title: Metabolic functionalization of recombinant glycoproteins.
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.
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720, USA.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Luchansky
authors.forename: Sulabha
authors.initials: S
authors.lastname: Argade
authors.forename: Bradley K
authors.initials: BK
authors.lastname: Hayes
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D015394:Molecular Structure; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001386:Azides; D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D019158:N-Acetylneuraminic Acid