Humanizing Non-Mammalian Glycosylation Systems

The post-translational modifications of proteins in insect cells differ from those observed in mammalian cells. In insect cells, the N-linked oligosaccharides attached to proteins are truncated and terminate in mannose (Man) or N-acetylglucosamine (GlcNAc) instead of sialic acid (SA, Neu5Ac) or galactose termini as observed in mammalian glycoproteins. This results in differences in biological and pharmacokinetic activities, and often allergenic reactions to humans when used as a therapeutic.

Some of the reasons for the inability of insect cells to produce glycoproteins with advanced mammalian-type glycosylational modifications are:

1. Lack of sufficient glycosyltransferases
2. Inability to produce CMP-N-acetylneuraminic acid, the precursor nucleotide sugar
3. Presence of enzymes such as ß-N-acetylglucosaminidase which removes GlcNAc from the Mana(1,3) branch

(click image to enlarge)

As can be seen in the figure, the initial steps of glycosylation in insect cells are similar to those in mammalian cells. After these steps, the GlcNacT-II in mammalian cells adds GlcNAc to the oligosaccharide. In contrast, a very low amount of GlcNAcT-II activity has been observed in insect cells. Also, the ß-N-acetylglucosaminidase activity observed in insect cells removes GlcNAc from the Mana(1,3) branch. At times, additional Man groups might be removed due to subsequent mannosidase activity. As a result, insect cells generate paucimannosidic and hybrid N-glycan structures. Later in the pathway, in mammalian cells, the presence of galactosyltransferase and sialyltransferase leads to the addition of galactose and sialic acid respectively. However, these steps do not take place in insect cells due to the lack of the above mentioned enzymatic activities. The insect cells also lack the UDP-GlcNAc-2-epimerase/ ManNAc kinase, required for converting UDP-GlcNAc to ManNAc-6-phosphate. In addition sialic acid synthase and CMP-sialic acid synthase activities needed for the synthesis of the donor nucleotide CMP-Neu5Ac are also most often absent. These are essential for the sialylation the glycan structure.

Glycosylation determines the functional properties and the in vivo clearance rates of the glycoproteins. Thus developing a technology that will enable production of mammalian-type glycoproteins from insect cells is highly desirable.

Our lab has applied metabolic engineering approach to achieve this goal. The glycosyltransferases have been incorporated into the insect cells. This has enabled the production of fully galactosylated and some sialylated glycoproteins from insect cell lines. In addition, genes responsible for the metabolic pathways involved in the generation of all the necessary nucleotide sugar precursors has enabled insect cells to generate full sialylation (Lawrence et al., 2001).