Structural analysis of N-glycans in medaka gut exposed to silver and titanium dioxide nanoparticles.

Nanomaterials are in general use in a broad range of industries. However, there are concerns that their intense use leads to heavy damage to the aquatic environment, and their discharge harms many aquatic organisms. N-Glycans are widely distributed in eukaryotic organisms and are intimately involved in most life phenomena. However, little is known about N-glycans in aquatic organisms exposed to nanomaterials. In this study, we investigated how nanomaterials affect N-glycans in the gut of adult female medaka. We found that silver nanoparticles exposure had little effect on gut N-glycans, whereas titanium dioxide nanoparticles (TiO2NPs) exposure increased the relative levels of several N-glycans in comparison with control. Structural analysis showed high levels of N-glycans of the high-mannose type, of which five N-glycans were free N-glycans with one ß-N-acetylglucosamine residue on the reducing end. The levels of free N-glycans are closely related to protein quality control in the endoplasmic reticulum and cytosol. Our results suggest that TiO2NPs exposure increases the levels of misfolded glycoproteins, resulting in generation of considerable amounts of free N-glycans. Our findings also suggest that TiO2NPs exposure suppresses cytosolic α-mannosidase trimming. This study provides new evidence for the effect of TiO2NPs on medaka gut from the aspect of environmental glycobiology.

Preparation of neocarzinostatin apoprotein mutants and the randomized library on the chromophore-binding cavity.

W39F, F52Y, S98G, S98A, and S98C mutants of the neocarzinostatin apoprotein (apo-NCS) were newly prepared and investigated their physicochemical properties. The circular dichroism (CD) spectra of F78W, F52Y, S98A, S98G, S98C were superimposable with that of wild type 1R49 protein although the minor spectral change seemed to be in the ellipticity of W39F. The results suggest that position 52, 78, and 98 involving natural chromophore binding do not play a major role in the inducing overall structural changes of the protein. Conversely, the position 39 would be affected slightly. Ethidium bromide (EtdBr) binding to mutants was also evaluated by the monitoring of total fluorescence intensity and fluorescence polarization (FP). The observed dissociation constant in the FP study was 4.4 microM for wild type, 2.2 microM for S98A, 1.3 microM for S98G, 9.7 microM for S98C, respectively. When S98G and F52Y, the calculated maximum change of the total fluorescence intensity was increased, suggesting that the EtdBr binding to S98G or F52Y were slightly improved compared with the wild type. Then, a total of 14 amino acids randomly substituted phage displayed library of apo-NCS was successfully prepared, because substitution of the amino acid structured the chromophore-binding cavity were not change the overall structural features. The phages which bound glycyrrhetic acid conjugated bovine serum albumin were enriched from this library using phage display technique as the pilot experiments. Although more precision investigation still needs, it should be possible to select variants that have new functions not found in nature.