Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics

Abstract

Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In this study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared with those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, approximately 1/3 were decreased in the PC1/3 null mice relative to wild-type mice, approximately 1/3 showed no change, and approximately 1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC.

Figure 1

Comparison of results from analyses of PC1/3 null mice versus PC2 null mice. Neuropeptides detected in both the present analysis of PC1/3 null mice and previously published analysis of PC2 null mice are shown. The relative levels of peptides in PC1/3 and PC2 null mice, compared to their respective WT counterparts in each experiment, are indicated. The individual peptides used for this analysis are listed in supplementary Table S1, supplement.

Figure 2

Cleavage site analysis for PC1/3 null and PC2 null mice. Graphs show frequency at which peptides are cleaved at specific residues in peptides that either decrease or show no change in PC1/3 null or PC2 null mice relative to wild-type mice. Graph A indicates these frequencies in P2 and P1 positions. “Single R” refers to cleavages with a non-basic residue in the P2 position, but does not take into account if there is a basic residue in the P4 or P6 positions. Graph B indicates frequencies of selected residues in the P1’ and P2’ positions. The individual peptides used for this analysis are listed in Table 1, and the numbers of peptides in each category are indicated in Table S2, supplement.