“Probing the molecular basis of the binding properties of rMUP mutants”

The mouse Major Urinary Proteins (MUPs) define a class of lipocalins involved in pheromonal signaling and that, known as Mus m 1 urinary allergen complex (1,2), present immunological properties. Objective: this study consists in the analysis of a recombinant urinary protein (rMUP) mutated with the aim of determining the conformational features critical for its ligand binding properties and immunoreactivity. Methods: the structural analysis of the mutants has been carried out by circular dichroism spectroscopy; binding properties were investigated by fluorescence spectroscopy using N-phenyl-naphtylamine (NPN, ref. 3), a specific probe of the hydrophobic calyx of rMUP; immunological assessment of mutant reactivity was performed using a specific, rabbit polyclonal Ab based ELISA for rMus m 1. This assay was highly sensitive (200pg/ml Mus m 1) and specific for mouse lipocalin. Results: the data indicate that 1) the mutant (C138S) is a more stable variant of rMUP with binding parameters and a reactivity with polyclonal antibodies similar to the original rMUP. However, differently from the native form, it exhibits a complete refolding after thermal denaturation; 2) two variants (Y120F, Y120A), where the mutations involve the "cavity", show that the progressive loss of structural stability parallels the reduction of the binding ability and the loss of immunoreactivity. Conclusion: this structural and functional approach turns out to be an effective way to interpret the conformational features that underline the binding capability of the target proteins and provide further information on the antigenic determinants of MUP. References: 1) Ass. Occup. Hyg. (2002), 46(1), pp. 61-68. 2) J. Allergy Clin. Immunol. (2000), 106, pp. 1075-80. 3) Protein Science (2001), 10, pp.411-417.