Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines

Abstract

In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic enzymes responsible for generating the essential donor sugar nucleotide, CMP- N -acetylneuraminic acid, required for sialylation. Inhibition of N -acetylglucosaminidase has also been applied to alter N-glycan processing in insect cells. This review summarizes current knowledge on N-glycan processing in lepidopteran insect cell lines, and recent progress in glycoengineering lepidopteran insect cells to produce glycoproteins containing complex N-glycans.

The molecular structure of the Toll-like receptor 3 ligand-binding domain

“…Of the 15 predicted N-glycosylation sites, 11 are observed to be glycosylated (Table 1). Because TLR3-ECD protein was expressed in insect cells, the glycans lack the high mannose content characteristic of N-linked carbohydrate in mammalian cells (36). Two of the observed glycans are located on the ␤-sheet of the solenoid, at Asn-252 (LRR 9) and Asn-413 (LRR 15), a surface that in other LRR proteins is involved in ligand binding and recognition (10,37).…”

Design of homogeneous, monopegylated erythropoietin analogs with preserved in vitro bioactivity

“…Epo expressed in mammalian cells is extensively glycosylated with highly branched, complex sugars and the commercial form of the protein is selected for high sialic acid content to maximize its half-life [5]. In contrast, insect cells attach only simple sugars to proteins and do not appear to add terminal sialic acids to N-linked sugars [29]. These glycosylation differences may account, at least in part, for the rapid clearance of BV Epo in rats.…”

Structural Flexibility of the Macrophage Dengue Virus Receptor CLEC5A

“…This array included a wide range of N-glycan-related probes. Among these were paucimannose N-glycans with or without the ␣1,6-linked core fucose typically found on insect-derived glycoproteins and an N-glycan with the alternative ␣1,3-linked core fucosylation found in insects (45). Also included were high mannose-type N-glycans, many of which are common to mammals and insects.…”