Amino-acid substitution in the disordered loop of blood group B-glycosyltransferase enzyme causes weak B phenotype.

Background: Few studies have investigated the reaction kinetics and interactions with nucleotide donor and acceptor substrates of mutant human ABO glycosyltransferases. Previous work identified a B(w) allele featuring a 556G>A polymorphism giving rise to a weak B phenotype. This polymorphism is predicted to cause a M186V amino-acid mutation within a highly conserved series of 16 amino acids present both in both blood group A- and blood group B-synthesizing enzymes. These residues are known as the disordered loop because their location cannot be determined in the crystal structure of the enzyme. Another patient has been identified with a 556G>A B(w) allele and the kinetics of the resulting mutant glycosyltransferase were studied.

Study Design And Methods: Serologic testing with murine and human reagents, amplification of the coding regions of exons 6 and 7, and DNA sequencing were performed with standard protocols. Enzyme kinetic studies utilized a model of human GTB M186V expressed in Escherichia coli with radiolabeled UDP-galactose and UDP-N-acetylgalactosamine as donor substrates and synthetic H-disaccharide as acceptor following standard protocols.

Results: The patient's red blood cells demonstrated a weak, but not mixed-field, B phenotype. Kinetic studies on the mutant enzyme revealed diminished activity (k(cat) = 0.15 per sec with UDP-galactose compared to 5.1 per sec for wild-type GTB) and elevated K(m) values for all substrates.

Conclusion: This enzyme with a mutation in the disordered loop produces weak B antigen expression because of greatly decreased enzyme activity and reduced affinity for B-donor and acceptor substances.