Molecular cloning and expression of GDP-D-mannose-4,6-dehydratase, a key enzyme for fucose metabolism defective in Lec13 cells

Abstract

Subsets of mammalian cell surface oligosaccharides contain specific fucosylated moieties expressed in lineage- and/or temporal-specific patterns. The functional significance of these fucosylated structures is incompletely defined, although there is evidence that subsets of them, represented by the sialyl Lex determinant, are important participants in leukocyte adhesion and trafficking processes. Genetic deletion of these fucosylated structures in the mouse has been a powerful tool to address functional questions about fucosylated glycans. However, successful use of such approaches can be problematic, given the substantial redundancy in the mammalian alpha-1,3-fucosyltransferase and alpha-1,2-fucosyltransferase gene families. To circumvent this problem, we have chosen to clone the genetic locus encoding a mammalian GDP-D-mannose-4,6-dehydratase (GMD). This enzyme generates GDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which is then converted by the FX protein (GDP-4-keto-6-D-deoxymannose epimerase/GDP-4-keto-6-L-galactose reductase) to GDP-L-fucose. GMD is thus imperative for the synthesis of all fucosylated oligosaccharides. An expression cloning approach and the GMD-deficient CHO host cell line Lec13 were used to generate a population of cDNA molecules enriched in GMD cDNAs. This enriched plasmid population was then screened using a human expressed sequence tag (EST AA065072) with sequence similarity to an Arabidopsis thaliana GMD cDNA. This approach, together with 5'-rapid amplification of cDNA ends, yielded a human cDNA that complements the fucosylation defect in the Lec13 cell line. Northern blot analyses indicate that the GMD transcript is absent in Lec13 cells, confirming the genetic deficiency of this locus in these cells. By contrast, the transcript encoding the FX protein, which forms GDP-L-fucose from the ketosugar intermediate produced by GMD, is present in increased amounts in the Lec13 cells. These results suggest that metabolites generated in this pathway may participate in the transcriptional regulation of the FX protein and possibly the GMD protein. The results also suggest that the genomic structure encoding GMD in Lec13 cells likely has a defect different from a point mutation in the coding region.