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 i n the screening of mature and immature thymocytes as well as in the detecti on of cancerous malignancies. The basis for these applications is the remar kably strong affinity of the lectin for the tumor-associated Thomsen-Friede nreich 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 pea nut 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 carbon yl oxygen of the acetamido group of the second hexopyranose ring of the sug ar molecule. Site directed mutational changes were introduced at this resid ue with the objective of probing the role of this residue in T-antigen bind ing 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 affinitie s of the mutants can be readily explained on the basis of the crystal struc ture of the complex and simple modeling. In particular, the change of aspar agine to glutamine could lead to a direct interaction of the side chain wit h the sugar while at the same time retaining the water bridge. This study s trengthens the theory that in lectins the nonprimary contacts generally mad e through water bridges are involved in imparting exquisite specificity.