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
| Autor: | Steffen Liedtke, Hans-Dieter Klenk, E. Kretzschmar, Rudolf Geyer, Hildegard Geyer, Ralf Wagner |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Glycan
Glycosylation viruses Molecular Sequence Data Hemagglutinins Viral Hemagglutinin (influenza) Sf9 Spodoptera N-Acetylglucosaminyltransferases Biochemistry Epitope Cell Line chemistry.chemical_compound Polysaccharides Glycosyltransferase Carbohydrate Conformation Animals Humans Cloning Molecular DNA Primers Base Sequence biology biology.organism_classification Molecular biology Recombinant Proteins Carbohydrate Sequence chemistry Influenza A virus Cell culture biology.protein |
| Zdroj: | Glycobiology. 6:165-175 |
| ISSN: | 1460-2423 0959-6658 |
| Popis: | Spodoptera frugiperda (Sf9)-cells differ markedly in their protein glycosylation capacities from vertebrate cells in that they are not able to generate complex type oligosaccharide side chains. In order to improve the oligosaccharide processing properties of these cells we have used baculovirus vectors for expression of human (beta 1,2-N-acetylglucosaminyltransferase I (hGNT-I), the enzyme catalysing the crucial step in the pathway leading to complex type N-glycans in vertebrate cells. One vector (Bac/GNT) was designed to express unmodified GNT-I protein, the second vector (Bac/tagGNT) to express GNT-I protein with a tag epitope fused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virus a protein of about 50 kDa representing hGNT-I was detected with an antiserum directed against the tag epitope. HGNT-I activity was increased at least threefold in lysates of infected cells when N-acetylglucosamine (GlcNAc)-free ovalbumine was used as substrate. To monitor hGNT-I activity in intact Sf9-cells, the glycosylation of coexpressed fowl plague virus hemagglutinin (HA) was investigated employing a galactosylation assay and chromatographic analysis of isolated HA N-glycans. Coexpression of hGNT-I resulted in an at least fourfold increase of HA carrying terminal GlcNAc-residues. The only structure detectable in this fraction was GlcNAcMan3GlcNAc2. These results show that hGNT-I is functionally active in Sf9-cells and that the N-glycans of proteins expressed in the baculovirus/insect cell system are elongated by coexpression of glycosyltransferases of vertebrate origin. Complete complex type oligosaccharide side chains were not observed when hGNT-I was overexpressed, thus supporting the concept that Sf9-cells do not contain glycosyltransferases acting after hGNT-I. |
| Databáze: | OpenAIRE |
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