LEC12 and LEC29 Gain-of-Function Chinese Hamster Ovary Mutants Reveal Mechanisms for Regulating VIM-2 Antigen Synthesis and E-selectin Binding
Journal of Biological Chemistry
LEC12 and LEC29 are two gain-of-function Chinese hamster ovary glycosylation mutants that express the Fut9 gene encoding ␣(1,3)fucosyltransferase IX (␣(1,3) Fuc-TIX). Both mutants express the Lewis X (Le X ) determinant Gal␤(1,4)[Fuc␣(1,3)]GlcNAc, and LEC12, but not LEC29 cells, also express the VIM-2 antigen SA␣(2,3)-Gal␤(1,4)GlcNAc␤(1,3)Gal␤(1,4)[Fuc␣(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. Semiquantitative reverse transcription-PCR showed that LEC12 has 10-to 20-fold less Fut9 gene transcripts than LEC29. However, Western analysis revealed that LEC12 has ϳ20 times more Fut9 protein than LEC29. The latter finding was consistent with our previous observation that LEC12 has ϳ40 times more in vitro ␣(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 ␣(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 .