Production of a Sialylated N‐Linked Glycoprotein in Insect Cells: Role of Glycosidases and Effect of Harvest Time on Glycosylation

Abstract

Using a nonengineered Trichoplusia ni insect cell line, Tn-4s, infected with an Autographa californica recombinant baculovirus, 20% sialylation of human secreted placental alkaline phosphatase (SEAP) was observed. In contrast to this level of sialylation, intermediate complex forms with terminal galactose or N-acetylglucosamine were found in low proportions (<3% and <1%, respectively). We tested whether time of harvest or degradation of intermediate complex forms is responsible for this distribution of glycoforms. Spinner-flask cultures were infected with the SEAP baculovirus expression vector, and the cultures were harvested 48, 72, and 96 h post-infection. Structural analysis revealed that the glycoform distribution of SEAP was very similar at the different times of harvest, indicating that the cellular machinery was not significantly affected by the progress of infection and that the glycoforms obtained were stable. High levels of beta-galactosidase and N-acetylglucosaminidase activity were detected throughout infection. In contrast, sialidase activity was below detection level both in cell extracts and in supernatants. These levels of glycosidases activities raise the possibility that intermediate complex glycoforms may be degraded while sialylated forms should not experience significant degradation in this cell line. However, culture in the presence of extracellular beta-galactosidase and N-acetylglucosaminidase inhibitors did not significantly improve glycosylation, suggesting that extracellular degradation processes are not taking place. Instead, results suggest that the intracellular machinery of the Tn-4s cells tends to either shunt the glycans to paucimannosidic forms or drive them completely to sialylation.

Molecular cloning and expression profile analysis of a novel β‐D‐N‐acetylhexosaminidase of domestic silkworm (Bombyx mori)

“…Previous studies also reported supportive results: (i) sialylated N ‐glycans were detected on both native and recombinant proteins produced by insect cells (Davidson & Castellino 1991; Davis & Wood 1995; Ogonah et al. 1996; Joosten & Shuler 2003); (ii) significant activity of GlcNAcase was observed in insoluble fractions of lepidopteran cell homogenates (Kimura 1974, 1976; Altmann et al. 1993, 1995).…”

“…Meanwhile, we previously reported that baculovirus-infected moth cells, Sf21AE (S. frugiperda origin) and TN5-5B1-4 (TN5; T. ni origin), synthesized N-glycans with sialic acid modification under b-D-N-acetylglucosaminidase (GlcNAcase)-inhibited conditions (Watanabe et al 2002). Previous studies also reported supportive results: (i) sialylated N-glycans were detected on both native and recombinant proteins produced by insect cells (Davidson & Castellino 1991;Davis & Wood 1995;Ogonah et al 1996;Joosten & Shuler 2003); (ii) significant activity of GlcNAcase was observed in insoluble fractions of lepidopteran cell homogenates (Kimura 1974(Kimura , 1976Altmann et al 1993Altmann et al , 1995. Despite of these findings, until recently, any GlcNAcase activity associated with the synthesis pathway of N-glycans was identified in the species.…”

Effect of Production Method and Gene Amplification on the Glycosylation Pattern of a Secreted Reporter Protein in CHO Cells

“…We speculate that the cell death and lysis observed during the last 3 days of batch culture resulted in the release of glycan structures from the Golgi that had not completed the full biochemical synthesis necessary for formation of complex structures. The predominance of high mannose glycan structures has also been observed for cultures of insect cells (28)(29)(30) where the cells are known to undergo lytic cell death as a result of the baculovirus expression system.…”

Molecular Cloning and Functional Characterization of a Lepidopteran Insect β4-N-Acetylgalactosaminyltransferase with Broad Substrate Specificity, a Functional Role in Glycoprotein Biosynthesis, and a Potential Functional Role in Glycolipid Biosynthesis

“…In addition, several studies had shown that T. ni cells could produce recombinant glycoproteins with terminally galactosylated N-glycans when infected with baculovirus expression vectors (32,35,38,39). Thus, it was theoretically possible to isolate a ␤4-galactosyltransferase family member from this organism.…”

“…However, more recent reports have shown that lepidopteran insect cell lines actually do have low levels of some of the glycosyltransferase activities mediating elongation of N-glycan termini, including N-acetylglucosaminyltransferase I (28,29), N-acetylglucosaminyltransferase II (28), ␤4-galactosyltransferase (30,31), and ␤4-N-acetylgalactosaminyltransferase (30). In addition, various reports have documented the presence of terminal N-acetylglucosamine, galactose, N-acetylgalactosamine, and even sialic acid residues on N-glycans produced by insect cells (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). Finally, putative N-acetylglucosaminyltransferase, galactosyltransferase, and sialyltransferase genes have been identified in the D. melanogaster data bases (44), and the biochemical functions of the putative fly N-acetylglucosaminyltransferase I (45) and sialyltransferase (46) gene products have been experimentally confirmed in published studies.…”