Hydrolysis by cathepsin B of fluorescent peptides derived from human prorenin.
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.
Almeida
P C
PC
Department of Biophysics, Escola Paulista de Medicina, Såo Paulo, Brazil.
Oliveira
V
V
Chagas
J R
JR
Meldal
M
M
Juliano
M A
MA
Juliano
L
L
D000818:Animals; D002401:Cathepsin B / Q000378:metabolism*; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D012083:Renin / Q000737:chemistry / Q000378:metabolism*
C410765:Abz-FSQPMKRLTLGNTTQ-EDDnp; D004792:Enzyme Precursors; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002401:Cathepsin B; D012083:Renin