Drosophila Transgenic Lines Expressing the Mammalian N-acetylglucosaminyltransferase II and β 1,4-galactosyltransferase
Abstract
Nlinked glycosylated proteins play a role in different biological processes. The initial steps of protein modifications with glycan in eukaryotic cells are identical. Both mammalian and insect N-glycosylation pathway share the intermediate complex, (For review see: Jarvis 2003; Rendić et al., 2008). The N-glycosylation process starts with trimming three glucose residues from the high mannose structure "Glu3Man9GlcNac2-N-Asn", followed by further removing of five mannose residues. One residue of N-acetylglocusamine is added to the structure and the common intermediate structure is achieved by trimming two mannose residues. In insect cells removal of further residues is continue by trimming the terminal N-acetylglucosamine residue forming the major N-glycan product in insect cells, the paucimannose, "Man3GlcNAc2-N-Asn" (Altmann et al., 1995). However, in some cases, only extremely low levels of terminal glycosyltransferase activities have been detected in insect cells (Jarvis, 2003). In contrast, the mammalian glycosylation process tends to elongate this intermediate structure and producing more complicated complex of N-glycans with terminal sialic acids (Beyer et al., 1979).
The differences in glycosylation pathways between insect and mammalian cells limit the ability of insect-based baculovirus expression systems to produce broad spectrum varieties of N-glycoproteins. In early attempts to overcome such problematic differences, lepidopteran cells were transformed with mammalian genes to shift the insect glycoslation process toward mammalian pathway. The Spodoptera frugiperda cells (Sf9) were engineered to express the mammalian β1,4-galactosyltransferase (GalT) and α2,6-sialyltransferase (ST6). The resulted cells, Sf4GalT/ST6, produced a recombinant glycoprotein with mono-antennary structure with only α1,3 arm elongated (Hollister and Jarvis, 2001; Hollister et al., 2002). Further transformion with N-acetylglucoseaminyl-transferase II gene created SfSWT-1 cells which produced a recombinant glycoprotein with bi-antennary sialylated N-glycans (Hollister et al., 2002).
Our goal is to apply the knowledge gained from engineered insect cells to “humanize” the glycoprotein processing pathway in intact insects. Here we report the generation of transgenic Drosophila lines expressing the human Nacetylglucosaminyltransferase II and a bovine β1,4-galactosyltransferase.
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