O-glycosylation potential of lepidopteran insect cell lines

The enzyme activities involved in O-glycosylation have been studied in thre e insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The st ructural features of O-glycoproteins in these insect cells were investigate d using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc :p olypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta 1,3-galac tosyltransferase, 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 :polypep tide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two differe nt UDP-GalNAc :polypeptide N-acetylgalactosaminyltransferases in these inse ct cells, Only some O-linked GalNAc residues are further processed by the a ddition 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 bindi ng 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 o f 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 t hat the culture medium influences the O-glycosylation potential of each cel l line. (C) 1999 Elsevier Science B.V. All rights reserved.