Mutational analysis at Asn-41 in peanut agglutinin. A residue critical for the binding of the tumor-associated Thomsen-Friedenreich antigen.
Peanut agglutinin is a clinically important lectin due to its application in the screening of mature and immature thymocytes as well as in the detection of cancerous malignancies. The basis for these applications is the remarkably strong affinity of the lectin for the tumor-associated Thomsen-Friedenreich antigen (T-antigen) and more so due to its ability to distinguish T-antigen from its cryptic forms. The crystal structure of the complex of peanut agglutinin with T-antigen reveals the basis of this specificity. Among the contacts involved in providing this specificity toward T-antigen is the water-mediated interaction between the side chain of Asn-41 and the carbonyl oxygen of the acetamido group of the second hexopyranose ring of the sugar molecule. Site-directed mutational changes were introduced at this residue with the objective of probing the role of this residue in T-antigen binding and possibly engineering an altered species with increased specificity for T-antigen. Of the three mutants tested, i.e. N41A, N41D, and N41Q, the last one shows improved potency for recognition of T-antigen. The affinities of the mutants can be readily explained on the basis of the crystal structure of the complex and simple modeling. In particular, the change of asparagine to glutamine could lead to a direct interaction of the side chain with the sugar while at the same time retaining the water bridge. This study strengthens the theory that in lectins the nonprimary contacts generally made through water bridges are involved in imparting exquisite specificity.
Adhikari
P
P
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Bachhawat-Sikder
K
K
Thomas
C J
CJ
Ravishankar
R
R
Jeyaprakash
A A
AA
Sharma
V
V
Vijayan
M
M
Surolia
A
A
D015295:Antigens, Tumor-Associated, Carbohydrate / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019887:Peanut Agglutinin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020135:Peptide Nucleic Acids
D015295:Antigens, Tumor-Associated, Carbohydrate; D019887:Peanut Agglutinin; D020135:Peptide Nucleic Acids; C016467:Thomsen-Friedenreich antigen; D001216:Asparagine