Hydrolysis by Cathepsin B of Fluorescent Peptides Derived From Human Prorenin

Abstract

Abstract —Cathepsin B is a lysosomal thiolprotease that, because of its colocalization with renin and its ability to activate prorenin, has been proposed as a prorenin processing enzyme. To characterize the biochemical aspect of this potential cathepsin B activity in more detail, we synthesized and assayed with human cathepsin B the internally quenched fluorescent peptide Abz-FSQPMKRLTLGNTTQ-EDDnp (Abz, ortho- aminobenzoic acid fluorescent group and EDDnp, N-[2,4-dinitrophenyl]-ethylenediamine quencher group) that contains 7 amino acids for each side of the R-L bond that is the processing site of human prorenin. Human cathepsin B hydrolyzed this peptide at the correct site (R-L bond), with k cat /K m =75 mmol/L −1 s −1 . Analogues of this peptide obtained by Ala scanning at positions P 5 to P 5 ’ were also synthesized and assayed as substrates for human cathepsin B. The obtained specificity constant (k cat /K m ) values have a significant parallel with the previous data of prorenin activation by AtT-20 cells and in vitro by cathepsin B. In addition, we demonstrated the presence of cathepsin B–like activity in rat mesangial cells and the ability of its whole soluble fraction lysates, as well as that of purified cloned rat cathepsin B, to hydrolyze Abz-IKKSSF-EDDnp at the K-S bond, which contains 6 amino acids of rat prorenin processing site. The specificity data of cathepsin B toward peptides derived from prorenin processing site support the view that human or rodent cathepsin B could be involved in the intracellular processing of prorenin that is locally synthesized or taken up from the extracellular compartment.