Cathepsin H functions as an aminopeptidase in secretory vesicles for production of enkephalin and galanin peptide neurotransmitters

Abstract

Peptide neurotransmitters function as key intercellular signaling molecules in the nervous system. These peptides are generated in secretory vesicles from proneuropeptides by proteolytic processing at dibasic residues, followed by removal of N- and/or C-terminal basic residues to form active peptides. Enkephalin biosynthesis from proenkephalin utilizes the cysteine protease cathepsin L and the subtilisin-like prohormone convertase 2 (PC2). Cathepsin L generates peptide intermediates with N-terminal basic residue extensions, which must be removed by an aminopeptidase. In this study, we identified cathepsin H as an aminopeptidase in secretory vesicles that produces (Met)enkephalin (ME) by sequential removal of basic residues from KR-ME and KK-ME, supported by in vivo knockout of the cathepsin H gene. Localization of cathepsin H in secretory vesicles was demonstrated by immunoelectron microscopy and immunofluorescence deconvolution microscopy. Purified human cathepsin H sequentially removes N-terminal basic residues to generate ME, with peptide products characterized by nano-LC-MS/MS tandem mass spectrometry. Cathepsin H shows highest activities for cleaving N-terminal basic residues (Arg and Lys) among amino acid fluorogenic substrates. Notably, knockout of the cathepsin H gene results in reduction of ME in mouse brain. Cathepsin H deficient mice also show a substantial decrease in galanin peptide neurotransmitter levels in brain. These results illustrate a role for cathepsin H as an aminopeptidase for enkephalin and galanin peptide neurotransmitter production.

Figure 1. Cathepsin H in secretory vesicles illustrated by western blot and immunoelectron microscopy

(a) Western blot of cathepsin H in secretory vesicles isolated from bovine adrenal medulla. Secretory vesicles isolated from adrenal medulla were subjected to anti-cathepsin H western blots. Results indicate the presence of a 26-29 kDa band that likely corresponds to the single chain form of cathepsin H with calculated molecular weight of 28 kDa (Kirschke, 2004). (b) Immunoelectron microscopy of cathepsin H in isolated secretory vesicles. Cathepsin H in isolated secretory vesicles from bovine adrenal medulla was illustrated by immunoelectron microscopy. Cathepsin H is indicated by the 6 nm gold labeled particles conjugated to anti-rabbit to detect anti-cathepsin H rabbit (see methods).

Figure 2. Cathepsin H localization with (Met)enkephalin (ME) in secretory vesicles illustrated by immunofluorescence confocal microscopy of neuronal-like chromaffin cells

The colocalization of cathepsin H (Cat.H, green immunofluorescence) with ME ((Met)enk, red immunofluorescence) in secretory vesicles is shown by the merged yellow immunofluorescence areas (indicated by arrows) within neuronal-like chromaffin cells. The merged yellow immunofluorescence shows partial localization of cathepsin H with ME. Quantitation of the cathepsin H colocalized with (Met)enkephalin was assessed by measurement of the Pearson’s correlation coefficient (Rr value) of 0.77 + 0.024 (n = 12 cells), which indicates partial colocalization. An Rr value of ‘1’ indicates complete colocalization and a value of ‘0’ indicates no specific colocalization.

Figure 3. Cathepsin H removes NH₂-terminal Lys and Arg from Lys-Arg-(Met)enkephalin (KR-ME)

(a) KR-ME substrate conversion to R-ME and M: total ion chromatogram. Cathepsin H was incubated with KR-ME and nano-LC-MS/MS was conducted to identify peptide cleavage products. The total ion chromatogram (TIC) shows the presence of KR-ME substrate, and the products R-ME and ME, based on their masses (shown in supplemental Table 1). (b) R-ME identified by tandem mass spectrometry. R-ME peptide (RYGGFM) was identified by MS/MS tandem mass spectrometry. The MS/MS spectra is illustrated here; those for KR-ME and ME are shown in supplemental Figure A. (c) Time-course of cathepsin H production of R-ME and ME from KR-ME. The time course of cathepsin H conversion of KR-ME to R-ME and ME is illustrated by the open bars, hatched bars, and black solid bars, respectively. Using (Met)enkephalin standard peptide, LC peptide peak integrations vary by less than 1%, which we have previously demonstrated (Hwang et al., 2007).

Figure 4. Cathepsin H removes NH₂-terminal Lys from Lys-Lys-(Met)enkephalin (KK-ME)

(a) KK-ME substrate conversion to K-ME and M: total ion chromatogram. Cathepsin H was incubated with KK-ME and nano-LC-MS/MS was conducted to identify peptide cleavage products. The total ion chromatogram (TIC) shows the presence of KK-ME substrate, and the products K-ME and ME, based on their masses (shown in supplemental Table 1). (b) K-ME identified by tandem mass spectrometry. R-ME peptide (RYGGFM) was identified by MS/MS tandem mass spectrometry. The MS/MS spectra is illustrated here; those for KK-ME and ME are shown in supplemental Figure A. (c) Time-course of producing K-ME and ME from KK-ME by cathepsin H. The time course of cathepsin H conversion of KK-ME to K-ME and ME is illustrated by the open bars, hatched bars, and black solid bars, respectively. Using (Met)enkephalin standard peptide, LC peptide peak integrations vary by less than 1%, which we have previously demonstrated (Hwang et al., 2007).

Figure 5. Knockout of the cathepsin H gene results in decreased brain levels of (Met)enkephalin

(Met)enkephalin in brain cortex of cathepsin H knockout (KO) and wild-type (WT) mice were measured by radioimmunoassay (RIA) of brain extracted fractions. Brain tissue was prepared as an acetic acid extract, fractionated by RP-HPLC, and fractions corresponding to the elution position of (Met)enkephalin were assayed for content of (Met)enkephalin by RIA. Brain (Met)enkephalin is expressed as the pg per μg brain extract protein, calculated as the mean + s.e.m (standard error of the mean). *Statististically significant (p < 0.05, n = 8 per group).

Figure 6. Cathepsin H knockout results in a substantial decrease in galanin

Galanin in brain cortex extracts of cathepsin H knockout (KO) and wild-type (WT) mice were measured by radioimmunoassay (RIA). Galanin is expressed as pg per μg brain extract protein, calculated as the mean + s.e.m (standard error of the mean). *Statististically significant (p < 0.05, n = 8 per group).

Figure 7. Cathepsin H functions with the cathepsin L and prohormone convertase protease pathways for producing enkephalin and galanin peptide neurotransmitters

(a) Proenkephalin and progalanin proneuropeptides. The proneuropeptide precursors are schematically illustrated for proenkephalin and progalanin that undergo proteolytic processing to generate active enkephalin and galanin peptide neurotransmitters (neuropeptides). Active neuropeptides are typically flanked by dibasic residue processing sites within the precursor proteins. (b) Protease pathways for neuropeptide production: cathepsin L and prohormone convertase pathways. It is proposed that cathepsin H functions as an aminopeptidase, subsequent to the endoproteolytic action of secretory vesicle cathepsin L. Cathepsin L cleaves proneuropeptides at dibasic residues, at the NH₂-terminal side or between the dibasic residues. Cathepsin H participates as an exopeptidase to remove NH₂-terminal basic residues from peptide intermediates; aminopeptidase B (AP-B) also functions as a Lys/Arg aminopeptidase (Hwang et al., 2007) with cathepsin H. The carboxypeptidase E (CPE) exopeptidase removes COOH-terminal basic residues from peptide intermediates generated by cathepsin L, as well as by the subtilisin-like prohormone convertases (PC1/3 and PC2). Thus, cathepsin H participates in proneuropeptide processing achieved by the cathepsin L and prohormone convertase protease pathways.