title: Chemical Synthesis of Glycoproteins with the Specific Installation of Gradient-Enriched15N-Labeled Amino Acids for Getting Insights into Glycoprotein Behavior.
abstract: Elucidating the effects of oligosaccharides on glycoprotein properties, such as local conformational changes, stability, and dynamics, has still been challenging. In this paper, a novel partial15N-labeling method for the amide backbone of a synthetic glycoprotein is proposed. Using solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL), thirteen15N-labeled amino acids were inserted at specific positions of the protein backbone, while intentionally varying the enrichment of15N atoms. This idea discriminated even the same type of amino acid based on the intensities of1H-15N HSQC signals, combined with classic homonuclear TOCSY and NOESY methods, thus allowing for understanding the dynamics of the local conformation of a synthetic homogeneous glycoprotein. Results suggested that the attachment of an oligosaccharide of either a bi-antennary complex-type or a high-mannose-type did not disturb protein conformation. However, T1values suggested that the oligosaccharide influenced dynamics at the local conformation. Temperature-varied circular dichroism (CD) spectra and T1values clearly indicated that oligosaccharides appeared to inhibit protein fluctuation or, in other words, stabilize protein structure. This insight into oligosaccharide behavior suggests some further effects on binding affinity between a glycoprotein and its receptor.
authors.affiliation: Deprtment of Chemistry, Osaka University, 1-1, Machikaneyama, Toyonaka, 560-0043, Japan.|Current address: Department of Chemistry, Da Nang University of Education, the University of Da Nang, Vietnam.
authors.forename: Nguyen
authors.initials: N
authors.lastname: Minh Hien
authors.affiliation: Deprtment of Chemistry, Osaka University, 1-1, Machikaneyama, Toyonaka, 560-0043, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Izumi
authors.affiliation: Bruker BioSpin K.K., 3-9, Moriya-cho, Kanagawa-ku, Yokohama, Kanagawa <postCode/221-0022, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Sato
authors.affiliation: Deprtment of Chemistry, Osaka University, 1-1, Machikaneyama, Toyonaka, 560-0043, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Okamoto
authors.affiliation: Deprtment of Chemistry, Osaka University, 1-1, Machikaneyama, Toyonaka, 560-0043, Japan.
authors.forename: Yasuhiro
authors.identifier: http://orcid.org/0000-0002-6656-2394
authors.initials: Y
authors.lastname: Kajihara
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006020:Glycopeptides / Q000032:analysis; D006023:Glycoproteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D008358:Mannose / Q000737:chemistry; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D060327:Solid-Phase Synthesis Techniques; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000596:Amino Acids; D006020:Glycopeptides; D006023:Glycoproteins; D009587:Nitrogen Isotopes; C000615243:Nitrogen-15; D009844:Oligosaccharides; D008358:Mannose
keywords: NMR spectroscopy; glycoprotein synthesis; glycoproteins; oligosaccharides; stable isotope labeling