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 Bw 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 Bw 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 (kcat = 0.15 per sec with UDP‐galactose compared to 5.1 per sec for wild‐type GTB) and elevated Km 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.