Functional neoglycopeptides: synthesis and characterization of a new class of MUC1 glycoprotein models having core 2-based O-glycan and complex-type N-glycan chains.
An efficient protocol for the construction of MUC1-related glycopeptide analogues having complex O-glycan and N-glycan chains was established by integrating chemical and enzymatic approaches on the functional polymer platforms. We demonstrated the feasibility of sortase A-mediated ligation between two glycopeptide segments by tagging with signal peptides, LPKTGLR and GG, at each C- or N-terminal position. Structural analysis of the macromolecular N,O-glycopeptides was performed by means of ESI-TOFMS (MS/MS) equipped with an electron-captured dissociation device. Immunological assay using MUC1 glycopeptides synthesized in this study revealed that N-glycosylation near the antigenic O-glycosylated PDTR motif did not disturb the interaction between the anti-MUC1 monoclonal antibody and this crucial O-glycopeptide moiety. NMR study indicated that the N-terminal immunodominant region [Ala-Pro-Asp-Thr(O-glycan)-Arg] forms an inverse gamma-turn-like structure, while the C-terminal region composed of N-glycopeptide and linker SrtA-peptide was proved to be an independently random structure. These results indicate that the bulky O- and N-glycan chains can function independently as disease-relevant epitopes and ligands for carbohydrate-binding proteins, when both are combined by an artificial intervening peptide having a possible effect of separating N- and C-terminal regions. The present strategy will greatly facilitate rapid synthesis of multiply functionalized complex neoglycopeptides as new types of convenient tools or models for the investigation of thhe structure-function relationship of various glycoproteins and development of novel class glycopeptide-based biopharmaceuticals, drug delivery systems, and biomedical materials.
Matsushita
Takahiko
T
Division of Advanced Chemical Biology, Graduate School of Life Science, Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University, N21, W11, Kita-ku, Sapporo 001-0021, Japan.
Sadamoto
Reiko
R
Ohyabu
Naoki
N
Nakata
Hideki
H
Fumoto
Masataka
M
Fujitani
Naoki
N
Takegawa
Yasuhiro
Y
Sakamoto
Takeshi
T
Kurogochi
Masaki
M
Hinou
Hiroshi
H
Shimizu
Hiroki
H
Ito
Takaomi
T
Naruchi
Kentarou
K
Togame
Hiroko
H
Takemoto
Hiroshi
H
Kondo
Hirosato
H
Nishimura
Shin-Ichiro
S
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry; D001667:Binding, Competitive / Q000276:immunology; D055162:Biocatalysis; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry; D006023:Glycoproteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D018396:Mucin-1 / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D011134:Polysaccharides / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry* / Q000276:immunology; D013211:Staphylococcus aureus / Q000201:enzymology; D053719:Tandem Mass Spectrometry
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D006023:Glycoproteins; C060192:MUC1 protein, human; D018396:Mucin-1; D011134:Polysaccharides; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases