A single point mutation reverses the donor specificity of human blood group B-synthesizing galactosyltransferase.
Blood group A and B antigens are carbohydrate structures that are synthesized by glycosyltransferase enzymes. The final step in B antigen synthesis is carried out by an alpha1-3 galactosyltransferase (GTB) that transfers galactose from UDP-Gal to type 1 or type 2, alphaFuc1-->2betaGal-R (H)-terminating acceptors. Similarly the A antigen is produced by an alpha1-3 N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine from UDP-GalNAc to H-acceptors. Human alpha1-3 N-acetylgalactosaminyltransferase and GTB are highly homologous enzymes differing in only four of 354 amino acids (R176G, G235S, L266M, and G268A). Single crystal x-ray diffraction studies have shown that the latter two of these amino acids are responsible for the difference in donor specificity, while the other residues have roles in acceptor binding and turnover. Recently a novel cis-AB allele was discovered that produced A and B cell surface structures. It had codons corresponding to GTB with a single point mutation that replaced the conserved amino acid proline 234 with serine. Active enzyme expressed from a synthetic gene corresponding to GTB with a P234S mutation shows a dramatic and complete reversal of donor specificity. Although this enzyme contains all four "critical" amino acids associated with the production of blood group B antigen, it preferentially utilizes the blood group A donor UDP-GalNAc and shows only marginal transfer of UDP-Gal. The crystal structure of the mutant reveals the basis for the shift in donor specificity.
Marcus
Sandra L
SL
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
Polakowski
Robert
R
Seto
Nina O L
NO
Leinala
Eeva
E
Borisova
Svetlana
S
Blancher
Antoine
A
Roubinet
Francis
F
Evans
Stephen V
SV
Palcic
Monica M
MM
D000017:ABO Blood-Group System / Q000235:genetics*; D000116:Acetylgalactosamine / Q000378:metabolism; D001782:Blood Donors; D005690:Galactose / Q000378:metabolism; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010641:Phenotype; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014961:X-Ray Diffraction
D000017:ABO Blood-Group System; D011994:Recombinant Proteins; D005700:Galactosyltransferases; C023155:blood-group-substance alpha-D-galactosyltransferase; D000116:Acetylgalactosamine; D005690:Galactose