Identification of genes encoding N‐glycan processing β‐N‐acetylglucosaminidases in Trichoplusia ni and Bombyx mori: Implications for glycoengineering of baculovirus expression systems

Abstract

Glycoproteins produced by non-engineered insects or insect cell lines characteristically bear truncated, paucimannose N-glycans in place of the complex N-glycans produced by mammalian cells. A key reason for this difference is the presence of a highly specific N-glycan processing β-N-acetylglucosaminidase in insect, but not in mammalian systems. Thus, reducing or abolishing this enzyme could enhance the ability of glycoengineered insects or insect cell lines to produce complex N-glycans. Of the three insect species routinely used for recombinant glycoprotein production, the processing β-N-acetylglucosaminidase gene has been isolated only from Spodoptera frugiperda. Thus, the purpose of this study was to isolate and characterize the genes encoding this important processing enzyme from the other two species, Bombyx mori and Trichoplusia ni. Bioinformatic analyses of putative processing β-N-acetylglucosaminidase genes isolated from these two species indicated that each encoded a product that was, indeed, more similar to processing β-N-acetylglucosaminidases than degradative or chitinolytic β-N-acetylglucosaminidases. In addition, over-expression of each of these genes induced an enzyme activity with the substrate specificity characteristic of processing, but not degradative or chitinolytic enzymes. Together, these results demonstrated that the processing β-N-acetylglucosaminidase genes had been successfully isolated from Trichoplusia ni and Bombyx mori. The identification of these genes has the potential to facilitate further glycoengineering of baculovirus-insect cell expression systems for the production of glycosylated proteins.

Substrate Specificities and Intracellular Distributions of Three N-glycan Processing Enzymes Functioning at a Key Branch Point in the Insect N-Glycosylation Pathway

“…The reactions were incubated at 37°C for 15 min and frozen until separated by RP-HPLC, which was performed essentially as described previously (34). For pH optima, cofactor requirement, and acceptor substrate specificity determinations, the enzymes were added to achieve ϳ50% conversion (compared with MM for Sf-GNT-I and MGn for Sf-GNT-II), and efficiencies were calculated by dividing the amount of product formed compared with the amount of input glycan compared with the reference conversion.…”

Modifying an Insect Cell N-Glycan Processing Pathway Using CRISPR-Cas Technology

“…An unusual ß-N-acetylglucosaminidase, designated fused lobes (FDL) for its phenotype in mutant flies 36 , catalyzes the trimming reaction that yields paucimannosidic N-glycan products in insect cells [37][38][39][40] . FDL is a resident Golgi enzyme with a neutral pH optimum and a high level of substrate specificity, as it only removes the terminal, non-reducing N-acetylglucosamine residue from the α1,3-branch of GlcNAcMan 3 GlcNAc 2 or GlcNAc 2 Man 3 GlcNAc 2 37-41 .…”

CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus–insect cell system

“…Nevertheless, this genetic capacity is not expressed in Sf9 or High Five cells. Insect cells also have a trimming enzyme, absent in mammalian cells, which antagonizes N-glycan elongation (18)(19)(20)(21). This enzyme, which is a specific, processing β-N-acetylglucosaminidase called fused lobes (FDL), removes a terminal N-acetylglucosamine residue from trimmed N-glycanprocessing intermediates.…”