β-(1→4)-Galactosyltransferase activity in native and engineered insect cells measured with time-resolved europium fluorescence

Abstract

To evaluate the ability of insect cells to produce complex-type N-glycans, β-(1→4)-galactosyltransferase (β4GalT) activity in several insect cell lines was analyzed. For this purpose, we developed a simple and highly sensitive assay for β-(1→4)-galactosyltransferase (β4GalT) activity, which is based on time-resolved fluorometry of europium. Bovine serum albumin (BSA) modified with GlcNAc (GlcNAc 44–BSA) was used as the acceptor. GlcNAc 44–BSA was coated on a 96-well microplate, and after incubation with the enzyme sample in the presence of UDP-Gal, Eu-labeled RCA 120 ( Ricinus communis aggutin I), was added. RCA 120 binds to the Galβ(1→4)GlcNAc structure in the product, and the bound Eu-RCA 120 was measured by the fluorescence of europium. When bovine β4Gal-T-I was used as a standard reference enzyme, a linear relationship between enzyme activity and fluorescent signal was obtained over the range of 0–1000 μUnits (IU). Using this system, we were able to measure a low but significant level of β4GalT activity in Trichoplusia ni cells (‘High Five’). In contrast, no endogenous β4GalT activity was detected in a Spodoptera frugiperda (Sf-9) cell line. However, Sf-9 cells stably transfected with the bovine β4GalT-I gene and ‘High Five’ cells infected with a baculovirus containing the same gene produced activity levels that were comparable to or greater than those found in Chinese hamster ovary cells. We also showed that the β4GalT activity level observed in the baculovirus-infected T. ni cells under the control of immediate early promoter was highly dependent on the post-infection time, suggesting that galactosylation level may also be variable during the infection period.