Title :
Asparagine Asparagine-linked oligosaccharide processing in lepidopteran insect cells
Abstract :
- 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
- 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