Murine Prorenin Tertiary Structure Visualization

The tertiary structure of murine prorenin (PDB ID: 5MKT) was depicted by using graphical imaging software CueMol2 (Molecular Visualization Framework; http://www.cuemol.org/) (Fig 1A). Prorenin consists of renin (white ribbons) and the prosegment (colored ribbon), which covers the enzymatic active site on renin [1 (link)]. From the 43 amino acids of prosegment, we selected three different amino acid sequences (Fig 1B): E1 (L^1PPTRTATFERIPLKKMP^17P) and E2 (T^7PFERIPLKKMP^17P), both of which contains the HR I^11PPLKK^15P, and E3 (M^16PPSVREILEER^26P), which does not contain the HR. E1, E2, and E3 were conjugated to keyhole limpet hemocyanin (KLH) [11 (link)]. For each mouse, we prepared 100 μl of peptide vaccine solution, which contained 20 μg of peptide vaccine diluted in 50 μl of normal saline and 50 μl of adjuvant, as described below. We used Freund’s complete adjuvant (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in the first vaccine and Freund’s incomplete adjuvant (Wako Pure Chemical Industries, Ltd.) in the subsequent one [15 (link), 16 (link), 20 (link), 21 (link)].