Proteomics of dense core secretory vesicles reveal distinct protein categories for secretion of neuroeffectors for cell-cell communication

Abstract

Regulated secretion of neurotransmitters and neurohumoral factors from dense core secretory vesicles provides essential neuroeffectors for cell-cell communication in the nervous and endocrine systems. This study provides comprehensive proteomic characterization of the categories of proteins in chromaffin dense core secretory vesicles that participate in cell-cell communication from the adrenal medulla. Proteomic studies were conducted by nano-HPLC Chip MS/MS tandem mass spectrometry. Results demonstrate that these secretory vesicles contain proteins of distinct functional categories consisting of neuropeptides and neurohumoral factors, protease systems, neurotransmitter enzymes and transporters, receptors, enzymes for biochemical processes, reduction/oxidation regulation, ATPases, protein folding, lipid biochemistry, signal transduction, exocytosis, calcium regulation, as well as structural and cell adhesion proteins. The secretory vesicle proteomic data identified 371 proteins in the soluble fraction and 384 membrane proteins, for a total of 686 distinct secretory vesicle proteins. Notably, these proteomic analyses illustrate the presence of several neurological disease-related proteins in these secretory vesicles, including huntingtin interacting protein, cystatin C, ataxin 7, and prion protein. Overall, these findings demonstrate that multiple protein categories participate in dense core secretory vesicles for production, storage, and secretion of bioactive neuroeffectors for cell-cell communication in health and disease.

Figure 1. Purity of isolated chromaffin granules (secretory vesicles) evaluated with (Met)enkephalin as a marker for chromaffin granules and with acid phosphatase as a marker for lysosomes

(a) Preparation of purified chromaffin granules by differential density centrifugation. The flow chart illustrates the purification scheme for chromaffin granules from bovine adrenal medulla homogenate, achieved by differential centrifugation. The homogenate (in 0.32 M sucrose buffer) is centrifuged at 365 × g to remove nuclei (P1, pellet 1) from the supernatant (S1, soluble fraction 1) that represents a crude fraction of chromaffin granules. The Granules of pelleted by centrifugation at 12,000 × g and washed three times in 0.32 M sucrose buffer to obtain enriched chromaffin granules (P5 fraction) that undergoes purification on a 1.6/0.32 M sucrose gradient subjected to ultracentrifugation (120,000 × g) to obtained a pellet of purified chromaffin granules. (b) Analyses of crude fraction of chromaffin granules on multi-step sucrose gradient. The crude chromaffin granule fraction (P2) was analyzed on a multi-step sucrose gradient of 2.2 M to 1.2 M sucrose as described in the methods. Gradient fractions were assayed for (Met)enkephalin (●) that is present in chromaffin granules, and for the lysosomal enzyme marker acid phosphatase (○). The crude P2 fraction of chromaffin granules contains enkephalin and acid phosphatase. (c) Analyses of purified chromaffin granules on multi-step sucrose gradient. The purified chromaffin granules were analyzed on the multi-step sucrose gradient of 2.2 M to 1.2 M sucrose as described in the methods. Gradient fractions were assayed for (Met)enkephalin and acid phosphatase. The presence of the purified chromaffin granules is indicated by the peak of (Met)enkephalin. The multi-step gradient showed no peak of acid phosphatase, indicating effective removal of lysosomes. These data document the purity of the chromaffin granule preparation. (It is noted that cytosolic proteins may possibly associate with the outside of the granule membrane during homogenization, and after freeze-thawing, such cytosolic proteins may become present in the soluble fraction giving the interpretation that they might be luminal proteins of the granules. Nonetheless, cytosolic proteins are likely to have importance because cellular function of the chromaffin granule must involve cytoplasmic proteins for regulated movement to achieve exocytosis.)

Figure 2. Removal of abundant chromogranin A protein from soluble and membrane fractions of purified chromaffin granules

Chromaffin granule soluble and membrane fractions were each subjected to calmodulin affinity chromatography to remove the most abundant protein consisting of full-length CgA. The soluble fraction of these chromaffin granules is illustrated (lane 1), showing full-length CgA of ~66–70 kDa that has been identified by mass spectrometry in previous studies (10, 137). After affinity chromatography on calmodulin-Sepharose conducted two times, removal of the full-length CgA is illustrated (lane 2). Equal relative volumes (5 μl) of soluble chromaffin granule sample was applied to lanes 1 and 2 (corresponding to ~2 μg and ~6.5 μg protein, respectively). The CgA depletion step recovered ~30–35% of the original proteins of the soluble chromaffin granule sample. CgA also exists as cleaved proteolytic fragments in the chromaffin granules which presumably are largely removed by the calmodulin-Sepharose affinity step. After the affinity step, the overall pattern of protein bands (lane 2) resembles that of the soluble granule sample before the affinity step, with the exception of removal of CgA protein(s).

Figure 3. Venn diagram of common and different proteins in soluble and membrane fractions of chromaffin granules

This Venn diagram illustrates the the majority of the chromaffin secretory vesicle proteins identified in this study using nano-HPLC Chip MS/MS, combined with several proteins identified in in earlier proteomic studies using gel electrophoresis for protein enrichment [43]. The soluble fraction contained 371 distinct proteins and the membrane fraction contained 384 distinct proteins. Proteins common to both soluble and membrane fractions are illustrated as the intersecting area of the Venn diagram, indicating 69 proteins that were present in both soluble and membrane compartments of these secretory vesicles. The soluble and membrane fractions contained a total of 686 unique proteins in chromaffin secretory vesicles.

Figure 4. Comparison of soluble and membrane proteins by pie charts

The relative portion of proteins in each functional category are compared for the soluble (panel A) and membrane (panel B) fractions of chromaffin secretory vesicles. Each functional category of the pie chart is shown as a distinct color.

Figure 5. Presence of cystatin C, huntingtin interacting protein, ataxin 7, and prion protein in chromaffin secretory vesicles demonstrated by western blots

Purified chromaffin secretory vesicles were subjected to western blots for analyses of cystatin C (panel a), huntingtin interactin protein (HIP) (panel b), ataxin 7 (panel c), and prion protein (panel d). Cystatin C of ~12–14 kDa in these vesicles (panel a) is similar in MW (molecular weight) to that reported in other studies [132, 133]. Huntingtin-interacting proteins (3 bands) in the area of ~ 150–250 kDa were observed (panel b). Ataxin 7 of about 98–100 kDa (panel c) is similar to that found in prior studies [49]. Prion protein of several apparent molecular weights of ~30 kDa, 36–40 kDa, and 50–60 kDa were observed (lane d).

Figure 6. Cellular localization of cystatin C with enkephalin-containing secretory vesicles of chromaffin cells in primary culture

The localization of cystatin C to secretory vesicles that contain the enkephalin neuropeptide was observed by immunofluorescence confocal microscopy of chromaffin cells primary culture. Colocalization of enkephalin (green fluorescence) and cystatin C (red fluorescence) was demonstrated by the yellow fluorescence of merged images. Examples of secretory vesicle colocalization of cystatin C and enkephalin are indicated by the arrows.

Figure 7. Multiple protein categories for biosynthesis, storage, and regulated secretion of neuroeffectors for cell-cell communication in health and disease

The chromaffin granule proteome consists of distinct functional categories of proteins utilized for secretory vesicle production, storage, and regulated secretion of neuroeffector molecules. The architecture of proteins of the soluble (blue area) and membrane (gray area) function in the initial biogenesis and subsequent maturation of secretory vesicles, which produce and store bioactive chemical molecules for secretion. The secreted neurotransmitters and hormones mediate cell-cell communication among physiological target organs.