Human prorenin has "gate and handle" regions for its non-proteolytic activation

We investigated the mechanism for non-proteolytic activation of human prorenin using five kinds of antibodies. Each of the antigens, (LPTDTTTFKRI11P)-P-1P, (TFKRIFLKRMP17P)-F-7P, (IFLKRMPSIRESLKER26P)-F-11P, (MPSIRESLKER26P)-P-16P, and G(27P)VDMARLGPEWSQP-M-41P, was designed from the tertiary structure of predicted prorenin. These antibodies were labeled anti-01/ 06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41, respectively, for their binding specificities. Inactive recombinant human prorenin (0.1 nM) bound to various concentrations of anti-01/ 06, anti-11/26, and anti-27/41 antibodies at 4 degreesC with equilibrium dissociation constants of 138, 41, and 22 nM, respectively. However, intact prorenin ( 0.1 nM) did not show significant binding to 200 nM anti-07/10 and anti-16/26 antibodies for 20 h. Ninety percent of prorenin ( 0.1 nM) was found to be non-proteolytically activated by incubation with anti-11/26 antibodies ( 200 nM) at 4 degreesC for 20 h. Prorenin was not active even under complex with either anti-01/ 06 or anti-27/41 antibodies. Prorenin was also reversibly activated at pH 3.3 and 4 degreesC for 25 h. The acid-activated prorenin bound to anti-07/10 and anti-16/26 antibodies as well as to anti-01/ 06, anti-11/15, and anti-27/41 antibodies at neutral pH and 4 degreesC in 2 h. Their dissociation constants were 13, 40, 8.6, 3.6, and 14 nM, respectively. The acid-activated prorenin was re-inactivated by incubation at pH 7.4 and 4 degreesC in 50 h. Anti-07/10 and anti-11/26 antibodies inhibited such re-inactivation at 25 degreesC by more than 90% and 50%, respectively, whereas other kinds of antibodies did not prevent the re-inactivation at 25 degreesC. These results indicate that prorenin has "gate" ((TFKR10P)-F-7P) and "handle" ((IFLKR15P)-F-11P) regions critical for its nonproteolytic activation.