Imparting exquisite specificity to peanut agglutinin for the tumor-associated Thomsen-Friedenreich antigen by redesign of its combining site.
Lectins from legumes constitute one of the most thoroughly studied families of proteins, yet the absence of a rigorous framework to explain their carbohydrate binding specificities appears to have prevented a rational approach to alter their ligand binding activity. Studies reported here deal with the redesign of the recognition propensity of peanut agglutinin (PNA), an important member of the family. PNA is extensively used as a tool for recognition of the tumor-associated Thomsen-Friedenrich antigen (T-antigen; Galbeta1-3GalNAc) on the surfaces of malignant cells and immature thymocytes. PNA also recognizes N-acetyllactosamine (LacNAc; Galbeta1-4GlcNAc), which is present at the termini of several cell-surface glycoproteins. The crystal structure of the PNA-lactose complex revealed, in addition to the expected interactions with the residues constituting the binding site, the presence of leucine 212 at a position close enough to be in steric contact with the acetamido group on LacNAc. We report here two leucine mutants, one to asparagine (L212N) and the other to alanine (L212A), that exhibit distinct preference for T-antigen and N-acetyllactosamine, respectively. Carbohydrate binding studies reveal that mutant L212N does not recognize LacNAc at high concentrations, thus making it an exquisitely specific cell-surface marker compared with its wild-type counterpart.
Sharma
V
V
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Vijayan
M
M
Surolia
A
A
D000951:Antigens, Neoplasm / Q000378:metabolism*; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000378:metabolism*; D010367:Arachis / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D019887:Peanut Agglutinin; D037121:Plant Lectins
D000951:Antigens, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D002241:Carbohydrates; D037102:Lectins; D009838:Oligodeoxyribonucleotides; D019887:Peanut Agglutinin; D037121:Plant Lectins; C016467:Thomsen-Friedenreich antigen