title: Asparagine-linked oligosaccharide processing in lepidopteran insect cells. Temporal dependence of the nature of the oligosaccharides assembled on asparagine-289 of recombinant human plasminogen produced in baculovirus vector infected Spodoptera frugiperda (IPLB-SF-21AE) cells.
abstract: Previous studies from this laboratory have established that lepidopteran insect cells possess the glycosylation machinery needed to assemble N-linked complex-type oligosaccharides on Asn289 of recombinant human plasminogen (r-HPg). In the present paper, we show that the nature of N289-linked glycosylation of [R561E]r-HPg expressed in Spodoptera frugiperda (IPLB-SF-21AE) cells is dependent upon the length of time of infection of the cells with the recombinant baculovirus/HPg-cDNA construct. At the earliest postinfection (p.i.) time period studied, i.e., 0-20 h, virtually all (96%) of the oligosaccharides released with glycopeptidase F from N289 of the expressed r-HPg were of the high-mannose type and comprised nearly the full range of such structures, containing 3-9 mannose units. At a time window of 60-96 h, p.i., essentially all of the oligosaccharides (92% of the total) assembled on N289 of rHPg were of the biantennary, triantennary, and tetraantennary complex classes, with varying extents of outer arm completion. At an intermediate time period window, of 20-60 h, p.i., a mixture of complex-type oligosaccharides, totaling approximately 77% of the glycans, with various levels of branching and outer arm completion, and high-mannose type of oligosaccharides, totaling approximately 23% of the glycans, was assembled on N289 of the r-HPg produced. These studies demonstrate that lepidopteran insect cells contain the glycosyltransferase genes required for assembly of N-linked complex oligosaccharide and that these transferases are utilized under proper conditions. The time dependency of the assembly of complex-type oligosaccharides on r-HPg indicates that an activation of the appropriate glycosyl transferases and/or transferase genes can take place. Thus, one consequence of the infective process with the recombinant baculovirus/HPg-cDNA construct is to alter the normal glycosylation characteristics of insect cells and to allow complex-type oligosaccharide processing to occur.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
authors.forename: D J
authors.initials: DJ
authors.lastname: Davidson
authors.forename: F J
authors.initials: FJ
authors.lastname: Castellino
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D015870:Gene Expression; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008358:Mannose / Q000737:chemistry; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D009844:Oligosaccharides / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010958:Plasminogen / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins; D013997:Time Factors
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D001216:Asparagine; D010958:Plasminogen; D004247:DNA; D006602:Hexosyltransferases; C018660:asparagine-N-glycosyltransferase; D006026:Glycoside Hydrolases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose