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

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.