Glycoengineering of human IgG1-Fc through combined yeast expression and in vitro chemoenzymatic glycosylation.
The presence and precise structures of the glycans attached at the Fc domain of monoclonal antibodies play an important role in determining antibodies' effector functions such as antibody-dependent cell cytotoxicity (ADCC), complement activation, and anti-inflammatory activity. This paper describes a novel approach for glycoengineering of human IgG1-Fc that combines high-yield expression of human IgG1-Fc in yeast and subsequent in vitro enzymatic glycosylation, using the endoglycosidase-catalyzed transglycosylation as the key reaction. Human IgG1-Fc was first overproduced in Pichia pastoris. Then the heterogeneous yeast glycans were removed by Endo-H treatment to give the GlcNAc-containing IgG1-Fc as a homodimer. Finally, selected homogeneous glycans were attached to the GlcNAc-primer in the IgG1-Fc through an endoglycosidase-catalyzed transglycosylation, using sugar oxazolines as the donor substrates. It was found that the enzymatic transglycosylation was efficient with native GlcNAc-containing IgG1-Fc homodimer without the need to denature the protein, and the reaction could proceed to completion to give homogeneous glycoforms of IgG1-Fc when an excess of oligosaccharide oxazolines was used as the donor substrates. The binding of the synthetic IgG1-Fc glycoforms to the FcgammaIIIa receptor was also investigated. This novel glycoengineering approach should be useful for providing various homogeneous, natural or synthetic glycoforms of IgG1-Fc for structure-function relationship studies, and for future clinical applications.
Wei
Yadong
Y
Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Li
Cishan
C
Huang
Wei
W
Li
Bing
B
Strome
Scott
S
Wang
Lai-Xi
LX
D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry
D015815:Cell Adhesion Molecules; D017931:DNA Primers; C106607:FCGBP protein, human; D007074:Immunoglobulin G; D011134:Polysaccharides; D011994:Recombinant Proteins
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