The contributions of individual galactosyltransferases to protein specific N-glycan processing in Chinese Hamster Ovary cells

Publication date: Available online 11 July 2018Source: Journal of BiotechnologyAuthor(s): Nina Bydlinski, Daniel Maresch, Valerie Schmieder, Gerald Klanert, Richard Strasser, Nicole BorthAbstractGalactosylation as part of N-glycan processing is conducted by a set of beta-1,4-galactosyltransferases (B4 GALTs), with B4 GALT1 as the dominant isoenzyme for this reaction. Nevertheless, the exact contributions of this key-player as well as of the other isoenzymes involved in N-glycosylation, B4 GALT2, B4 GALT3 and B4 GALT4, have not been studied in-depth.To increase the understanding of the protein- and site-specific activities of individual galactosyltransferases in Chinese Hamster Ovary cells, a panel of triple deletion cell lines was generated that expressed only one isoform of B4 GALT each. Two model proteins were selected for this study to cover a large spectrum of possible N-glycan structures: erythropoietin and deamine-oxidase. They were expressed as Fc-fusion constructs (EPO-Fc and Fc-DAO) and their N-glycan processing status was analyzed by site-specific mass spectrometry. The sole activity of B4 GALT1 resulted in a decrease of 15 - 21 % of fully galactosylated structures for erythropoietin, emphasizing the involvement of other isoenzymes. Interestingly, the contributions of B4 GALT2 and B4 GALT3 differed for the two model proteins. Unexpectedly, removal of galactosyltransferases influenced the overall process of N-glycan maturation, with the result of a ...
Source: Journal of Biotechnology - Category: Biotechnology Source Type: research