Differential Neuropeptidomes of Dense Core Secretory Vesicles (DCSV) Produced at Intravesicular and Extracellular pH Conditions by Proteolytic Processing

Abstract

Neuropeptides mediate cell-cell signaling in the nervous and endocrine systems. The neuropeptidome is the spectrum of peptides generated from precursors by proteolysis within dense core secretory vesicles (DCSV). DCSV neuropeptides and contents are released to the extracellular environment where further processing for neuropeptide formation may occur. To assess the DCSV proteolytic capacity for production of neuropeptidomes at intravesicular pH 5.5 and extracellular pH 7.2, neuropeptidomics, proteomics, and protease assays were conducted using chromaffin granules (CG) purified from adrenal medulla. CG are an established model of DCSV. The CG neuropeptidome consisted of 1239 unique peptides derived from 15 proneuropeptides that were colocalized with 64 proteases. Distinct CG neuropeptidomes were generated at the internal DCSV pH of 5.5 compared to the extracellular pH of 7.2. Class-specific protease inhibitors differentially regulated neuropeptidome production involving aspartic, cysteine, serine, and metallo proteases. The substrate cleavage properties of CG proteases were assessed by multiplex substrate profiling by mass spectrometry (MSP-MS) that uses a synthetic peptide library containing diverse cleavage sites for endopeptidases and exopeptidases. Parallel inhibitor-sensitive cleavages for neuropeptidome production and peptide library proteolysis led to elucidation of six CG proteases involved in neuropeptidome production, represented by cathepsins A, B, C, D, and L and carboxypeptidase E (CPE). The MSP-MS profiles of these six enzymes represented the majority of CG proteolytic cleavages utilized for neuropeptidome production. These findings provide new insight into the DCSV proteolytic system for production of distinct neuropeptidomes at the internal CG pH of 5.5 and at the extracellular pH of 7.2.

Keywords: Neuropeptidome; cathepsin; chromaffin granule; extracellular; peptidomics; protease.

Figure 1

Neuropeptidome production by proteolysis by chromaffin granules (CG) at pH 5.5 within DCSV and at pH 7.2 of the extracellular environment. The production of the spectrum of diverse neuropeptides, the neuropeptidome, was analyzed in DCSV modeled by purified CG from adrenal medulla (bovine) by (A) proteomics identification of proneuropeptides and proteases, (B) neuropeptidomics analyses of neuropeptides generated during incubation of CG at the intravesicular pH 5.5 within DCSV and at the extracellular pH of 7.2, conducted in the absence and presence of class-specific protease inhibitors, and (C) proteolytic cleavage specificity analyses of CG proteases using a synthetic peptide substrate library by multiplex substrate profiling by mass spectrometry (MSP-MS) assays.

Figure 2

Diverse proteases and neuropeptides identified in CG by proteomics and peptidomics. (A) CG proteases numbering 64 were identified by proteomics and were categorized into four different classes. (B) Proteases were quantified by NSAF (normalized spectral abundance factor), and abundances are illustrated by the pie chart. (C) Proneuropeptides were identified by proteomics and quantified by NSAF. (D) Peptidomics identified 1981 unique peptides of which 1239 were derived from 12 different proneuropeptides. (E) Sequence logo shows the frequency of amino acids at the amino and carboxyl termini of proneuropeptide-derived peptides (shown by the gray area) identified by peptidomics.

Figure 3

Neuropeptidome production after incubation of CG at pH 5.5 and pH 7.2 analyzed by peptidomics. (A) The Venn diagram shows the number of neuropeptides that were significantly increased or decreased after 90 min of incubation (37 °C) at pH 5.5 and pH 7.2, compared to nonincubated control CG samples. (B) Comparison of neuropeptide abundances that were significantly increased (p < 0.05) after 90 min of incubation at pH 5.5 and pH 7.2 conditions. (C, D) Sequence logos show the relative frequencies of amino acid residues flanking the amino and carboxy termini of the peptide cleavage products derived from proneuropeptides after 90 min incubation at pH 5.5 (C) and at pH 7.2 (D).

Figure 4

Effect of protease inhibitors on CG neuropeptidome production. The heatmap shows the abundance of newly formed CG neuropeptides after 90 min of incubation with and without class-specific inhibitors (DMSO vehicle, pepstatin, E64c, AEBSF, and EDTA) and time 0 min control. Each row represents a neuropeptide. Peptide abundance was log₂ transformed and normalized within each peptide across all conditions. Neuropeptides colored in red are at higher abundance, and the ones colored in blue are at lower abundance. Hierarchical clustering was performed to group neuropeptides based on similar protease inhibition profiles. Data were shown as the average of three biological replicates.

Figure 5

Neuropeptidomes generated from chromogranin A (CHGA) and proenkephalin (PENK) proneuropeptides in the presence of class-specific protease inhibitors. For each amino acid of proneuropeptides, the height of the green bars is proportional to the number of peptide amino acids overlapping the indicated region. The darkness of the color is proportional to the sum of the quantified peptide intensities. These heat maps illustrate peptides generated from CHGA and PENK that were sensitive to each of the indicated protease inhibitors of pepstatin, E64c, AEBSF, or EDTA.

Figure 6

MSP-MS reveals distinct substrate cleavage properties of CG proteases at pH 5.5 compared to pH 7.2. (A) MSP-MS (multiplex substrate profiling mass spectrometry) was performed at pH 5.5 and pH 7.2 to characterize CG protease activities at the internal DCSV pH of 5.5 and at the extracellular pH of 7.2 into which CG contents are released. (B, C) Substrate cleavage specificity profiles of CG proteases at pH 5.5 (B) and pH 7.2 (C). (D, E) The bar graphs summarize the cleavage frequency at each peptide bond within the 14-mer peptide substrates of the library at pH 5.5 (D) and pH 7.2 (E).

Figure 7

Inhibitor-sensitive protease cleavage profiles of CG neuropeptidome production at pH 5.5 and pH 7.2. (A) Substrate cleavage profile of pepstatin-sensitive aspartic proteolytic activity at pH 5.5. (B, C) Substrate cleavage profile of E64c-sensitive cysteine proteolytic activity at pH 5.5 (B) and pH 7.2 (C). (D, E) Substrate cleavage profile of AEBSF-sensitive serine proteolytic activity at pH 5.5 (D) and pH 7.2 (E). (F) Substrate cleavage profile of EDTA-sensitive proteolytic activity at pH 7.2.

Figure 8

MSP-MS cleavage profiles of recombinant cathepsins A, B, C, D, as well as CPE. Recombinant, purified enzymes were subjected to substrate cleavage profiling analysis by MSP-MS. Sequence logos show the preferred amino acid adjacent to the cleavage site (P1–P1′), and the frequency of cleavages at each of the peptide bonds within the 14-mer peptides of the 228 peptide library is shown for (A) cathepsin A at pH 5.5, (B) cathepsin A at pH 7.2, (C) cathepsin B at pH 5.5, (D) cathepsin B at pH 7.2, (E) cathepsin C at pH 5.5, (F) cathepsin C at pH 7.2, (G) cathepsin D at pH 5.5, (H) cathepsin L at pH 5.5, (I) carboxypeptidase E (CPE) at pH 5.5, and (J) CPE at pH 7.2.

Figure 9

Parallel cleavage profiles of CG proteolytic activity utilized for neuropeptidome production with that of selected recombinant proteases analyzed by MSP-MS. Comparison of CG protease cleavage profiles with that of selected recombinant proteases identified in CG (by proteomics), conducted by MSP-MS analysis, was assessed at pH 5.5 (A) and pH 7.2 (B). Data show the matching cleavage profiles of endogenous CG proteolytic activity with that of purified recombinant enzymes consisting of cathepsins A, B, C, D, L, and CPE, assessed for each of the 13 cleavage sites of 14-mer peptides (228 total) of the substrate peptide library.