which was prepared by crosslinking Galß1-4Fuc-O-(CH2)2-SH and BSA. The binding specificity of the antibody was analyzed by frontal affinity chromatography, and it was confirmed that it recognizes naturally occurring N-glycans containing the Galß1-4Fuc unit linked to the reducing-end GlcNAc via α1-6 linkage. By western blotting analysis, the antibody was also found to bind to (Galß1-4Fuc)n-BSA but not to BSA or asialofetuin, which has N-glycan chains containing Galß1-4GlcNAc. Western blotting experiments also revealed presence of stained proteins in crude extracts of C. elegans, the parasitic nematode Ascaris suum, and the allergenic mite Dermatophagoides pteronyssinus, while those from Drosophila melanogaster, Mus musculus, and the allergenic mites Dermatophagoides farinae and Tyrophagus putrescentiae were negative. This antibody should be a very useful tool for research on the distribution of the Galß1-4Fuc disaccharide unit in glycans in a wide range of organisms.

Frontal affinity chromatography (FAC): theory and basic aspects.

Frontal affinity chromatography (FAC) is a versatile analytical tool for determining specific interactions between biomolecules and is particularly useful in the field of glycobiology. This article presents its basic aspects, merits, and theory.

Crosslinking of Cys-mutated human galectin-1 to the model glycoprotein ligands asialofetuin and laminin by using a photoactivatable bifunctional reagent.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. In our previous study, we showed that a human galectin-1 (hGal-1) mutant, in which a cysteine residue was introduced at Lys(28), forms a covalently cross-linked complex with the model glycoprotein ligands asialofetuin and laminin by using the photoactivatable sulfhydryl reagent benzophenone-4-maleimide (BPM). In the present study, we used several hGal-1 mutants in which single cysteine residues were introduced at different positions and examined their ability to form a covalent complex with asialofetuin or laminin by using BPM. We found that the efficiency of formation of the cross-linked products differed depending on the positions of the cysteine introduced and also on the ligand used for crosslinking. Therefore, by using different cysteine hGal-1 mutants, the chances of isolating different ligands for hGal-1 should increase depending on the systems and cells used.

Affinity probe capillary electrophoresis of insulin using a fluorescence-labeled recombinant Fab as an affinity probe.

Affinity probe CE (APCE) separates and detects a target molecule as a complex using a fluorescence-labeled affinity probe (AP) by CE. The electrophoretic separation of the complex ensures accurate identification of a specific signal among nonspecific ones, which often compromises the credibility of immunoassays. APCE of insulin using a recombinant Fab (rFab) as an AP was demonstrated as a model system in this report. Anti-insulin rFab was expressed in Escherichia coli and labeled at a cysteine residue in the hinge region with a thiol-reactive rhodamine dye. Electrophoretically pure labeled rFab was recovered from a focused band in slab-gel IEF and used as an AP. A mixture of standard insulin and the AP with carrier ampholyte was introduced into a neutral-polymer coated fused silica capillary (50 µm id, 120 mm long). IEF was carried out at 500 V/cm, and the capillary was scanned for laser-induced fluorescence under focusing conditions. The insulin-AP complex focused at pH 6.6 within 6 min along with the free AP at pH 7.6. The complex peak decayed according to the first-order reaction kinetics with a half life of 3.8 min. A linear calibration line was obtained for standard insulin at a concentration range of 20 pM to 5 nM using the AP at 50 nM. These results demonstrate that rFab is useful for the preparation of an AP for APCE.

Galectin LEC-1 plays a defensive role against damage due to oxidative stress in Caenorhabditis elegans.

LEC-1 is a major galectin in Caenorhabditis elegans and contains two carbohydrate recognition domains (CRDs), N-CRD and C-CRD. To determine the role of LEC-1, we examined the phenotypes of a mutant C. elegans strain lacking lec-1. We observed negligible differences in embryogenesis, morphogenesis and egg laying at 20 °C between the mutant and the wild-type. Furthermore, the life spans of the mutant and the wild-type were equivalent at either 20 °C or 25 °C. However, the lec-1 mutant showed a greater susceptibility to H2O2 and paraquat than the wild-type. This result suggests an increased susceptibility to oxidative stress, with the phenotypes being similar to those of lec-10 deletion mutants as previously described. To understand the molecular mechanism underlying this phenotype, C. elegans proteins bound by either the LEC-1 N-CRD or C-CRD were isolated and identified using a nano liquid chromatography-tandem mass spectrometry technique. MIG-6 was identified as a major binding partner of LEC-1 with both N- and C-CRD. From these results and previous reports, we speculate that interaction of LEC-1 and MIG-6 in the pharynx may affect susceptibility to paraquat and that LEC-10 has different functions from LEC-1 in regulating H2O2 and paraquat resistance in the intestine.

Mammalian galectins bind galactoseß1-4fucose disaccharide, a unique structural component of protostomial N-type glycoproteins.

Galactoseß1-4Fucose (Galß1-4Fuc) 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 Galß1-4Fuc compared to Galß1-4GlcNAc, 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 Galß1-4Fuc disaccharide. Further, we assessed the inhibitory effect of Galß1-4Fuc, Galß1-4Glc, and Gal on the interaction between hGal-1 and its model ligand glycan, and found that Galß1-4Fuc is the most effective. Although the biological significance of galectin-Galß1-4Fuc interaction is obscure, it might be possible that Galß1-4Fuc disaccharide is recognized as a non-self-glycan antigen. Furthermore, Galß1-4Fuc could be a promising seed compound for the synthesis of novel galectin inhibitors.

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 ß-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-ß1,4-fucose (Galß1-4Fuc) 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 Galß1-4Fuc unit. Frontal affinity chromatography analysis indicated that the affinities of E67D and E67A mutants for Galß1-4Fuc are lower than that of wild-type LEC-6. Furthermore, the affinities of Glu67 mutants for an endogenous oligosaccharide, which contains a Galß1-4Fuc 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.