Pro-rat atrial natriuretic peptide-mimicking peptides as substrates for rat kallikreins rK2 (tonin) and rK9

Abstract

Investigation of the substrate specificity of rat tissue kallikreins has shown the importance of an extended site of interaction, and that the proform of rat natriuretic peptides, pro-ANP, could be a substrate for two members of the family, rK2 (tonin) and rK9 (Moreau et al. (1992) J. Biol. Chem. 267, 10045–10051). Synthetic peptide substrates that reproduce the sequence of rat pro-ANP in the region of the activation sites were used to further assess the specificity of these two proteinases. Peptides 95–107 (AGPRSLRRSSCFG) and 91–107 (RALLAGPRSLRRSSCFG) of the rat pro-ANP sequence, which include all the cleavage sites for generating natriuretic peptides (R 98, R 101, R 102), were synthesized and assayed as kallikrein substrates. Despite their homology, the two peptides had different susceptibilities to cleavage by rK2 and rK9. Peptide 91–107 was rapidly and specifically cleaved by both kallikreins, with a single cleavage site at the R 98-S 99 bond, which is the primary cleavage site in pro-ANP for generating ANP[1–28]. The k cat/ K m values were 289 000 M −1s −1 for rK2 and 39 000 M −1s −1 for rK9. The N-terminally truncated peptide (95–107) was also cleaved at that bond by both proteinases, but far less rapidly than peptide 91–107, and additional cleavages appeared at secondary sites i.e those generating atriopeptin III (R101) and auriculin (R102) in rat pro-ANP. A commercial fluorogenic tetrapeptide substrate reproducing the sequence of rat pro-ANP was slowly hydrolysed under the same conditions. The kinin-releasing kallikrein rK1 did not cleave synthetic peptides at the R 98-S 99 bond, further demonstrating the different specificities of tissue kallikreins. The results indicate that residues in positions P5 to P8 with respect to the cleavage site in the substrate, are essential for the substrate binding and specificity of kallikreins rK2 and rK9. They also show that long peptide substrates should be used to identify biological substrates of kallikreins from the investigation of their kinetic properties. The biological significance of pro-ANP processing by these proteinases, remains, however, to be proven.