Identification of genes encoding N-glycan processing beta-N-acetylglucosaminidases in Trichoplusia ni and Bombyx mori: Implications for glycoengineering of baculovirus expression systems.
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 beta-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 beta-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 beta-N-acetylglucosaminidase genes isolated from these two species indicated that each encoded a product that was, indeed, more similar to processing beta-N-acetylglucosaminidases than degradative or chitinolytic beta-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 beta-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.
Geisler
Christoph
C
Dept. of Molecular Biology, University of Wyoming, 1000 E. University Avenue, Laramie, WY 82071, USA.
Jarvis
Donald L
DL
D000118:Acetylglucosaminidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D029721:Drosophila Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D009036:Moths / Q000235:genetics* / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D013379:Substrate Specificity
D029721:Drosophila Proteins; D011134:Polysaccharides; D000118:Acetylglucosaminidase; C509916:fdl protein, Drosophila