DELETION OF 2 EXONS FROM THE LYMNAEA-STAGNALIS BETA-1-]4-N-ACETYLGLUCOSAMINYLTRANSFERASE GENE ELEVATES THE KINETIC EFFICIENCY OF THE ENCODED ENZYME FOR BOTH UDP-SUGAR DONOR AND ACCEPTOR SUBSTRATES

Lymnaea stagnalis UDP-GlcNAc:GlcNAc beta-R beta 1-->4-N-acetylglucosam inyltransferase (beta 4-GlcNAcT) is an enzyme with structural similari ty to mammalian UDP-Gal: GlcNAc beta-R beta 1-->4-galactosyltransferas e (beta 4-GalT). Here, we report that also the exon organization of th e genes encoding these enzymes is very similar. The beta 4-GlcNAcT gen e (12.5 kilobase pairs, spanning 10 exons) contains four exons, encomp assing sequences that are absent in the beta 4-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 beta 4-GalT gene, corresponding to beta 4 -GlcNAcT exon 6, encodes a region that has been proposed to be involve d in the binding of UDP-Gal. The question therefore arose, whether the repeating sequences encoded by exon 7 and 8 of the beta 4-GlcNAcT gen e 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 an d 8 resulted in an enzyme with an elevated kinetic efficiency for both UDP-sugar donors, as well as for its acceptor substrates. These resul ts suggest that at least part of the donor and acceptor binding domain s of the beta 4-GlcNAcT are structurally linked and that the region en compassing the insertion contributes to acceptor recognition as well a s to UDP-sugar binding and specificity.