Engineering the Protein N-Glycosylation Pathway in Insect Cells for Production of Biantennary, Complex N-Glycans

Abstract

Insect cells, like other eucaryotic cells, modify many of their proteins by N-glycosylation. However, the endogenous insect cell N-glycan processing machinery generally does not produce complex, terminally sialylated N-glycans such as those found in mammalian systems. This difference in the N-glycan processing pathways of insect cells and higher eucaryotes imposes a significant limitation on their use as hosts for baculovirus-mediated recombinant glycoprotein production. To address this problem, we previously isolated two transgenic insect cell lines that have mammalian beta1,4-galactosyltransferase or beta1,4-galactosyltransferase and alpha2,6-sialyltransferase genes. Unlike the parental insect cell line, both transgenic cell lines expressed the mammalian glycosyltransferases and were able to produce terminally galactosylated or sialylated N-glycans. The purpose of the present study was to investigate the structures of the N-glycans produced by these transgenic insect cell lines in further detail. Direct structural analyses revealed that the most extensively processed N-glycans produced by the transgenic insect cell lines were novel, monoantennary structures with elongation of only the alpha1,3 branch. This led to the hypothesis that the transgenic insect cell lines lacked adequate endogenous N-acetylglucosaminyltransferase II activity for biantennary N-glycan production. To test this hypothesis and further extend the N-glycan processing pathway in Sf9 cells, we produced a new transgenic line designed to constitutively express a more complete array of mammalian glycosyltransferases, including N-acetylglucosaminyltransferase II. This new transgenic insect cell line, designated SfSWT-1, has higher levels of five glycosyltransferase activities than the parental cells and supports baculovirus replication at normal levels. In addition, direct structural analyses showed that SfSWT-1 cells could produce biantennary, terminally sialylated N-glycans. Thus, this study provides new insight on the glycobiology of insect cells and describes a new transgenic insect cell line that will be widely useful for the production of more authentic recombinant glycoproteins by baculovirus expression vectors.

Binding Properties of the N-Acetylglucosamine and High-Mannose N-Glycan PP2-A1 Phloem Lectin in Arabidopsis

“…It is possible, therefore, that PP2-A1-His 6 recognizes phloem sap glycoprotein. Man 3 GlcNAc 2 is also the major processed N-glycan produced by insect cells (Hollister et al, 2002;Tomiya et al, 2004); insects are thus an obvious potential target (see below).…”

The Drosophila fused lobes Gene Encodes an N-Acetylglucosaminidase Involved in N-Glycan Processing

“…One of the main targets in this quest has been to increase the amount of structures carrying a GlcNAc residue linked to the ␣1,3-mannose of the N-glycan core, which would, in turn, allow further elongation of this antenna. To this end, several approaches have been explored, ranging from the use of N-acetylglucosaminidase inhibitors (11) to the expression of mammalian glycosyltransferases capable of transfer of galactose and sialic acid to the nascent antennae to prevent removal of GlcNAc-1 by hexosaminidase(s) (6,25). Although the N-glycan analysis of the Drosophila fdl mutant shows that, at least in the case of whole flies, the complete absence of the enzyme mainly responsible for the occurrence of paucimannosidic structures does not lead to the obvious synthesis of N-glycan structures with mammalian-like extended antennae, the considerable increase in structures carrying GlcNAc-1 indicates that the ablation of FDL expression could prove to be an elegant way toward facilitating the synthesis of mammalian-like N-glycans in insect cells.…”

“…For instance, the heterologous overexpression of mammalian GlcNActransferase I was performed, but, because of the competition with the endogenous hexosaminidase, most of the N-glycans still did not carry GlcNAc-1 (24). Another approach is to overexpress ␤1,4-galactosyltransferase (25), which has the effect of "capping" the substrate for the hexosaminidase, or to inhibit the latter using inhibitors (26). However, the most elegant approach would be to specifically knock down or knock out the hexosaminidase gene.…”

Molecular Cloning and Functional Characterization of a Lepidopteran Insect β4-N-Acetylgalactosaminyltransferase with Broad Substrate Specificity, a Functional Role in Glycoprotein Biosynthesis, and a Potential Functional Role in Glycolipid Biosynthesis

“…In Vivo Function of Tn␤4GalNAcT-The postulated in vivo role of Tn␤4GalNAcT in N-glycoprotein biosynthesis was examined by co-expressing the full-length, untagged enzyme together with a secreted N-glycoprotein, GST-SfManI, which we have used as a reporter in several previous insect N-glycan processing studies (10,72,85,86). Sf9 cells were co-infected with a conventional baculovirus encoding GST-SfManI plus an immediate early recombinant baculovirus encoding either Tn␤4GalNAcT or bovine ␤4GalT-I as a control, and then the model glycoprotein was affinity-purified from each infected culture and analyzed by lectin blotting, as described under "Experimental Procedures."…”