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.


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.


CONCLUSIONS
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.
Yazer
Mark H
MH
Institute for Transfusion Medicine and Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Denomme
Greg A
GA
Rose
Natisha L
NL
Palcic
Monica M
MM
D000017:ABO Blood-Group System / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000328:Adult; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D003198:Computer Simulation; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D005260:Female; D005700:Galactosyltransferases / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D010641:Phenotype; D011110:Polymorphism, Genetic; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
D000017:ABO Blood-Group System; D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C023155:blood-group-substance alpha-D-galactosyltransferase