Characterization of human prorenin expressed in mammalian cells from cloned cDNA.

Abstract

Human preprorenin was synthesized in Chinese hamster ovary (CHO) cells transfected with an expression vector containing renin cDNA sequences. These cells secrete an inactive form of renin (EC 3.4.23.15) that can be activated by trypsin. This inactive renin is precipitable by antibody generated against purified human renal renin and also by antisera generated to a synthetic peptide derived from the amino acid sequence of the pro segment of preprorenin (anti-propeptide), indicating that the secreted inactive enzyme is a form of prorenin. Analysis of [35S]methionine-labeled proteins immunoprecipitated from CHO cell conditioned culture medium indicates that prorenin is expressed in CHO cells as two distinct forms that differ in their degree of glycosylation. In vitro trypsin activation of prorenin cleaves approximately 4.5 kDa from the protein, rendering it unreactive with the antipropeptide antiserum but still recognizable by anti-renal renin antibody. These results show directly that the prorenin expressed by CHO cells is an inactive enzyme that is activated by trypsin cleavage of the pro segment. The ability to express human renin in this form will allow for the purification of both active and inactive forms of the enzyme in quantities sufficient for detailed physiological and structural studies.The renin-angiotensin system plays a pivotal role in blood pressure homeostasis and in the regulation of extracellular fluid volume. Renin (EC 3.4.23.15) is an aspartyl protease that catalyzes the first and rate-limiting step in the sequence of reactions that leads to the production of the potent pressor substance angiotensin II (1). Renin is found in highest concentration in kidney (2) from which it is secreted into the bloodstream; however, the enzyme has also been detected in several extrarenal tissues (3). Additionally, human kidney, plasma, and amniotic fluid contain an inactive form of renin that can be activated by a variety of treatments, e.g., incubation with trypsin (4). Although the inactive form is usually the predominant species in human plasma, accounting for up to 90% of the total renin (4), the molecular identity of inactive renin has been a matter of considerable dispute. Many biochemical and physiological questions relating to renin and prorenin remain unanswered, in part, due to the difficulty in obtaining the necessary quantities of these proteins in pure form.Amino acid (5) and cDNA (6) sequence analyses of mouse submaxillary gland renin show that this enzyme is synthesized as a precursor, containing a proposed 18-amino acid amino-terminal signal sequence and a 45-amino acid pro segment followed by the active enzyme. cDNA (7) and genomic DNA sequence analyses (8-10) of human renin suggest that the human enzyme is also synthesized as preprorenin [with a 20-amino acid signal sequence and 46-amino acid pro segment (43)]. It has often been suggested that plasma and tissue inactive renins may be prorenin (4, 11), and this view is supported by experiments (12)(13)(14) showing that inactive renin fro...

Prorenin is sorted into the regulated secretory pathway independent of its processing to renin in mouse pituitary AtT‐20 cells

“…Expression plasmids coding for human preprorenin and a mutant of its processing site were introduced into AtT-20 cells which process prorenin to renin and have both constitutive and regulated pathways [8,9]. Since the activity of renin promoter is tissue-specific, human preprorenin cDNA was placed under the Rous sarcoma virus enhancerlpromotor which is active in AtT-20 cells RSVLTR, ROW sarcoma virus long terminal repeat; SVE Intron Poly(A), the intron and the polyadenylation signal from SV40 early gene; Ori, the replication origin of ColEi; Pl, the pBR322 Pl promotor; Ptk, the thymidine kinase promotor from herpes simplex virus; G418/Km, Tn5 derived neomycin phosphotransferase gene; tk Poly(A), the polyadenylation signal from the thymidine kinase gene.…”

“…A further investigation on the cellular pathway of prorenin and renin secretion was carried out by using 8-bromo-CAMP (B-Br-CAMP) to stimulate secretion via the regulated pathway [8,9,13]. After the labeling period, cells were chased for 3 h with unlabeled medium (first chase period) and, finally, replicated cultures were incubated for a further 3 h in unlabeled medium with or without 5 mM 8-Br-CAMP (second chase period).…”

“…Alternatively, each cleaved product may possess a sorting signal and may be sorted individually after processing. In order to clarify this, the native prorenin and the mutant of its processing site were expressed in AtT-20 cells which process prorenin to renin and have both regulated and constitutive secretory pathways [8,9].…”

“…To solve this problem, a native human prorenin and a mutant of its processing site (Arg-43 to Gln) were expressed in AtT-20 cells which are known to process prorenin to renin and have both regulated and constitutive pathways [8,9].…”

Molecular Biology of Human Renin and Its Gene

“…CLONING AND STRUCTURAL ANALYSIS OF THE HUMAN RENIN GENE To obtain human renin gene sequences, we earlier cloned the human renin chromosomal gene [10], using a mouse cDNA probe [27], and later cloned a human renin cDNA [28]. The chromosomal gene was sequenced in the 5'-and 3'-flanking DNA, the exons, and portions of the introns.…”