O-glycosylation potential of lepidopteran insect cell lines.

The enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc :polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta 1,3-galactosyltransferase, CMP-NeuAc :Gal beta 1-3GalNAc alpha 2,3-sialyltransferase, and UDP-Gal:Gal beta 1-3GalNAc alpha 1,4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAc alpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc :polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc :polypeptide N-acetylgalactosaminyltransferases in these insect cells, Only some O-linked GalNAc residues are further processed by the addition of beta 1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta 1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin Bq to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal :Gal beta 1-3GalNAc alpha 1,4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line. (C) 1999 Elsevier Science B.V. All rights reserved.