Functional analysis of a mutation occurring between the two in-frame AUG codons of human angiotensinogen

Angiotensinogen (ANG) is the specific substrate of the renin-angiotensin sy stem, a major participant in blood pressure control, We have identified a n atural mutation at the -30 amino acid position of the angiotensinogen signa l peptide, in which an arginine is replaced by a proline (R-30P), Heterozyg ous individuals with R-30P showed a tendency to lowered plasma angiotensino gen level (1563 ng of ANG I/ml (range 1129-1941)) compared with normal indi viduals in the family (1892 ng of ANG I/ml (range 1603-2072)). Human angiot ensinogen mRNA has two in-phase translation initiation codons (AUG) startin g upstream 39 and 66 nucleotides from the cap site. R-30P occurs in a clust er of basic residues adjacent to the first AUG codon that may affect intrac ellular sorting of the nascent protein, Pulse-chase experiments in transien tly transfected cultured cells revealed that the R-30P mutation was associa ted with reduced amounts of both intra- and extracellular protein, In a cel l-free system, we found that two forms of native angiotensinogen were gener ated by alternative initiation of translation at either AUG codon, Alterati on of either the first or second AUG codons abolished the synthesis of the longer and the shorter form of native angiotensinogen, respectively, Furthe rmore, the rate of secretion of the shorter form was lower than that of the longer form. By transplanting angiotensinogen signal peptide onto green fl uorescence protein, however, we found that both forms of the signal peptide could target green fluorescence protein, normally localized in the cytopla sm, to the secretory pathway, Although the R-30P mutation may not affect in tracellular sorting of angiotensinogen in a qualitative manner, it leads to a quantitative reduction in the net secretion of mature angiotensinogen th rough decreased translocation or increased residence time in the endoplasmi c reticulum.