The whole genome approach enables the characterization of all components of any given biological pathway. Moreover, it can help to uncover all the metabolic routes for any molecule. Here we have used the genome of Drosophila melanogaster to search for enzymes involved in the metabolism of fucosylated glycans. Our results suggest that in the fruit fly GDP-fucose, the donor for fucosyltransferase reactions, is formed exclusively via the de novo pathway from GDP-mannose through enzymatic reactions catalyzed by GDP-D-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (GMER, also known as FX in man). The Drosophila genome does not have orthologs for the salvage pathway enzymes, i.e. fucokinase and GDP-fucose pyrophosphorylase synthesizing GDP-fucose from fucose. In addition we identified two novel fucosyltransferases predicted to catalyze alpha1,3- and alpha1,6-specific linkages to the GlcNAc residues on glycans. No genes with the capacity to encode alpha1,2-specific fucosyltransferases were found. We also identified two novel genes coding for O-fucosyltransferases and a gene responsible for a fucosidase enzyme in the Drosophila genome. Finally, using the Drosophila CG4435 gene, we identified two novel human genes putatively coding for fucosyltransferases. This work can serve as a basis for further whole-genome approaches in mapping all possible glycosylation pathways and as a basic analysis leading to subsequent experimental studies to verify the predictions made in this work.

While most humans express an alpha(1,3)-fucosyltransferase in plasma, 9% of individuals on the isle of Java (Indonesia) do not express this enzyme. Ninety-five percent of these plasma alpha(1,3)-fucosyltransferase-deficient individuals have Lewis negative phenotype on red cells, suggesting strong linkage disequilibrium between these two traits. To define the molecular basis for this plasma deficiency and to determine which of two candidate human alpha(1,3)-fucosyltransferase genes encode this enzyme (FUT5 and FUT6), we cloned and analyzed alleles at these two loci from an Indonesian individual deficient in plasma alpha(1,3)-fucosyltransferase activity. Single base pair changes were identified in the coding region of each gene, relative to previously published wild type alleles. These changes in turn yield three codon changes in FUT5 and three in FUT6. The codon changes in the FUT5 gene do not yield detectable diminutions in alpha(1,3)-fucosyltransferase activity when tested by expression in transfected COS-1 cells, and none of the FUT5 alleles co-segregate with plasma alpha(1,3)-fucosyltransferase deficiency in Indonesian pedigrees. By contrast, two of the codon changes in the FUT6 alleles inactivate this gene when tested by expression in transfected COS-1 cells. One of these inactivating changes is a missense mutation (Glu-247-->Lys) within the enzyme's catalytic domain. The other inactivating mutation represents a nonsense mutation (Tyr-315-->stop) that truncates the COOH terminus of the enzyme by 45 amino acids. The Glu-247-->Lys missense mutation is present in double dose in the nine plasma alpha(1,3)-fucosyltransferase-deficient individuals tested, whereas the nonsense mutation at tyrosine 315 is present in double dose in just one of these persons. These results demonstrate that the alpha(1,3)-fucosyltransferase activity in human plasma is encoded by the FUT6 gene and that the missense mutation within codon 247 of this gene is responsible for deficiency of this activity in these Indonesian families.

While most humans express an alpha(1,3)-fucosyltransferase in plasma, 9% of individuals on the isle of Java (Indonesia) do not express this enzyme. Ninety-five percent of these plasma alpha(1,3)-fucosyltransferase-deficient individuals have Lewis negative phenotype on red cells, suggesting strong linkage disequilibrium between these two traits. To define the molecular basis for this plasma deficiency and to determine which of two candidate human alpha(1,3)-fucosyltransferase genes encode this enzyme (FUT5 and FUT6), we cloned and analyzed alleles at these two loci from an Indonesian individual deficient in plasma alpha(1,3)-fucosyltransferase activity. Single base pair changes were identified in the coding region of each gene, relative to previously published wild type alleles. These changes in turn yield three codon changes in FUT5 and three in FUT6. The codon changes in the FUT5 gene do not yield detectable diminutions in alpha(1,3)-fucosyltransferase activity when tested by expression in transfected COS-1 cells, and none of the FUT5 alleles co-segregate with plasma alpha(1,3)-fucosyltransferase deficiency in Indonesian pedigrees. By contrast, two of the codon changes in the FUT6 alleles inactivate this gene when tested by expression in transfected COS-1 cells. One of these inactivating changes is a missense mutation (Glu-247-->Lys) within the enzyme's catalytic domain. The other inactivating mutation represents a nonsense mutation (Tyr-315-->stop) that truncates the COOH terminus of the enzyme by 45 amino acids. The Glu-247-->Lys missense mutation is present in double dose in the nine plasma alpha(1,3)-fucosyltransferase-deficient individuals tested, whereas the nonsense mutation at tyrosine 315 is present in double dose in just one of these persons. These results demonstrate that the alpha(1,3)-fucosyltransferase activity in human plasma is encoded by the FUT6 gene and that the missense mutation within codon 247 of this gene is responsible for deficiency of this activity in these Indonesian families.

The whole genome approach enables the characterization of all components of any given biological pathway. Moreover, it can help to uncover all the metabolic routes for any molecule. Here we have used the genome of Drosophila melanogaster to search for enzymes involved in the metabolism of fucosylated glycans. Our results suggest that in the fruit fly GDP-fucose, the donor for fucosyltransferase reactions, is formed exclusively via the de novo pathway from GDP-mannose through enzymatic reactions catalyzed by GDP-D-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (GMER, also known as FX in man). The Drosophila genome does not have orthologs for the salvage pathway enzymes, i.e. fucokinase and GDP-fucose pyrophosphorylase synthesizing GDP-fucose from fucose. In addition we identified two novel fucosyltransferases predicted to catalyze alpha1,3- and alpha1,6-specific linkages to the GlcNAc residues on glycans. No genes with the capacity to encode alpha1,2-specific fucosyltransferases were found. We also identified two novel genes coding for O-fucosyltransferases and a gene responsible for a fucosidase enzyme in the Drosophila genome. Finally, using the Drosophila CG4435 gene, we identified two novel human genes putatively coding for fucosyltransferases. This work can serve as a basis for further whole-genome approaches in mapping all possible glycosylation pathways and as a basic analysis leading to subsequent experimental studies to verify the predictions made in this work.

The whole genome approach enables the characterization of all components of any given biological pathway. Moreover, it can help to uncover all the metabolic routes for any molecule. Here we have used the genome of Drosophila melanogaster to search for enzymes involved in the metabolism of fucosylated glycans. Our results suggest that in the fruit fly GDP-fucose, the donor for fucosyltransferase reactions, is formed exclusively via the de novo pathway from GDP-mannose through enzymatic reactions catalyzed by GDP-D-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (GMER, also known as FX in man). The Drosophila genome does not have orthologs for the salvage pathway enzymes, i.e. fucokinase and GDP-fucose pyrophosphorylase synthesizing GDP-fucose from fucose. In addition we identified two novel fucosyltransferases predicted to catalyze alpha1,3- and alpha1,6-specific linkages to the GlcNAc residues on glycans. No genes with the capacity to encode alpha1,2-specific fucosyltransferases were found. We also identified two novel genes coding for O-fucosyltransferases and a gene responsible for a fucosidase enzyme in the Drosophila genome. Finally, using the Drosophila CG4435 gene, we identified two novel human genes putatively coding for fucosyltransferases. This work can serve as a basis for further whole-genome approaches in mapping all possible glycosylation pathways and as a basic analysis leading to subsequent experimental studies to verify the predictions made in this work.