Organization and regulation of Chinese hamster alpha(1,3)fucosyltransferase genes

Abstract

Recognition of selectins by their ligands initiates leukocyte recruitment to sites of inflammation, and sialyl-Lewis X (sLe{dollar}\sp{lcub}\rm x{rcub}{dollar})-type structures serve as the indispensable glycan determinants of selectin ligands. Since the addition of fucose is the last step in the biosynthesis of sLe{dollar}\sp{lcub}\rm x{rcub}{dollar}, {dollar}\alpha{dollar}(1,3)fucosyltransferases ({dollar}\alpha{dollar}(1,3)Fuc-Ts) are crucial regulatory enzymes in the construction of selectin ligands, and much attention has been focused on the characterization of genes that encode {dollar}\alpha{dollar}(1,3)Fuc-Ts. LEC11 and two other independent Chinese hamster ovary cell isolates, LEC11A and LEC11B, generate sLe{dollar}\sp{lcub}\rm x{rcub}{dollar} on glycoproteins due to the expression of {dollar}\alpha{dollar}(1,3)Fuc-T activity not present in parental CHO cells. A Chinese hamster (Cricetulus griseus) gene (cgFUTA) was cloned, the coding region of which, when transfected into parental CHO cells, caused expression of cell surface sLe{dollar}\sp{lcub}\rm x{rcub}{dollar}. Based on substrate specificity of the {dollar}\alpha{dollar}(1,3)Fuc-T and the deduced protein sequence, cgFUTA is the Chinese hamster homologue of human gene FUT5 or FUT6. Northern blot analysis and RNase protection assays revealed that cgFUTA or a related gene is expressed in each LEC11 mutant, and is absent from parental CHO cells. Northern analysis and transferase assays of somatic cell hybrids between mutant and parental CHO as well as between different LEC11 mutants, together with Southern blot analysis of genomic DNA from each mutant, provided evidence that the expression level of a cgFUT gene was elevated by two distinct mechanisms in LEC11, LEC11A, and LEC11B mutants. The gene in LEC11 and LEC11A is rearranged, suggesting that the expression level became elevated by a cis mechanism. By contrast, the LEC11B gene expression level is elevated via a trans mechanism, apparently due to the loss of a trans-acting factor of gene expression.;Using a RACE strategy, a 180-bp 5{dollar}\sp\prime{dollar} untranslated region (UTR) and the 3{dollar}\sp\prime{dollar} end of the cgFUT gene LEC11B transcript were obtained. Surprisingly, the 5{dollar}\sp\prime{dollar}UTR mapped downstream of the cloned cgFUTA gene locus, and the 3{dollar}\sp\prime{dollar}UTR had a completely different sequence from that of the cgFUTA gene, indicating that another copy of the cgFUT gene exists 3{dollar}\sp\prime{dollar} to cgFUTA on the same genomic fragment. Southern analysis following digestion of genomic DNA with XbaI, confirmed this hypothesis and showed that two cgFUT genes with similar coding sequences are closely linked within an 8-Kb region in the Chinese hamster genome. This second gene designated cgFUTB, was cloned using RT-PCR from LEC11B mRNA. Sequence analysis disclosed that cgFUTA and cgFUTB are closely related to each other differing by only 8 nucleotides. Northern blot analysis using gene-specific 3{dollar}\sp\prime{dollar}UTR probes revealed that cgFUTA is expressed in LEC11A cells, whereas cgFUTB is expressed in both LEC11 and LEC11B cells. Two active alleles of {dollar}\alpha{dollar}(1,3)Fuc-T genes linked in an 8 kb piece of Chinese hamster genomic DNA, provides the first piece of evidence showing that a duplication event in the Lewis FUT subfamily occurred in lower mammals, which changes the current evolutionary model of FUT genes.