Deletion of two exons from the Lymnaea stagnalis beta1-->4-N-acetylglucosaminyltransferase gene elevates the kinetic efficiency of the encoded enzyme for both UDP-sugar donor and acceptor substrates.
Lymnaea stagnalis UDP-GlcNAc:GlcNAcbeta-R beta1-->4-N-acetylglucosaminyltransferase (beta4-GlcNAcT) is an enzyme with structural similarity to mammalian UDP-Gal:GlcNAcbeta-R beta1-->4-galactosyltransferase (beta4-GalT). Here, we report that also the exon organization of the genes encoding these enzymes is very similar. The beta4-GlcNAcT gene (12.5 kilobase pairs, spanning 10 exons) contains four exons, encompassing sequences that are absent in the beta4-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 beta4-GalT gene, corresponding to beta4-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 beta4-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 beta4-GlcNAcT are structurally linked and that the region encompassing the insertion contributes to acceptor recognition as well as to UDP-sugar binding and specificity.
Bakker
H
H
Department of Medical Chemistry, Vrije Universiteit, 1081 BT Amsterdam, The Netherlands.
Van Tetering
A
A
Agterberg
M
M
Smit
A B
AB
Van den Eijnden
D H
DH
Van Die
I
I
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D005091:Exons; D006801:Humans; D007700:Kinetics; D008195:Lymnaea / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics*; D017384:Sequence Deletion
D017351:N-Acetylglucosaminyltransferases; C090677:UDP-GlcNAc-GlcNAc-R 1-4-N-acetylglucosaminyltransferase