Angiotensinogen: Definition, Mechanism, Structure and Function of Angiotensinogen Protein

It is a component of the renin-angiotensin system (RAS), a hormonal system that regulates blood pressure and fluid balance.

It is also known as a renin substrate and is a non-inhibitory member of the serpin family of proteinase inhibitors (MEROPS I4 family of inhibitors, clan ID, MEROPS identifier I04.953).

Mechanism

Angiotensinogen is catalytically cleaved by renin to produce angiotensin I in response to decreased blood pressure.

Angiotensin-converting enzyme (ACE) subsequently removes a dipeptide to produce angiotensin II, the physiologically active peptide that regulates body fluids’ volume and mineral balance.

Angiotensin I and angiotensin II can be further processed to generate angiotensin III, which stimulates the release of aldosterone and angiotensin IV. Angiotensin 1-9 is cleaved from angiotensin-1 by ACE2 and can be further processed by ACE to produce angiotensin 1-7, angiotensin 1-5, and angiotensin 1-4.

Angiotensinogen is synthesized in the liver and secreted in plasma.

Angiotensinogen appears to be associated with a predisposition to essential hypertension; It is also associated with pregnancy-induced (e.g.) hypertension (preeclampsia), a heterogeneous disorder that complicates 5-7% of all pregnancies and remains a leading cause maternal, fetal, and neonatal morbidity and mortality.

 

The renin-angiotensin system (RAS) is essential for regulating blood pressure and water and sodium homeostasis through the actions of angiotensin II (AngII).

Many other bioactive angiotensin peptides have recently been identified. RAS is now recognized as an essential regulator for various physiological and pathophysiological functions.

Angiotensinogen (AGT) is the sole precursor of all angiotensin peptides. Human AGT is 485 amino acids long, including a 33 amino acid signal peptide. The 10 N-terminal amino acids are cleaved by renin to provide angiotensin I (Angi), the source of several active angiotensin peptides.

The removal of Angi leaves behind a protein called des (Angi) AGT. Although des (Angi) AGT is 98% of the original protein, its biological properties and fate are mainly unknown.

Even fundamental questions such as the relative concentrations of intact AGT vs.. des (Angi) AGT in plasma and tissues have not been determined.

AGT is increasingly recognized as more than just a passive substrate for RAS. Recent studies have provided information on the new roles of AGT in both the peptide-dependent angiotensin and the independent forms.

Structure and function of the angiotensinogen protein

AGT is a non-inhibitory serpin (serine protease inhibitor) superfamily member.

Other serpin family members include alpha1 antitrypsin, alpha1 antichymotrypsin, and antithrombin III. The N-terminus of AGT, which encodes Angi, represents a unique extension compared to other members of the Serpin family.

An initial model of AGT structure was developed by sequence alignment with ovalbumin, which is also a non-inhibitory serpin.

The basic principles of this proposed structural model were confirmed when AGT crystallized and the structure resolved for non-glycosylated recombinant proteins from mice, rats, and humans.

Non-glycosylated AGT has a molecular weight of 53 kDa and can be present in states of up to 75 kDa, depending on the degree of glycosylation.

The variability of these glycosylation sites contributes to the difficulty of obtaining crystals of sufficient quality to allow X-ray diffraction.

Cleavage of AGT by renin is the rate-limiting step to release AngI.

The initial structural study implies that the efficiency of renin cleavage may be facilitated by interactions with domains beyond the N-terminal amino acids of AGT.

This speculation is partially based on the lower Km for AGT interactions with renin (2.6 μm) compared to an alpha1 antitrypsin chimera and the 17 N-terminal amino acids of AGT that interact with renin (47.5 μm).

This Km is also lower than the N-terminal tetradecapeptide isolated from AGT. This initial analysis suggests that there are angiotensin-dependent roles in other regions beyond the N-terminus of AGT.

Renin cleavage of AGT also exhibits species specificity. For example, human AGT cannot be easily cleaved with mouse renin. This has been shown in mice that carry a human AGT transgene that requires co-expression of human renin to facilitate AngII production.

It is assumed that the presence of Leu11-Tyr12 in mouse vs. AGT. Val11-Ile12 in human AGT leads to altered enzyme kinetics of mouse renin to cleave human AGT.

However, no studies have directly determined whether the substitution of Leu11-Tyr12 in mouse AGT for Val11-Ile12 can impair mouse renin cleavage in vivo.

Conserved Cys18-Cys138 disulfide bond in AngII-dependent functions

Initial structural analysis has noted that cysteines at positions 18 and 138 of human AGT (Cys18-Cys137 in mice) have the potential to form an intermolecular disulfide bond that is conserved in all species.

A subsequent study has confirmed the presence of this disulfide bond. The AGT protein is secreted with the disulfide bond, which an unknown mechanism can reduce.

Resolution of the crystal structure of the AGT protein found that the renin cleavage site of AGT was buried in the N-terminal tail of AGT. The conformational rearrangement that makes this site accessible for proteolysis has been revealed in human AGT and the renin complex.

The disulfide bridge was predicted to expose the N-terminus of AGT via a redox-dependent mechanism. Changes in Km during in vitro comparison of reduced vs. the oxidized states were improved by including the prorenin receptor protein in the reaction mixture.

This structural analysis anticipates that the redox state of AGT represents an AngII-dependent blood pressure regulation mechanism.

Proof of principle for this prediction was provided by the higher proportion of oxidized vs. Reduced AGT, determined by western blot, in women with preeclampsia.

The role of AGT disulfide bonding in AngII release in vivo was determined in mice with a substantial deficiency of endogenous AGT repopulated with native or mutated AGT using an adeno-associated viral (AAV) approach.

Approximately 60% of plasma AGT is oxidized in humans, but plasma AGT is almost completely oxidized in mice.

Therefore, the repopulation of AGT-depleted mice with a virus-derived native AGT should be optimized for renin cleavage. In contrast, repopulation of AGT in which both cysteines are mutated to serines would produce renin less efficiently in the spin-off of AGT.

However, a comparison of mice repopulated with native and mutated forms revealed no differences in AngII release or AngII-dependent effects, such as blood pressure and atherosclerosis.

The findings of this animal study cannot negate the potential role of this disulfide bond in the release of Angi in humans; However, the results in women with preeclampsia have to be validated by measuring both angiotensin II oxidize vs. reduced forms of AGT in a more precise quantitative method.

Potentially functional conserved sequences in the central serine domain of AGT

Analysis of conservation in AGT provides significant insight into functionally essential regions of the protein. For example, the eight amino acids encoding AngII are highly conserved in many species.

In addition to the highly encoded sequence that encodes AngII, the central hydrophobic residues that stabilize the protein structure are also highly conserved.

Also, the comparison of the AGT protein sequence conservation has revealed exciting aspects on the surface of the protein that interacts with distal and proximal renin.

AGT encodes the central serine domain, which contains other significant regions of conserved residues as mapped on the protein’s surface using Consurf, a web server for surface mapping phylogenetic information.

A conserved region is proximal to the Angi sequence, which is in direct contact with renin.

Although it is predicted to contribute to the AngII-dependent functions of AGT, there is no direct experimental evidence for this hypothesis. Two highly conserved regions are also present on the distal side of the renin-binding surface.

Due to the remote location, this face is unlikely to contribute to Angi release or AGT-renin interaction.

Some studies show that AGT exerts independent effects of AngII, and des (AngI) AGT has direct biological properties.

These include effects on kidney function, the blood-brain barrier, angiogenesis, adipose expansion, and hepatic steatosis.

The functional determination of these conserved regions and the differences between species in vivo will provide information to understand the structural contribution of AGT to its catabolic fate and biological functions dependent and independent of the release of Angi.