Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human β1,2-N-acetylglucosaminyltransferase I

Wagner, Ralf; Liedtke, Steffen; Kretzschmar, Evelyne; Geyer, Hildegard; Geyer, Rudolf; Klenk, Hans‐Dieter

doi:10.1093/glycob/6.2.165

Cited by 81 publications

(58 citation statements)

References 51 publications

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“…It is noteworthy that this enzyme could not hydrolyze Man5Gn, 4 but acted only further "downstream" on MGn and GnGn where it specifically removed GlcNAc-1 (20). Subsequent cell culture experiments in the presence of hex-osaminidase inhibitors (7,11,21) or of recombinant ␤1,4-galactosyltransferase (22,23) corroborated the putative existence of a processing hexosaminidase. It is obvious, therefore, that this enzyme interferes in strategies to produce glycoproteins with human-like glycosylation in insect cells.…”

mentioning

confidence: 77%

“…The most common substitution is the presence of terminal GlcNAc on the ␣1,3-linked mannosyl residue (GlcNAc-1) 3 added by GlcNAc-transferase I, although in a few cases further modifications such as galactose, N-acetylgalactosamine, fucose, and aminoethylphosphonate (i.e. phosphorylethanolamine) have been found linked to this GlcNAc residue (7)(8)(9)(10)(11). The occurrence of larger and sialylated N-glycans has been reviewed elsewhere (1,4).…”

mentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Léonard

Rendić

Rabouille

et al. 2006

Journal of Biological Chemistry

147

152

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Most processed, e.g. fucosylated, N-glycans on insect glycoproteins terminate in mannose, yet the relevant modifying enzymes require the prior action of N-acetylglucosaminyltransferase I. This led to the hypothesis that a hexosaminidase acts during the course of N-glycan maturation. To determine whether the Drosophila melanogaster genome indeed encodes such an enzyme, a cDNA corresponding to fused lobes (fdl), a putative ␤-N-acetylglucosaminidase with a potential transmembrane domain, was cloned. When expressed in Pichia pastoris, the enzyme exhibited a substrate specificity similar to that previously described for a hexosaminidase activity from Sf-9 cells, i.e. it hydrolyzed exclusively the GlcNAc residue attached to the ␣1,3-linked mannose of the core pentasaccharide of N-glycans. It also hydrolyzed p-nitrophenyl-N-acetyl-␤-glucosaminide, but not chitooligosaccharides; in contrast, Drosophila HEXO1 and HEXO2 expressed in Pichia cleaved both these substrates but not N-glycans. The localization of recombinant FDL tagged with green fluorescent protein in Drosophila S2 cells by immunoelectron microscopy showed that this enzyme transits through the Golgi, is present on the plasma membrane and in multivesicular bodies, and is secreted. Finally, the N-glycans of two lines of fdl mutant flies were analyzed by mass spectrometry and reversed-phase high-performance liquid chromatography. The ratio of structures with terminal GlcNAc over those without (i.e. paucimannosidic N-glycans) was drastically increased in the fdldeficient flies. Therefore, we conclude that the fdl gene encodes a novel hexosaminidase responsible for the occurrence of paucimannosidic N-glycans in Drosophila.Insect cells are considered to be an interesting alternative to mammalian, yeast, or bacterial cells for the expression of recombinant proteins (1, 2), because the potentially high levels of expression are associated with the ability to perform many eukaryotic post-translational modifications. Nevertheless, glycoproteins produced in insects and insect cells have been mainly found to carry paucimannosidic N-glycans, i.e. glycans consisting of the pentasaccharide core with or without ␣1,6-and/or ␣1,3-linked fucose (1, 3-5). In addition to the possible presence of the immunogenic core ␣1,3-linked fucose, the lack of complex type N-glycans with complete, sialylated antennae as found on mammalian glycoproteins makes insect cells unsuitable for the production of many therapeutic glycoproteins (6).Only in a few cases have substitutions of the terminal mannose residues of insect N-glycans been described. The most common substitution is the presence of terminal GlcNAc on the ␣1,3-linked mannosyl residue (GlcNAc-1) 3 added by GlcNAc-transferase I, although in a few cases further modifications such as galactose, N-acetylgalactosamine, fucose, and aminoethylphosphonate (i.e. phosphorylethanolamine) have been found linked to this GlcNAc residue (7-11). The occurrence of larger and sialylated N-glycans has been reviewed elsewhere (1, 4). In most instances, howe...

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confidence: 77%

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confidence: 99%

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confidence: 99%

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

Léonard

Rendić

Rabouille

et al. 2006

Journal of Biological Chemistry

147

152

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“…This idea led some investigators to attempt to modify the protein N-glycosylation pathway in baculovirus-infected insect cells by introducing exogenous glycosyltransferase activities into the system (for a general review, see Jarvis et al, 1998a). Wagner et al (1996b) demonstrated that the N-glycans of fowl-plague hemagglutinin expressed in Sf9 cells could be elongated by coexpression of human β1,2-Nacetylglucosaminyltransferase I (GNT-I). These authors used two different recombinant baculoviruses, one expressing the hemagglutinin and the other expressing the human GNT-I gene, to coinfect Sf9 cells.…”

Section: Engineering Glycoprotein Processing Pathways In the Baculovimentioning

confidence: 99%

Glycoproteins from Insect Cells: Sialylated or Not?

Marchal

Jarvis²,

Cacan³

et al. 2001

Biological Chemistry

159

122

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Our growing comprehension of the biological roles of glycan moieties has created a clear need for expression systems that can produce mammalian-type glycoproteins. In turn, this has intensified interest in understanding the protein glycosylation pathways of the heterologous hosts that are commonly used for recombinant glycoprotein expression. Among these, insect cells are the most widely used and, particularly in their role as hosts for baculovirus expression vectors, provide a powerful tool for biotechnology. Various studies of the glycosylation patterns of endogenous and recombinant glycoproteins produced by insect cells have revealed a large variety of O-and Nlinked glycan structures and have established that the major processed O-and N-glycan species found on these glycoproteins are (Galβ1,3)GalNAc-O-Ser/Thr and Man3(Fuc)GlcNAc2-N-Asn, respectively. However, the ability or inability of insect cells to synthesize and compartmentalize sialic acids and to produce sialylated glycans remains controversial. This is an important issue because terminal sialic acid residues play diverse biological roles in many glyco-conjugates. While most work indicates that insect cell-derived glycoproteins are not sialylated, some wellcontrolled studies suggest that sialylation can occur. In evaluating this work, it is important to recognize that oligosaccharide structural determination is tedious work, due to the infinite diversity of this class of compounds. Furthermore, there is no universal method of glycan analysis; rather, various strategies and techniques can be used, which provide gly-cobiologists with relatively more or less precise and reliable results. Therefore, it is important to consider the methodology used to assess glycan structures when evaluating these studies. The purpose of this review is to survey the studies that have contributed to our current view of glycoprotein sialylation in insect cell systems, according to the methods used. Possible reasons for the disagreement on this topic in the literature, which include the diverse origins of biological material and experimental artifacts, will be discussed. In the final analysis, it appears that if insect cells have the genetic potential to perform sialylation of glycoproteins, this is a highly specialized function that probably occurs rarely. Thus, the production of sialylated recombinant glycoproteins in the baculovirus-insect cell system will require metabolic engineering efforts to extend the native protein glycosylation pathways of insect cells.

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“…63 Two strategies have been used to "humanize" glycan structures in insect cells; integrating the missing glycosyltransferases into either the cellular genome 83 or the viral genome. 84 Using the latter approach, we have constructed a new baculovirus expressing GNT-I, GNT-II and β1-4 galactosyltransferase (Cérutti M, et al unpublished data). In order to obtain a stable genetic construct without any duplicated sequences, we chose to direct the gene expression under the control of RNA polymerase II heterologous promoters.…”

Section: Enhancing the Production And Secretion Of Recombinant Antibomentioning

confidence: 99%

Lepidopteran cells, an alternative for the production of recombinant antibodies?

Cérutti

Golay

2012

mAbs

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Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human β1,2-N-acetylglucosaminyltransferase I

Cited by 81 publications

References 51 publications

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

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

Glycoproteins from Insect Cells: Sialylated or Not?

Lepidopteran cells, an alternative for the production of recombinant antibodies?

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