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J. Biol. Chem., Vol. 279, Issue 48, 49716-49726, November 26, 2004

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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, and Pamela Stanley

From the Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461

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 20 times more Fut9 protein than LEC29. The latter finding was consistent with our previous observation that LEC12 has 40 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.

Received for publication, August 2, 2004 , and in revised form, September 2, 2004.

^* This work was supported by Grant NCI RO1 30645 from the National Institutes of Health (NIH) (to P. S.), and partial support was provided by Cancer Center Grant PO1 13330 from the NCI, NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY628212 and AY628213.

To whom correspondence should be addressed: Dept. of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., New York, NY 10461. Tel.: 718-430-3346; Fax: 718-430-8574; E-mail: stanley{at}aecom.yu.edu.

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