Functional Analysis of a Mutation Occurring between the Two In-frame AUG Codons of Human Angiotensinogen

J. Howard Pratt,Ituro Inoue,Toshiaki Nakajima,Laura J. Bloem,Andreas Rohrwasser,Jean-Marc Lalouel,Tong Cheng

doi:10.1074/jbc.274.50.35749

J. Howard Pratt, Ituro Inoue + Show 5 more

Open Access

https://doi.org/10.1074/jbc.274.50.35749

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Abstract

Angiotensinogen (ANG) is the specific substrate of the renin-angiotensin system, a major participant in blood pressure control. We have identified a natural mutation at the -30 amino acid position of the angiotensinogen signal peptide, in which an arginine is replaced by a proline (R-30P). Heterozygous individuals with R-30P showed a tendency to lowered plasma angiotensinogen level (1563 ng of ANG I/ml (range 1129-1941)) compared with normal individuals in the family (1892 ng of ANG I/ml (range 1603-2072)). Human angiotensinogen mRNA has two in-phase translation initiation codons (AUG) starting upstream 39 and 66 nucleotides from the cap site. R-30P occurs in a cluster of basic residues adjacent to the first AUG codon that may affect intracellular sorting of the nascent protein. Pulse-chase experiments in transiently transfected cultured cells revealed that the R-30P mutation was associated with reduced amounts of both intra- and extracellular protein. In a cell-free system, we found that two forms of native angiotensinogen were generated by alternative initiation of translation at either AUG codon. Alteration of either the first or second AUG codons abolished the synthesis of the longer and the shorter form of native angiotensinogen, respectively. Furthermore, the rate of secretion of the shorter form was lower than that of the longer form. By transplanting angiotensinogen signal peptide onto green fluorescence protein, however, we found that both forms of the signal peptide could target green fluorescence protein, normally localized in the cytoplasm, to the secretory pathway. Although the R-30P mutation may not affect intracellular sorting of angiotensinogen in a qualitative manner, it leads to a quantitative reduction in the net secretion of mature angiotensinogen through decreased translocation or increased residence time in the endoplasmic reticulum.

Highlights

** Present address: Laboratory of Molecular Genetics, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 3718512, Japan
Analysis of RϪ30P Mutation in Human Angiotensinogen Signal Peptide—While screening patients with essential hypertension, we identified a molecular variant, a G to A nucleotide substitution in exon 2 of angiotensinogen gene (AGT), which resulted in the replacement of arginine by proline residue at the Ϫ30 amino acid position (RϪ30P) in the angiotensinogen signal peptide (Fig. 1A)
The RϪ30P Mutation Leads to Decreased Secretion of Angiotensinogen—Supporting evidence is accumulating that molecular variants of angiotensinogen constitute genetic susceptibility of human essential hypertension (1–3)

Summary

Introduction

We have identified a mutation leading to the substitution of proline for arginine at the Ϫ30 amino acid position of the angiotensinogen signal peptide (RϪ30P). This substitution affects a cluster of basic residues, and it has been suggested that such motifs may play a direct role in the association between signal sequence and the membrane of the endoplasmic reticulum through electrostatic interaction with negatively charged phospholipids (9). Ii31 was transported rapidly out of the ER after synthesis, whereas Ii33 was maintained in the ER (10, 12) These authors suggested that the amino-terminal cluster of basic residues adjacent to the first AUG codon might serve as a signal for retention of Ii33 in the endoplasmic reticulum. We investigate the functional significance of the two AUG codons and the functional impact of the RϪ30P mutation on angiotensinogen processing in the secretory pathway

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