title: An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection.
abstract: The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T. gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH(2)-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.
authors.affiliation: Med. Zentrum für Hygiene und Medizinische Mikrobiologie, Philipps-Universität Marburg, Robert-Koch-Strasse 17, D-35037 Marburg, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Azzouz
authors.forename: M H
authors.initials: MH
authors.lastname: Kedees
authors.forename: P
authors.initials: P
authors.lastname: Gerold
authors.forename: S
authors.initials: S
authors.lastname: Becker
authors.forename: J F
authors.initials: JF
authors.lastname: Dubremetz
authors.forename: H D
authors.initials: HD
authors.lastname: Klenk
authors.forename: V
authors.initials: V
authors.lastname: Eckert
authors.forename: R T
authors.initials: RT
authors.lastname: Schwarz
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000096:biosynthesis / Q000235:genetics; D016367:Baculoviridae / Q000502:physiology*; D002460:Cell Line; D005822:Genetic Vectors; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D007915:Lepidoptera / Q000378:metabolism* / Q000821:virology*; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D014122:Toxoplasma; D014162:Transfection
chemical_list: D000953:Antigens, Protozoan; D017261:Glycosylphosphatidylinositols; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C047168:SAG1 antigen, Toxoplasma