α2u-globulin is the only member of the lipocalin protein superfamily that binds to hyaline droplet inducing agents

Abstract

The rate-limiting step in chemically induced, male rat-specific hyaline droplet nephropathy is the reversible binding of a xenobiotic to α2u-globulin. In this study, equilibrium saturation binding experiments were conducted to evaluate the in vitro binding of d-limonene-1,2-oxide (dLO) and 2,4,4-trimethyl-2-pentanol (TMP-OH) to α2u-globulin and members of the α2u-globulin protein superfamily. Both dLO and TMP-OH bound to α2u-globulin, with Scatchard analysis yielding dissociation constants of 5.6 and 6.4 × 10 −7 m, respectively. The B max for binding (nmol bound/mg protein) was 50.7 and 61.1 for dLO and TMP-OH, respectively, yielding a molar ratio of approximately 1 for both ligands. The ability of dLO and TMP-OH to bind to human-derived α1-acid glycoprotein, rat-derived retinol-binding protein, human protein-1, and bovine β-lactoglobulin was also studies. These superfamily proteins are generally abundant in plasma, are freely filtered across the glomerulus, and can bind a wide range of ligands. However, neither dLO nor TMP-OH bound to any of the superfamily proteins. In contrast, under identical experimental conditions, α1-acid glycoprotein did bind progesterone (K d = 10 −6 m), whereas both β-lactoglobulin and retinol-binding protein bound retinol ( K d = 10 −8 m for both proteins). These results indicate that, under conditions where α2u-globulin superfamily proteins bind to established ligands, the proteins do not interact with hyaline droplet inducing agents. Thus, the interaction between male rat-specific nephrotoxicants and α2u-globulin is unique to this protein. More importantly, these results provide direct evidence that the presence of the α2u-globulin superfamily proteins does not predispose humans to develop hyaline droplet nephropathy and renal cancer from this class of chemicals.