Glycosylation in lepidopteran insect cells: identification of a beta 1-->4-N-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains.
The choice for a heterologous expression system to produce glycoprotein therapeutics highly depends on its potential to perform mammalian-like posttranslational modifications such as glycosylation. To gain more insight into the glycosylation potential of the baculovirus mediated insect cell expression system, we have studied the expression of glycosyltransferases involved in complex-type N-glycosylation. Lepidopteran insect cell lines derived from Trichoplusia ni, Spodoptera frugiperda, and Mamestra brassicae were found to express a beta 1-->4- N-acetylgalactosaminyltransferase (beta 4-GalNAcT) that catalyzes the transfer of GalNAc from UDP-GalNAc to oligosaccharides and glycoproteins carrying a terminal beta-linked GlcNAc residue. These results suggest that Lepidopteran insect cells are capable of synthesizing complex-type carbohydrate chains containing GalNAc beta 1-->4GlcNAc (LacdiNAc) units. Baculovirus infection of the cells, however, resulted in a decrease in the activity of beta 4-GalNAcT from 80 to <1 pmol.min-1 mg-1 protein within 48 h post infection. Furthermore, considerable beta-N-acetylgalactosaminidase and beta-N-acetylglucosaminidase activity was observed in insect cells, whether or not infected with baculovirus, as well as in the culture medium. These enzyme activities could be responsible for degradation of complex-type oligosaccharide chains containing LacdiNAc units. Our findings provide an enzymatic basis for the observation that most recombinant glycoproteins produced by baculovirus infected insect cells carry oligomannosidic-type N-linked glycans, in spite of the fact that uninfected insect cells have the potential for the synthesis of mammalian-like complex-type glycans.
van Die
I
I
Department of Medical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands.
van Tetering
A
A
Bakker
H
H
van den Eijnden
D H
DH
Joziasse
D H
DH
D000818:Animals; D016367:Baculoviridae / Q000502:physiology; D002240:Carbohydrate Sequence; D002460:Cell Line; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D007915:Lepidoptera / Q000166:cytology / Q000821:virology; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000378:metabolism*; D009844:Oligosaccharides / Q000096:biosynthesis*; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D006023:Glycoproteins; D009844:Oligosaccharides; D017350:N-Acetylgalactosaminyltransferases; D001619:beta-N-Acetylhexosaminidases