LEC12 and LEC29 gain-of-function Chinese hamster ovary mutants reveal mechanisms for regulating VIM-2 antigen synthesis and E-selectin binding

Abstract

LEC12 and LEC29 are two gain-of-function Chinese hamster ovary glycosylation mutants that express the Fut9 gene encoding α(1,3) fucosyltransferase IX α(1,3) Fuc-TIX). Both mutants express the Lewis X (LeX) determinant Galβ(1,4)[Fucα(1,3)]GlcNAc, and LEC12, but not LEC29 cells, also express the VIM-2 antigen SAα(2,3)-Galβ(1, 4)GlcNAcβ(1,3)Galβ(1,4)[Fucα(1,3)]GlcNAc. Here we show that LEC29 cells transfected with a Fut9 cDNA express VIM-2, and thus LEC29 cells synthesize appropriate acceptors to generate the VIM-2 epitope. Semi-quantitative reverse transcription-PCR showed that LEC12 has 10- to 20-fold less Fut9 gene transcripts than LEC29. However, Western analysis revealed that LEC12 has ∼20 times more Fut9 protein than LEC29. The latter finding was consistent with our previous observation that LEC12 has ∼40 times more in vitro α(1,3)Fuc-T activity than LEC29. The basis for the difference in Fut9 protein levels was found to lie in sequence differences in the 5′-untranslated regions (5′-UTR) of LEC12 and LEC29 Fut9 gene transcripts. Whereas reporter assays with the respective 5′-UTR regions linked to luciferase did not indicate a reduced translation efficiency caused by the LEC29 5′-UTR, transfected full-length LEC29 Fut9 cDNA or in vitro-synthesized full-length LEC29 Fut9 RNA gave less Fut9 protein than similar constructs with a LEC12 5′-UTR. This difference appears to be largely responsible for the reduced α(1,3)Fuc-TIX activity and lack of VIM-2 expression of LEC29 cells. This could be of physiological relevance, because LEC29 and parent Chinese hamster ovary cells transiently expressing a Fut9 cDNA were able to bind mouse E-selectin, although they did not express sialyl-Le X.