Metabolic Functionalization of Recombinant Glycoproteins

2004/08/26 by Sarah J. Luchansky, Sulabha Argade, Bradley K. Hayes +1 · 25 citations
Biochemistry, Genetics and Molecular Biology · Chemistry · Medicine · #Glycosylation and Glycoproteins Research #Carbohydrate Chemistry and Synthesis #Monoclonal and Polyclonal Antibodies Research

paper · doi:10.1021/bi049274f

Abstract

Glycoproteins are essential for cellular communication and are the most rapidly growing class of therapeutic agents. Chemical modification of glycoproteins has been employed to improve their in vivo efficacy or to label them for detection. Methods for the controlled derivatization of glycoproteins are presently limited by the repertoire of natural amino acid side chain and carbohydrate functionalities. Here, we use metabolic oligosaccharide engineering to introduce a bioorthogonal functional group, the azide, into cellular and recombinant glycoproteins for subsequent chemical elaboration via Staudinger ligation. As most therapeutic glycoproteins are sialylated and require this saccharide for optimal pharmacokinetics, we targeted sialic acid as a host for azides using N-azidoacetylmannosamine (ManNAz) as a biosynthetic precursor. Metabolic conversion of ManNAz to N-azidoacetylsialic acid (SiaNAz) within membrane-bound and secreted glycoproteins was quantified in a variety of cell types. SiaNAz was found to comprise between 4% and 41% of total sialosides, depending on the system. Metabolic labeling of recombinant interferon-beta and GlyCAM-Ig was achieved, demonstrating the utility of the method for functionalizing N-linked and O-linked glycoproteins of therapeutic interest. More generally, the generation of recombinant glycoproteins containing chemical handles within their glycans provides a means for studying their behavior and for improving their in vivo efficacy.

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• Selective enrichment and identification of azide-tagged cross-linked peptides using chemical ligation and mass spectrometry. [europepmc]
• Bioorthogonal Chemistry-A Covalent Strategy for the Study of Biological Systems. [europepmc]
• Evaluation of alkyne-modified isoprenoids as chemical reporters of protein prenylation. [europepmc]
• Metabolism of diazirine-modified N-acetylmannosamine analogues to photo-cross-linking sialosides. [europepmc]
• Metabolic oligosaccharide engineering with N-Acyl functionalized ManNAc analogs: cytotoxicity, metabolic flux, and glycan-display considerations. [europepmc]
• Imaging the sialome during zebrafish development with copper-free click chemistry. [europepmc]
• Reversible labeling of native and fusion-protein motifs. [europepmc]
• Applications of azide-based bioorthogonal click chemistry in glycobiology. [europepmc]
• Experimental Design Considerations for In Vitro Non-Natural Glycan Display via Metabolic Oligosaccharide Engineering. [europepmc]
• Quantifying the Efficiency of N -Phenyl-D-mannosamine to Metabolically Engineer Sialic Acid on Cancer Cell Surface. [europepmc]
• Chemoenzymatic exchange of phosphopantetheine on protein and peptide. [europepmc]
• Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusion. [europepmc]
• Metabolic labeling of HIV-1 envelope glycoprotein gp120 to elucidate the effect of gp120 glycosylation on antigen uptake. [europepmc]
• Cell Line-, Protein-, and Sialoglycosite-Specific Control of Flux-Based Sialylation in Human Breast Cells: Implications for Cancer Progression. [europepmc]
• Metabolic Glycoengineering with Azide- and Alkene-Modified Hexosamines: Quantification of Sialic Acid Levels. [europepmc]
• Resources and Methods for Engineering "Designer" Glycan-Binding Proteins. [europepmc]
• Real-time monitoring of the sialic acid biosynthesis pathway by NMR. [europepmc]
• A framework for the simulation of individual glycan coordinates to analyze spatial relationships within the glycocalyx. [europepmc]
• GlycoRNA research: from unknown unknowns to known unknowns. [europepmc]
• Endothelial-erythrocyte glycocalyx exchange enables liquid biopsies of endothelial function. [europepmc]
• Metabolic Probing of Sialylated Glycoconjugates with Fluorine-Selenol Displacement Reaction (FSeDR) [europepmc]

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