title: 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.
authors.affiliation: Department of Molecular Biology, University of Wyoming, Laramie 82071, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Hollister
authors.forename: Eckart
authors.initials: E
authors.lastname: Grabenhorst
authors.forename: Manfred
authors.initials: M
authors.lastname: Nimtz
authors.forename: Harald
authors.initials: H
authors.lastname: Conradt
authors.forename: Donald L
authors.initials: DL
authors.lastname: Jarvis
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism / Q000821:virology; D002469:Cell Separation; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D008361:Mannosidases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008745:Methylation; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics* / Q000821:virology; D019076:Transgenes; D043323:alpha-Mannosidase
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D017351:N-Acetylglucosaminyltransferases; C063313:alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase; D005982:Glutathione Transferase; D008361:Mannosidases; D043323:alpha-Mannosidase