LEC12 and LEC29 Gain-of-Function Chinese Hamster Ovary Mutants Reveal Mechanisms for Regulating VIM-2 Antigen Synthesis and E-selectin Binding

Santosh K Patnaik,Barry Potvin,Pamela Stanley

doi:10.1074/jbc.m408755200

Abstract

LEC12 and LEC29 are two gain-of-function Chinese hamster ovary glycosylation mutants that express the Fut9 gene encoding alpha(1,3)fucosyltransferase IX (alpha(1,3) Fuc-TIX). Both mutants express the Lewis X (Le(X)) determinant Galbeta(1,4)[Fucalpha(1,3)]GlcNAc, and LEC12, but not LEC29 cells, also express the VIM-2 antigen SAalpha(2,3)-Galbeta(1,4)GlcNAcbeta(1,3)Galbeta(1,4)[Fucalpha(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 approximately 20 times more Fut9 protein than LEC29. The latter finding was consistent with our previous observation that LEC12 has approximately 40 times more in vitro alpha(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 alpha(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).

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY628212 and AY628213
The basis for the difference in Fut[9] protein levels was found to lie in sequence differences in the 5؅-untranslated regions (5؅-UTR) of LEC12 and LEC29 Fut[9] 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 Fut[9] cDNA or in vitro-synthesized full-length LEC29 Fut[9] RNA gave less Fut[9] protein than similar constructs with a LEC12 5؅-UTR

Summary

Introduction

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY628212 and AY628213. The Chinese hamster Fut[4] ortholog is expressed in the LEC30 CHO mutant and the Fut[9] ortholog is active in the LEC12, LEC29, and LEC30 CHO mutants (11) Neither of the latter mutants express the Fut[4] gene (11). CHO transfectants expressing a CHO Fut[9] cDNA behave like LEC12 cells, and bind both anti-LeX and anti-VIM-2 antibodies suggesting that LEC29 cells express a compromised. We identify 5Ј-untranslated region (UTR) sequence differences in Fut[9] cDNAs from LEC12 and LEC29 and provide evidence that they are largely responsible for the differences in Fut[9] levels and fucosylated antigen expression in LEC12 and LEC29. We show that regulation of Fut[9] levels may have physiological significance in terms of

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Conclusion