Deletion of two exons from the Lymnaea stagnalis ß164-N-acetylglucosaminyltransferase gene elevated the kinietic efficiency of the encoded enzyme for both UDP-sugar donor and acceptor substrates

H. Bakker, A. van Tetering, L. Agterberg, A.B. Smit, D.H. van den Eijnden, I.M. van Die

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Abstract

Lymnaea stagnalis UDP-GlcNAc:GlcNAcβ-R β1→4-N- acetylglucosaminyltransferase (β4-GlcNAcT) is an enzyme with structural similarity to mammalian UDP-Gal: GlcNAcβ-R β1→4-galactosyltransferase (β4-GAlT). Here, we report that also the exon organization of the genes encoding these enzymes is very similar. The β4-GlcNAcT gene (12.5 kilobase pairs, spanning 10 exons) contains four exons, encompassing sequences that are absent in the β4-GalT gene. Two of these exons (exons 7 and 8) show a high sequence similarity to part of the preceding exon (exon 6), suggesting that they have originated by exon duplication. The exon in the β4-GalT gene, corresponding to β4-GlcNAcT exon 6, encodes a region that has been proposed to be involved in the binding of UDP-Gal. The question therefore arose, whether the repeating sequences encoded by exon 7 and 8 of the β4-GlcNAcT gene would determine the specificity of the enzyme for UDP-GlcNAc, or for the less preferred UDP-GalNAc. It was found that deletion of only the sequence encoded by exon 8 resulted in a completely inactive enzyme. By contrast, deletion of the amino acid residues encoded by exons 7 and 8 resulted in an enzyme with an elevated kinetic efficiency for both UDP-sugar donors, as well as for its acceptor substrates. These results suggest that at least part of the donor and acceptor binding domains of the β4-GlcNAcT are structurally linked and that the region encompassing the insertion contributes to acceptor recognition as well as to UDP-sugar binding and specificity.
Original languageEnglish
Pages (from-to)18580-18585
JournalJournal of Biological Chemistry
Volume272
DOIs
Publication statusPublished - 1997

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