Peptide neurotransmitters are required for cell-cell signaling among neurons to transmit commands among neural circuits to regulate behavior and physiological functions. Such peptide neurotransmitters, also known as neuropeptides, mediate key functions of the nervous system. As an example, the enkephalin-endorphin neuropeptide family regulates pain relief and analgesia (Law et al. 2000; Gustein and Akil 2006). Galanin in brain participates in cognition for learning and memory (Steiner et al. 2001; Robinson 2004). The CRF (corticotropin-releasing factor) and dynorphin mediate stress responses related to addiction (Koob 2008; Logrip et al. 2011). NPY (neuropeptide Y) participates in brain regulation of feeding behavior and obesity (Ramos et al. 2005; Zhang et al. 2011). These and numerous other neuropeptides are required for health and are involved in human diseases (Kastin 2006; Kim et al. 2011).
Neuropeptides are generated from inactive precursor proteins by proteolytic processing (Hook et al. 2008; Seidah et al. 2008; Mbikay and Seidah 2011) in secretory vesicles, which store the neuropeptides for activity-dependent, regulated secretion for their function in neurotransmission. Endoproteolytic processing of proneuropeptides occurs at paired basic residues, as well as monobasic and multibasic residues. Proneuropeptide processing in secretory vesicles is achieved by the cysteine protease cathepsin L (Hook et al. 2008; Funkelstein et al. 2010) and the subtilisin-like prohormone convertases 1 and 2 (PC1/3 and PC2) (Seidah et al. 2008; Mbikay and Seidah 2011). Protease gene knockout mouse studies have demonstrated the prominent roles of the cathepsin L and prohormone convertase protease pathways in the biosynthesis of peptide neurotransmitters (Miller et al. 2003a, 2003b; Scamuffa et al. 2006; Hook et al. 2008; Mbikay and Seidah 2011). The well-established PC1/3 and PC2 proteases cleave dibasic residues at their COOH-terminal side, resulting in peptide intermediates with basic residues at their COOH-termini, which are removed by carboxypeptidase E (Fricker 1988). The more recently discovered cathepsin L cysteine protease cleaves dibasic residues at their N-termini, as well as between the dibasic residues, that flank neuropeptide sequences within their precursor proteins (Hook et al. 2008; Funkelstein et al. 2010). Peptide products generated by cathepsin L will then require removal of NH2- and COOH-terminal basic residues by aminopeptidase activity and carboxypeptidase E.
Because of the requirement for aminopeptidase activity to remove basic residues from peptide intermediates for neuropeptide production, this study investigated the hypothesis that cathepsin H, a cysteine protease, participates in removing basic residues from peptides to form the active enkephalin opioid neuropeptide. The rationale for this hypothesis is the knowledge that cathepsin H possesses aminopeptidase activity for NH2-terminal basic residues (Rothe and Dodt 1992; Kirschke 2004), combined with the finding in this study that cathepsin H is present in neuropeptide-producing secretory vesicles. Neuropeptide-generating aminopeptidase activity of cathepsin H was demonstrated by its sequential removal of NH2-terminal Lys-Arg and Lys-Lys residues from peptide substrates to generate the active (Met)enkephalin neuropeptide, illustrated by HPLC and mass spectrometry. Notably, cathepsin H knockout mice show significant reduction in brain levels of (Met)enkephalin and galanin neuropeptides, indicating the functional role of cathepsin H to produce peptide neurotransmitters. These findings support the hypothesis for participation of secretory vesicle cathepsin H in neuropeptide biosynthesis.
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- Materials and methods
- Conflict of interest
- Supporting Information
Results of this study show that cathepsin H participates in the in vivo production of the peptide neurotransmitter (Met)enkephalin, by functioning as an aminopeptidase to remove NH2-terminal basic residues from peptide intermediates to generate active ME in neuropeptide secretory vesicles. Cathepsin H knockout mouse brains contain reduced levels of ME, that were approximately 50% lower than wild-type control mouse brains. Cathepsin H removes basic residues (Lys, Arg) from the NH2-termini of neuropeptide intermediates, to generate mature ME. Cathepsin H is colocalized with ME in secretory vesicles, the primary subcellular site of neuropeptide production. Western blots showed the presence of cathepsin H in secretory vesicles from adrenal medulla; immunoelectron microscopy demonstrated localization of cathepsin H within neuropeptide secretory vesicles, and immunofluorescent microscopy of neuronal-like chromaffin cells illustrated the colocalization of cathepsin H and ME. Furthermore, cathepsin H knockout mice showed a substantial decrease in brain levels of galanin, another peptide neurotransmitter. Interestingly, cathepsin H knockout mouse brains showed no change in dynorphin A, β-endorphin, or CRF neuropeptides, indicating the selectivity of cathepsin H for biosynthesis of enkephalin and galanin. These findings support the hypothesis that cathepsin H in secretory vesicles particpates in the production of selected peptide neurotransmitters.
The findings of this study support the hypothesis that cathepsin H provides Lys/Arg aminopeptidase activity for removal of NH2-terminal basic residues from peptide intermediates generated from the proneuropeptide by secretory vesicle cathepsin L, which cleaves at the NH2-terminal side of dibasic residues, as well as between dibasic residues (Hook et al. 2008; Funkelstein et al. 2010) (Fig. 7). Subsequent to cathepsin L, both Lys/Arg aminopeptidase and carboxypeptidase E (CPE) (Fricker 1988) remove basic residues from NH2- and COOH-termini of peptide intermediates, respectively. Cathepsin L functions jointly with the prohormone convertases 1 and 2 (PC1/3 and PC2), subtilisin-like proteases, which prefer to cleave at the COOH-terminal side of dibasic residues (Fig. 7). Peptide products generated by these PC enzymes then require CPE to remove COOH-terminal basic residues to produce the active neuropeptide.
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.
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Production of (Met)enkephalin from the proenkephalin precursor utilizes cathepsin L and PC2, based on data from protease gene knockout mice (Miller et al. 2003a; Yasothornsrikul et al. 2003). Participation of cathepsin L, generating peptide intermediates with basic residue extensions at their N-termini, implicates roles for aminopeptidases to generate mature (Met)enkephalin. Such aminopeptidase activity of cathepsin H may be involved in enkephalin production. Notably, brain levels of (Met)enkephalin were reduced by 50% in the cathepsin H knockout mice compared to wild-type control mice. This data indicates that cathepsin H participates in enkephalin production. However, there is likely another aminopeptidase that can generate the 50% (Met)enkephalin remaining in the cathepsin H knockout mouse brain. Previous studies have demonstrated aminopeptidase B (AP-B) as a processing protease in secretory vesicles for production of (Met)enkephalin and NPY peptide neurotransmitters (Hwang et al. 2007). Results show that AP-B and cathepsin H have similar time courses for cleaving basic residues to generate (Met)enkephalin (Hwang et al. 2007; Foulon et al. 2004). These findings indicate the presence of two Lys/Arg aminopeptidase in secretory vesicles for neuropeptide production, cathepsin H and AP-B (illustrated in Fig. 7). It will be of interest in future studies to investigate the distribution of cathepsin H and AP-B between brain and neuroendocrine tissue regions to compare their utilization among neuroendocrine cells.
The peptide neurotransmitter galanin is substantially reduced in cathepsin H knockout mouse brain cortex. These results implicate cathepsin L in processing progalanin. Indeed, cathepsin L knockout mice show significant reduction of galanin in brain that is decreased by about 80% compared to wild-type controls (Figure S6). These data support the hypothesis that cathepsin H participates in galanin production.
It is of interest that dynorphin A, β-endorphin, and CRF peptide neurotransmitter levels in cathepsin H knockout mouse brains were not altered, indicating the selectivity of cathepsin H for producing galanin and (Met)enkephalin. Although dynorphin A and β-endorphin utilize the endoprotease cathepsin L in their biosynthesis (Funkelstein et al. 2008; Minokadeh et al. 2010), it is hypothesized that subsequent removal of N-terminal basic residues may be achieved by aminopeptidase B or cathepsin H (Fig. 7). But as the cathepsin H knockout mice show no changes in brain levels of dynorphin A or β-endorphin, it is likely that AP-B may be involved in dynorphin and β-endorphin production. Results of this study indicate the selective nature of cathepsin H for producing (Met)enkephalin and galanin neuropeptides.
The biochemical properties of cathepsin H are compatible with its proposed function in secretory vesicles for neuropeptide production. The pH optimum of cathepsin H (pH 5.5–7.0) indicates that this enzyme is active within the internal pH environment of secretory vesicles of pH 5.5–6.5 (Hook et al. 2008). Cathepsin H activity occurs under reducing conditions (Kirschke 2004), which is compatible with the in vivo reducing conditions within secretory vesicles that contain reducing factors including ascorbic acid and glutathione (Yasothornsrikul et al. 1999). These pH and reducing conditions for cathepsin H resemble that of aminopeptidase B (AP-B) that also possesses Lys/Arg aminopeptidase activity. However, inhibitors distinguish cathepsin H from AP-B, as inhibitors of AP-B (bestatin and arphemenine A) have no effect on cathepsin H. Cathepsin H and AP-B, thus, represent distinct Arg/Lys aminopeptidases in secretory vesicles for neuropeptide production. It will be of interest in future studies to learn how natural occuring inhibitors of cathepsin H, the cystatins (Kirschke 2004), may be regulated in neuropeptide biosynthesis.
Results from this study illustrate a novel biological function for cathepsin H aminopeptidase activity in secretory vesicles, for production of an active peptide neurotransmitter. In addition to data of this study showing the presence of cathepsin H in secretory vesicles, an earlier investigation also demonstrated the presence of cathepsin H in secretory vesicles of neuroendocrine GH4C1 pituitary cells (Waguri et al. 1995). This biological function of cathepsin H contrasts with its known role in lysosomes for protein degradation (Turk et al. 1997; Kirschke 2004). In fact, human cathepsin H purified from brain has been shown to degrade mature neuropeptide substrates (Brguljan et al. 2003). It is apparent that the function of cathepsin H depends on its secretory vesicle or lysosomal location.
Additional new biological functions of cathepsin H have been illustrated in the cathepsin H knockout mice. Cathepsin H null mice, achieved by gene targeting in embryonic stem cells, results in impairment of lung surfactant based on the role of cathepsin H in production of the pulmonary surfactant protein B (SP-B) (Bühling et al. 2011). Cathepsin H can act as an additional progranzyme B convertase, in addition to cathepsin C (D’Angelo et al. 2010). Also, deletion of the cathepsin H gene perturbs angiogenic switching, vascularization, and growth of tumors in a mouse model of pancreatic islet cell cancer (Gocheva et al. 2010). These findings show that cathepsin H possesses biological functions in different physiological systems.
In summary, this study shows that cathepsin H functions as an aminopeptidase in secretory vesicles for the production of (Met)enkephalin and galanin. Cathepsin H is, thus, a new protease member of the cathepsin L pathway for processing proneuropeptides, together with the prohormone convertases (PC1/3 and PC2) for production of active peptide neurotransmitters.
- Top of page
- Materials and methods
- Conflict of interest
- Supporting Information
Table S1. Masses of KR-ME and KK-ME, intermediate peptides, and ME by mass spectrometry.
Table S2. Cathepsin H cleavage of aminopeptidase substrates.
Table S3. Cathepsin H activity: effects of inhibitors of aminopeptidases.
Figure S1. Anti-cathepsin H western blots detect cathepsin H, but not the cysteine cathepsins B, L, and V.
Figure S2. Control immunogold procedure with only secondary antibody lacks immunogold detection of cathepsin H.
Figure S3. Control immunofluorescence histochemistry with only secondary antibody lacks immunofluorescence signal.
Figure S4. Analyses of KK-ME peptide products by tandem mass spectrometry (MS/MS).
Figure S5. Analyses of KR-ME peptide products by tandem mass spectrometry (MS/MS).
Figure S6. Galanin is decreased in brains of cathepsin L knockout mice.
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