Characterization of chicken cystatin binding to rat renal brush-border membranes

Chicken cystatin, a homologue of human cystatin C, like other low-molecular-weight proteins is metabolized by renal proximal tubule cells. However, the precise mechanism(s) of this process has not been elucidated yet. To characterize chicken cystatin binding to renal brush-border membranes, the incubation of fluorescein labelled protein with rat cortical homogenate was performed. Saturation-dependent and reversible binding with low affinity (K(d)=3.67-4.07 microM) and high capacity (B(max)=2.32-2.79 nmol/mg) was observed. Bovine albumin was the most potent competitor (K(i)=0.7 microM) among other megalin/cubilin ligands tested. The presence of Ca(+2) ions was necessary to effective cystatin binding by brush-border membranes. Obtained data strongly support the hypothesis that chicken cystatin is a novel ligand for megalin/cubilin receptors tandem on proximal tubular cells.