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Biomolecules 2013, 3(1), 108-123; doi:10.3390/biom3010108

Application of Metabolic 13C Labeling in Conjunction with High-Field Nuclear Magnetic Resonance Spectroscopy for Comparative Conformational Analysis of High Mannose-Type Oligosaccharides

1
Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama Myodaiji, Okazaki 444-8787, Japan
2
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
3
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
4
The Glycoscience Institute, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan
5
Systems and Structural Biology Center, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan
6
GLYENCE Co., Ltd./2-22-8 Chikusa, Chikusa-ku, Nagoya 464-0858, Japan
These authors contributed equally to this work.
ǂ
Present address: Y.K., Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
*
Author to whom correspondence should be addressed.
Received: 19 December 2012 / Revised: 10 January 2013 / Accepted: 15 January 2013 / Published: 25 January 2013
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Abstract

High mannose-type oligosaccharides are enzymatically trimmed in the endoplasmic reticulum, resulting in various processing intermediates with exposed glycotopes that are recognized by a series of lectins involved in glycoprotein fate determination in cells. Although recent crystallographic data have provided the structural basis for the carbohydrate recognition of intracellular lectins, atomic information of dynamic oligosaccharide conformations is essential for a quantitative understanding of the energetics of carbohydrate–lectin interactions. Carbohydrate NMR spectroscopy is useful for characterizing such conformational dynamics, but often hampered by poor spectral resolution and lack of recombinant techniques required to produce homogeneous glycoforms. To overcome these difficulties, we have recently developed a methodology for the preparation of a homogeneous high mannose-type oligosaccharide with 13C labeling using a genetically engineered yeast strain. We herein successfully extended this method to result in the overexpression of 13C-labeled Man9GlcNAc2 (M9) with a newly engineered yeast strain with the deletion of four genes involved in N-glycan processing. This enabled high-field NMR analyses of 13C-labeled M9 in comparison with its processing product lacking the terminal mannose residue ManD2. Long-range NOE data indicated that the outer branches interact with the core in both glycoforms, and such foldback conformations are enhanced upon the removal of ManD2. The observed conformational variabilities might be significantly associated with lectins and glycan-trimming enzymes. View Full-Text
Keywords: nuclear magnetic resonance spectroscopy; high mannose-type oligosaccharide; stable isotope labeling; Saccharomyces cerevisiae; nuclear Overhauser effect nuclear magnetic resonance spectroscopy; high mannose-type oligosaccharide; stable isotope labeling; Saccharomyces cerevisiae; nuclear Overhauser effect
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MDPI and ACS Style

Kamiya, Y.; Yanagi, K.; Kitajima, T.; Yamaguchi, T.; Chiba, Y.; Kato, K. Application of Metabolic 13C Labeling in Conjunction with High-Field Nuclear Magnetic Resonance Spectroscopy for Comparative Conformational Analysis of High Mannose-Type Oligosaccharides. Biomolecules 2013, 3, 108-123.

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