Figure 4.

p331 is a novel protein named NSPA. (A) Preparative immunoprecipitation of neuronal anti-P target. Presynaptosomal fractions (P2) obtained from 50 rat brains were subjected to immunoprecipitation with affinity-purified anti-P antibodies. SDS-PAGE and Coomassie blue staining show two main bands (asterisks; IgG-HC, IgG–heavy chain) that were analyzed by mass spectrometry and microsequencing. The 180-kD protein corresponds to clathrin, whereas p331 corresponds to the human protein NP055928 of unknown function. (B) Sequence of p331-derived peptides, possible P epitopes, and the immunogenic NSPA peptide. Six out of six peptides derived from p331 (P1–6) matched perfectly with the putative human protein NP055928 at the indicated regions. We named this protein NSPA. The NSPA sequences ₆₄₄DDLG₆₄₇ and ₂₈₈₄GLFD₂₈₈₇ are homologous to the ribosomal DDMG and GLFD P epitopes within the 11 C-terminal region. The peptide sequence 2876–2886 used to immunize rabbits to obtain anti-NSPA antibodies is also shown.