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Paramagnetic-assisted NMR Analyses of Conformational Dynamics of Gangliosides
Ying Zhang,Takumi Yamaguchi,Sayoko Yamamoto,Yoshinori Uekusa,Koichi Kato 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
For better understanding the molecular basis of the mechanism underlying oligosaccharides functions, it is quite desirable to gain detail information on their conformational dynamics in solution. However, the conformational analysis of oligosaccharides is still a remaining challenge, because of their branched covalent structures and dynamic conformational multiplicities, which hinder conventional analytical methods. For conformational characterization of flexible oligosaccharides, their structures should not be dealt with as a single well-defined global free energy minimum but as an ensemble of low energy conformers. Hence, we have been developing a methodology for evaluating a dynamic ensemble of oligosaccharide conformations by employing paramagnetic-assisted NMR methods based on paramagnetic effects such as pseudocontact shift (PCS) and paramagnetic relaxation enhancement (PRE) in conjunction with molecular dynamics (MD) simulations [1,2]. We applied this approach to the conformational dynamics analyses of branched GM2 tetrasaccharide, βGalNAc-(1-4)-[αNeu5Ac-(2-3)]-βGal-(1-4)-βGlc, which share the common core structure of gangliosides forming an integral part of cellular membranes. We synthesized the GM2 tetrasaccharide covalently attached to a lanthanide chelating-tag (Fig. 1). Upon complexation with paramagnetic lanthanide ions, the tagged sugar exhibited NMR spectral changes due to PCSs. The observed PCS values were in excellent agreement with those back-calculated from the 3D ensemble models derived from the MD calculations. In addition, we have developed spin-labeling method of gangliosides for their NMR analyses in amphipathic environments. These methodologies open a new prospect for conformational analyses of dynamic structures of gangliosides toward decoding glycocodes from the 3D structural aspects.
Kotaro Yanagi,Yukiko Kamiya,Toshihiko Kitajima,Takumi Yamaguchi,Yasunori Chiba,Koichi Kato 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
High-mannose-type oligosaccharides are enzymatically trimmed in the endoplasmic reticulum, giving rise to various processing intermediates with exposure of specific glycotopes that are recognized by a series of lectins involved in glycoprotein-fate determination in cells. Atomic information of dynamic oligosaccharide conformations is essential for a quantitative understanding of energetics of the carbohydrate-lectin interactions. Although carbohydrate NMR spectroscopy is useful for characterizing such conformational dynamics, it is often hampered by poor spectral resolution and the lack of recombinant technique to produce homogeneous glycoforms. To overcome these difficulties, we have recently developed a methodology for preparation of a homogeneous high-mannose-type oligosaccharide with 13C labeling using genetically engineered yeast strain. We herein successfully extended this method to overexpression of 13C-lebeled Man9GlcNAc2 (M9) using a newly engineered yeast strain with deletion of four genes involved in N-glycan processing. This enabled high-field NMR analyses of 13C-labeld M9 in comparison with its processing product M8B, which lacks the terminal mannose residue ManD2. Long-range NOE data indicated that the outer branches can interact with the core in both glycoforms and such foldback conformations are enhanced upon the removal of ManD2. The observed conformational variabilities might be associated with the lectins and the glycan-trimming enzymes.
Yamazaki, Takeshi,Takeda, Hisato,Hagiya, Koichi,Yamaguchi, Satoshi,Sasaki, Osamu Asian Australasian Association of Animal Productio 2018 Animal Bioscience Vol.31 No.10
Objective: Because lactation periods in dairy cows lengthen with increasing total milk production, it is important to predict individual productivities after 305 days in milk (DIM) to determine the optimal lactation period. We therefore examined whether the random regression (RR) coefficient from 306 to 450 DIM (M2) can be predicted from those during the first 305 DIM (M1) by using a RR model. Methods: We analyzed test-day milk records from 85,690 Holstein cows in their first lactations and 131,727 cows in their later (second to fifth) lactations. Data in M1 and M2 were analyzed separately by using different single-trait RR animal models. We then performed a multiple regression analysis of the RR coefficients of M2 on those of M1 during the first and later lactations. Results: The first-order Legendre polynomials were practical covariates of RR for the milk yields of M2. All RR coefficients for the additive genetic (AG) effect and the intercept for the permanent environmental (PE) effect of M2 had moderate to strong correlations with the intercept for the AG effect of M1. The coefficients of determination for multiple regression of the combined intercepts for the AG and PE effects of M2 on the coefficients for the AG effect of M1 were moderate to high. The daily milk yields of M2 predicted by using the RR coefficients for the AG effect of M1 were highly correlated with those obtained by using the coefficients of M2. Conclusion: Milk production after 305 DIM can be predicted by using the RR coefficient estimates of the AG effect during the first 305 DIM.
Maho Yagi-Utsumi,Pornthip Boonsri,Yoshiki Yamaguchi,Koichi Kato 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
Glycolipids offer recognition sites of a variety of proteins such as antibodies and microbial toxins, thereby playing physiological and pathological roles on cell membrane surfaces.Therefore, these glycolipid-protein interaction systems could be potential therapeutic targets for various diseases, including bacterial infections and neurodegenerative disorders [1]. To elucidate the underlying mechanisms of molecular recognition between glycolipids and proteins, we investigatedthe interaction between sarcotoxin IA and lipid Aas a model system to characterize conformational transitions and intermolecular interactions of the membrane-binding peptides promoted on glycolipids. Lipid A is a major component of the outer membrane of Gram-negative bacteria and can be a recognition target in the innate immune system. This molecule can also serve as targets of sarcotoxin IA, which is a 39-residue cecropin-type antibacterial peptide from Sarcophaga peregrina. In order to obtain structural information at atomic level, we expressed sarcotoxin IA peptides in Escherichia coli to achieve 13C- and15N-labeling for detailed NMR analyses. We observed NMR spectral changes of sarcotoxin IA upon interacting with lipid A, which was embeded in micelles composed of dodecylphosphocholine. Our spectroscopic data revealed that the N-terminal segment of sarcotoxin IA was converted from random- coil to an amphiphilic α-helix upon specific binging to lipid A. By inspecting chemical shift perturbation data, we successfully identified key lysine residues involved in interaction with lipid A and consequent antibacterial activity.