Molecular determinants of human prorenin processing.

Abstract

In humans, active renin is generated by the removal of a 43-amino acid prosegment from the zymogen prorenin. This cleavage event is highly specific, occurring at only one of the seven pairs of basic amino acids in the body of preprorenin. This cleavage site selectivity is also displayed by a number of other proteases in vitro and in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector, suggesting that specificity of cleavage is directed in part by the primary sequence, the higher order structure, or both of prorenin itself. To test this hypothesis, single amino acid mutations were introduced in the region of human preprorenin surrounding the natural cleavage site, and the resultant recombinant proteins were expressed in cultured Chinese hamster ovary and AtT-20 cells. The results suggest that amino acids in addition to the pair of basic amino acids surrounding the cleavage site affect the ability of both trypsin and the endogenous AtT-20 processing enzyme to cleave prorenin. Notably, although a proline at position -4 is essential for processing of prorenin in AtT-20 cells and is correlated with predicted formation of a p-turn at this position, site-directed mutations suggest that this structural feature in addition to a pair of basic amino acids is not sufficient to lead to proteolytic activation of prorenin. Displacement of sequences surrounding the cleavage site to a position 10 amino acids toward the amino terminus led to partial processing of a mutated prorenin. Taken together, these results suggest that the ability of AtT-20 cells to cleave prorenin is directed primarily by specific amino acid recognition on the amino side of the sclssile bond and that additional determinants (perhaps structural) enhance the efficiency of the maturation step. (Hypertension 1992;20:782-787) KEY WORDS • enzyme precursors • renin • renin-angiotensin system R enin is an aspartyl protease and is the limiting component in the circulating renin-angiotensin system, an important modulator of blood pressure in mammals.

Two Activation States of the Prohormone Convertase PC1 in the Secretory Pathway

“…The mutation of lysine to alanine in the native Lys-Arg 42,43 cleavage site at the junction of the profragment and the renin moiety (Proren-K/A ϩ42 (19) ative to amino acid 1 of the prosegment) was carried out by overlap extension polymerase chain reaction (23). The cDNA for mPC1 (9) was inserted in the expression vector pRSVglobin (22).…”

“…Twenty-four h after transfection, the cells were glycerol shocked. After a 16-h secretion period, the supernatants were collected for determination of prorenin and renin levels by the angiotensin I generation assay described previously (19). Briefly, supernatants were incubated with an excess of the renin substrate angiotensinogen either directly (active renin content) or following an incubation with trypsin (total renin content ϭ prorenin ϩ renin).…”

Hydrolysis by Cathepsin B of Fluorescent Peptides Derived From Human Prorenin

“…Curiously, peptides 10 and 11 were hydrolyzed by cathepsin B, which has higher affinity; this indicates that positions P 4 Ј and P 5 Ј have significant influence on substrate binding. Peptides 12 and 13 were synthesized and assayed to verify the effects of Phe and Gly at the P 4 position on the hydrolysis of qf-peptides by cathepsin B, because these modifications were performed on site-mutated human prorenin 6 to force or release conformation restrictions at that position. Pro was described to be critical for prorenin processing, and mutants with Ala, Phe, or Gly impaired the capacity of AtT-20 cells to process prorenin.…”

“…The observed low k cat /K m values for hydrolysis of peptides 12 and 13 are in parallel with the resistance to be processed of P/F-mutated, P/G-mutated, and P/A-mutated prorenin mentioned above. 6 Table 2 also shows the kinetics parameters for hydrolysis by trypsin of those previously mentioned peptides with human prorenin sequence. All peptides were susceptible to hydrolysis by trypsin, and the R-L bond was essentially the cleavage bond except in peptide 6, in which Ala was substituted for Arg and the cleavage shifted to the K-A bond.…”

“…1 The sorting to regulated pathway depends on the presence of protease processing site, which is constituted in human prorenin of the basic amino acid pair Lys-Arg. 2 The protease that activates prorenin to renin in the regulated pathway is still unknown, although PC5 3,4 and cathepsin B [5][6][7] have been indicated as candidates. Prorenin released constitutively from renal and extrarenal tissues also circulates in the blood, and its plasma concentration is 10 times higher than that of active renin.…”