Human B-type natriuretic peptide is not degraded by meprin A

Abstract

B-type natriuretic peptide (BNP) combats cardiac stress by reducing blood pressure and ventricular fibrosis. Human BNP is inactivated by unknown cell surface proteases. N-terminal cleavage of mouse BNP by the renal protease meprin A was reported to increase inactivating degradation by a second protease named neprilysin. Since the sequence surrounding the meprin A cleavage site in BNP differs between species, we tested whether meprin A degrades human BNP. Using a recently developed proteolytic bioassay, the ability of various protease inhibitors to block the inactivation of BNP was measured. In rat kidney membranes, inhibitors of meprin A or neprilysin partially or completely blocked inactivation of rat BNP(1-32) when added individually or in combination, respectively. In contrast, neither inhibitor alone or in combination prevented the inactivation of human BNP(1-32) by human kidney membranes. Leupeptin, a serine protease inhibitor, totally blocked inactivation of human BNP by human membranes, substantially blocked the inactivation of rat BNP(1-32) by human membranes, but had no effect on the inactivation of rat BNP(1-32) by rat kidney membranes. Purified neprilysin reduced the bioactivity of rat BNP(1-32) and human BNP. Digestion with both meprin and neprilysis caused the greatest reduction in rat BNP(1-32) but had no effect on the bioactivity of human BNP(1-32). We conclude that meprin A does not degrade BNP in humans and should not be considered a pharmacologic target of the natriuretic peptide system.

FIGURE 1

Primary amino acid sequences of BNP from various species. The shaded areas indicate identical residues. The grey arrows indicate the location of the meprin-dependent cleavage. The black arrows indicate NEP cleavage sites.

FIGURE 2

Neither meprin nor NEP reduce the bioactivity of human BNP_1-32. Human BNP_1-32 was incubated with 40 μg human kidney membranes for 20 min at 37°C in the absence or presence of the indicated protease inhibitors and then the bioactivity of the resulting proteolyzed peptide was measured as described under Material and Methods. The values represent the mean +/− SEM with the indicated number of replicates. * indicates a significant difference between samples treated with membrane or membrane in the presence of the indicated inhibitor, P<0.0001. The p value was not significant (p<0.05) for all other treatments.

FIGURE 3

Effect of various protease inhibitors on the inactivation of rat BNP_1-32 by rat or human kidney membranes. Rat BNP_1-32 was incubated with 40 μg rat or human kidney membranes for 45 min. Bioactivity was determined as described under Material and Methods. The values represent the mean +/− SEM where N = 8. * = P < 0.05, ** = P < 0.005, *** = P < 0.0001

FIGURE 4

Human BNP_1-32 is resistant to degradation by rat kidney membranes. Human BNP_1-32 was incubated with 40 μg of rat kidney membranes for 45 min. The amount of peptide remaining was determined as described under Material and Methods. The values represent the mean +/− SEM where N = 4. None of the samples treated with membrane plus inhibitor were significantly different from membrane only treated samples.

FIGURE 5

Purified human meprin reduces rat but not human BNP activity. Rat or human BNP_1-32 were incubated at 37°C for 30 minutes with 40 ng recombinant human meprin, 50 ng of recombinant NEP or with both enzymes. Proteolysis was terminated with the addition of 0.5 N perchloric acid. An aliquot of the proteolysis sample was then assayed as described under Material and Methods. The values represent the mean +/− SEM where N = 6. Significance was determined by comparing the samples to peptide only, * = P<0.05, ** = P<0.005, *** = P<0.0001