Carbohydrate Engineering for Generating Sialylated Glycoproteins in Insect Cells
Michael J. Betenbaugh
Johns Hopkins University

Insect cells are used to generate of a variety of biotechnology products. Many of the most valuable biotechnology products are glycoproteins that include oligosaccharides attached to the protein at particular amino acids. These oligosaccharides can be extremely important to the therapeutic activity of biopharmaceuticals in humans.  Unfortunately, processing in insect cells yields glycoproteins with different oligosaccharides from those generated by human and other mammalian hosts. While mammalian cells produce complex oligosaccharides often terminating in the sugar, sialic acid, insect cells typically generate simplistic oligosaccharides terminating in mannose or N-acetylglucosamine.  Since these covalently-attached carbohydrates can significantly affect a protein's structure, stability, biological activity, and  in vivo circulatory half-life, the objective of this project is to manipulate carbohydrate-processing pathways in insect cells to generate complex sialylated glycoproteins. The sialylation reaction involves the addition of a donor substrate, cytidine monophosphate-sialic acid (CMP-SA) onto a specific acceptor carbohydrate via an enzymatic reaction in the Golgi apparatus. Evaluation of the nucleotide-sugars in Sf-9 and High Five insect cells grown in serum-free medium revealed negligible levels of CMP-SA to suggest a limitation in the donor substrate levels.   Consequently, the genes responsible for generating CMP-SA must be engineered into insect cells using metabolic engineering strategies.  Unfortunately, the mammalian genes were unknown so bioinformatics approaches were implemented to identify putative human genes based on known bacterial sequences.  When the enzymes encoded by these genes are expressed with baculovirus vectors, sialic acids and the donor substrate (CMP-SA) can be generated in insect cells at levels exceeding those typically observed in mammalian cell lines.  Furthermore, the enzymes have broad substrate specificities which may allow for the generation of glycoproteins with different sialic acid termini.  In addition to producing the donor substrate, CMP-SA, the correct acceptor carbohydrate acceptors must be generated in insect cells.  Collaborating scientists are generating correct carbohydrate acceptors by expressing favorable glycosyltransferase enzymes such as galactose transferase and by evaluating methods to inhibit unfavorable cleavage reactions.  The completion of the sialylation reaction will be obtained by expressing the catalyzing sialyltransferase enzyme in the presence of these correct acceptor and donor substrates.  Engineering the sialylation reaction into insect cells may increase the value of insect cell-derived products as vaccines, therapeutics, and diagnostics.   Humanizing insect cells and other recombinant DNA hosts will make expression systems more versatile and may ultimately lower biotechnology production costs. In the future a particular host may be chosen based on its efficiency of production rather than its capacity to generate particular oligosaccharide profiles.

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