Cathepsin L Expression Is Directed to Secretory Vesicles for Enkephalin Neuropeptide Biosynthesis and Secretion*

Proteases within secretory vesicles are required for conversion of neuropeptide precursors into active peptide neurotransmitters and hormones. This study demonstrates the novel cellular role of the cysteine protease cathepsin L for producing the (Met)enkephalin peptide neurotransmitter from proenkephalin (PE) in the regulated secretory pathway of neuroendocrine PC12 cells. These findings were achieved by coexpression of PE and cathepsin L cDNAs in PC12 cells with analyses of PE-derived peptide products. Expression of cathepsin L resulted in highly increased cellular levels of (Met)enkephalin, resulting from the conversion of PE to enkephalin-containing intermediates of 23, 18–19, 8–9, and 4.5 kDa that were similar to those present in vivo. Furthermore, expression of cathepsin L with PE resulted in increased amounts of nicotine-induced secretion of (Met)enkephalin. These results indicate increased levels of (Met)enkephalin within secretory vesicles of the regulated secretory pathway. Importantly, cathespin L expression was directed to secretory vesicles, demonstrated by colocalization of cathepsin L-DsRed fusion protein with enkephalin and chromogranin A neuropeptides that are present in secretory vesicles. In vivo studies also showed that cathepsin L in vivo was colocalized with enkephalin. The newly defined secretory vesicle function of cathepsin L for biosynthesis of active enkephalin opioid peptide contrasts with its function in lysosomes for protein degradation. These findings demonstrate cathepsin L as a distinct cysteine protease pathway for producing the enkephalin member of neuropeptides.

Proteases within secretory vesicles are required for conversion of neuropeptide precursors into active peptide neurotransmitters and hormones. This study demonstrates the novel cellular role of the cysteine protease cathepsin L for producing the (Met)enkephalin peptide neurotransmitter from proenkephalin (PE) in the regulated secretory pathway of neuroendocrine PC12 cells. These findings were achieved by coexpression of PE and cathepsin L cDNAs in PC12 cells with analyses of PE-derived peptide products. Expression of cathepsin L resulted in highly increased cellular levels of (Met)enkephalin, resulting from the conversion of PE to enkephalin-containing intermediates of 23, 18 -19, 8 -9, and 4.5 kDa that were similar to those present in vivo. Furthermore, expression of cathepsin L with PE resulted in increased amounts of nicotine-induced secretion of (Met)enkephalin. These results indicate increased levels of (Met)enkephalin within secretory vesicles of the regulated secretory pathway. Importantly, cathespin L expression was directed to secretory vesicles, demonstrated by colocalization of cathepsin L-DsRed fusion protein with enkephalin and chromogranin A neuropeptides that are present in secretory vesicles. In vivo studies also showed that cathepsin L in vivo was colocalized with enkephalin. The newly defined secretory vesicle function of cathepsin L for biosynthesis of active enkephalin opioid peptide contrasts with its function in lysosomes for protein degradation. These findings demonstrate cathepsin L as a distinct cysteine protease pathway for producing the enkephalin member of neuropeptides.
The biosynthesis of enkephalin opioid neuropeptides requires proteolytic processing of protein precursors within regulated secretory vesicles (1)(2)(3). Active enkephalin and related neuropeptides are stored in such secretory vesicles for regulated secretion that is induced by receptor-mediated mechanisms. Secreted enkephalin functions as an active peptide neurotransmitter in the control of analgesia for pain relief, behavioral responses, and related brain and physiological functions (4 -6).
Secretory vesicles represent the primary subcellular site for proteolytic processing of proenkephalin and other proneuropeptides (1,3). Secretory vesicles isolated from adrenal medullary chromaffin cells (also known as chromaffin granules) have provided a model system for identification of proteases for proneuropeptide processing, which includes PC1/3 and PC2 endopeptidases (7,8) and the exopeptidase carboxypeptidase E/H (9,10). These secretory vesicles contain (Met)enkephalin and its proenkephalin precursor (11,12), and, therefore, contain the appropriate processing proteases.
In vitro proneuropeptide processing assays have identified cathepsin L as a major proenkephalin-cleaving activity purified from neuropeptide-containing chromaffin granules (13,14). Cathepsin L generates (Met)enkephalin in vitro from enkephalin-containing peptide substrates, resulting from cleavage at paired basic residue processing sites, as well as monobasic residue sites.
It is notable that these findings for a potential secretory vesicle function of cathepsin L contrast with the well known lysosomal function of cathepsin L for protein degradation. Therefore, the important question of whether cathepsin L expression is directed to secretory vesicles for proteolytic processing of proenkephalin must be answered by direct cellular gene expression experiments. Therefore, this study evaluated (Met)enkephalin production during expression of cathepsin L in the regulated secretory pathway of neuroendocrine PC12 cells. PC12 cells have been extensively utilized as a model for regulated secretion (15)(16)(17)(18)(19)(20)(21)(22)(23)(24).
Results showed that expression of cathepsin L in PC12 cells resulted in the increased production of (Met)enkephalin, accompanied by the production of proenkephalin-derived intermediates that resemble those in vivo. Importantly, expression of cathepsin L resulted in elevated amounts of nicotineinduced secretion of (Met)enkephalin. Colocalization of cathepsin L with enkephalin in regulated secretory vesicles was demonstrated by immunofluorescent microscopy. Although expression of the cathepsin L-DsRed fusion protein was directed to neuropeptide-containing secretory vesicles, expression of the DsRed protein alone resulted in its localization throughout the cell. These novel findings demonstrate that the cysteine protease cathepsin L participates in the biosynthesis of the enkephalin peptide neurotransmitter in the regulated secretory pathway of neuroendocrine PC12 cells. These find-ings provide evidence for cathepsin L as a cysteine protease pathway for the biosynthesis of enkephalins and possibly other neuropeptides.

EXPERIMENTAL PROCEDURES
Construction of Prepro-cathepsin L cDNA in the pcDNA3.1 Vector-The pcDNA3.1 plasmid vector was used for expression of cathepsin L in PC12 cells. The bovine cDNA encoding prepro-cathepsin L was obtained by reverse transcription-PCR from total bovine pituitary RNA isolated by the TRIzol reagent (Invitrogen). The first strand cDNA was generated by Super-Script II reverse transcriptase with oligo(dT) [12][13][14][15][16][17][18] (Invitrogen) using conditions recommended by the manufacturer (Invitrogen). PCR with the first strand cDNA as template and Taq polymerase (Qiagen, Valencia, CA) utilized primers (0.4 M) consisting of 5Ј-AAAAGCTAGCATCCACCATGAATCCTTCATTCT-TCCTG ACTGT-3Ј (with NheI site, underlined) for the 5Јprimer and 5Ј-AAAAAGGATCCTCAAACAGTTGGATAG-CTGGCTGCTGT-3Ј (with BamHI site, underlined) for the 3Ј-primer, under PCR cycle conditions of 94°C for 60 s, 44°C for 60 s, and 72°C for 80 s, for a total of 30 cycles. The amplified prepro-cathepsin L cDNA fragment (size of about 1.0 kb) was double-digested with NheI/BamHI and ligated to NheI/BamHI digested pcDNA3.1 plasmid expression vector (Invitrogen). This construct was subjected to DNA sequencing (Davis DNA sequencing Inc., Davis, CA) to verify the nucleotide sequence and deduced primary amino acid sequence of the bovine prepro-cathepsin L cDNA.
Prepro-cathepsin L-DsRed Fusion Construct for Expression-A fusion construct of prepro-cathepsin L (bovine) with DsRed fused to the COOH terminus of cathepsin L was prepared in the pDsRed1-N1 vector (BD Biosciences). The prepro-cathepsin L without termination codon was obtained by PCR of the bovine prepro-cathepsin L cDNA with Pfx DNA polymerase (Invitrogen) with primers consisting of 5Ј-AAAAGCTAGCATCCA-CCATGAATCCTTCATTCTTCCTGACTGT-3Ј (with NheI site, underlined) and 5Ј-AAAAAGGATCCAACAGTTGGAT-AGCTGGCTGCTGT-3Ј (with BamHI site, underlined), using PCR cycling conditions (30 cycles total) with each cycle consisting of 94°C for 60 s, 44°C for 60 s, and 68°C for 80 s. The amplified DNA fragment was digested with BamHI/NheI and ligated to BamHI/NheI digested pDsRed2-N1 vector (BD Biosciences) to generate the prepro-cathepsin L-DsRed construct. DNA sequencing verified that the cathepsin L-DsRed sequence was obtained by PCR and subcloning.
Evaluation of PE Processing Products by Western Blotting and Radioimmunoassay (RIA)-Transfected cells were collected by centrifugation at 12,000 ϫ g for 15 min, and pelleted cells were subjected to homogenization in buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 10 M E64c, 10 M pepstatin A, 10 M chymostatin, 0.1 mM AEBSF (4-(2-aminoethyl)benzenesulfonylfluoride), 10 M leupeptin, 0.2% Tween 20, 0.2% Triton X-100, and 3 mM CHAPS. Cells were then disrupted by freeze-thawing (three times). Solubilized proteins were collected by centrifugation at 12,000 ϫ g for 15 min at 4°C. Protein concentration was determined by the Bradford reagent (Bio-Rad) according to the protocol provided by the manufacturer.
High molecular weight proenkephalin (PE)-derived products were analyzed by Western blots with anti-(Met)enkephalin and anti-synenkephalin (recognizing the NH 2 -terminal domain of PE that lacks enkephalin sequences). Twenty micrograms of protein from transfected cells was subjected to SDS-PAGE on 12% NuPAGE gels (Invitrogen), and Western blots were performed using anti-(Met)enkephalin (from Chemicon Inc., Temecula, CA) or anti-synenkephalin, as we have described previously (25). Processed (Met)enkephalin was measured by RIA as we have described previously (14). This RIA does not cross-react with PE, and therefore, measures processed (Met)enkephalin.
Regulated Secretion of (Met)enkephalin Induced by Nicotine-Subsequent to cotransfection of PC12 cells with both PE and cathepsin L cDNAs, as described above, evaluation of regulated secretion of (Met)enkephalin was conducted. Regulated secretion was induced by nicotine (10 M) by incubation of transfected cells with nicotine for 15 min, as described previously (26). Culture media were collected, and levels of (Met)enkephalin were measured by RIA.
Colocalization of Cathepsin L with Enkephalin in Secretory Vesicles of PC12 Cells by Confocal Immunofluorescent Microscopy-PC12 cells were transfected with the prepro-cathepsin L-DsRed construct as described above for transfection of prepro-cathepsin L/pcDNA3.1, and cathepsin L-DsRed immunofluorescence was examined 3-4 days after transfection. For cellular fixation, cells were harvested and reseeded at 100,000 -500,000 cells/well in poly-D-lysine-coated 2-well chamber slides for 4 h in culture media. Culture media were then aspirated, and cells were rinsed in PBS three times. Cells were then fixed in 3.7% paraformaldehyde at room temperature for 15 min, rinsed with PBS four times, and permeabilized with 0.3% Triton X-100. Colocalization of cathepsin L-DsRed with (Met)enkephalin was performed by immunostaining of cells with anti-(Met)-enkephalin (1:100 dilution, Chemicon Inc., Temecula, CA) in PBS containing 1% bovine serum albumin overnight at 4°C. Cells were washed and then incubated with Alexa Fluor 488 goat anti-mouse (Molecular Probes/Invitrogen) in PBS containing 1% bovine serum albumin for 1 h at room temperature. Finally, slides were mounted with VectaShield mounting medium containing 4Ј,6-diamidino-2-phenylindole staining and analyzed for immunofluorescence of cathepsin L-DsRed (red fluorescence) and enkephalin immunofluorescence (green fluorescence) using a Nikon Eclipse 800 microscope coupled to a PCM-2000 confocal system with image analyses by SimplePCI software. Alternatively, cathepsin L-DsRed colocalization was also examined with the secretory vesicle marker chromogranin A (1:100, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), visualized with anti-rabbit AgG/Alexa 488 (green fluorescence).
In Vivo Colocalization of Cathepsin L with Enkephalin in Adrenal Medulla (Bovine)-Fresh adrenal medullas (bovine) were obtained, and tissue slices were fixed as we have described previously (27). Cathepsin L localization with (Met)enkephalin was performed as described previously (27) with cathepsin L visualized by rabbit anti-cathepsin L (Athens Research & Technology, Athens, GA) and mouse anti-(Met)enkephalin (Chemicon, Temecula, CA). Cathepsin L was detected with anti-rabbit IgG-Alexa Fluor 488 (green fluorescence, Molecular Probes/Invitrogen), and (Met)enkephalin was detected with anti-mouse IgG-Alexa Fluor 594 (red fluorescence). Confocal immunofluorescence microscopy was conducted as described above for PC12 cells.

Cellular Cathepsin L Expression Generates (Met)enkephalin in the Regulated Secretory Pathway of PC12 Cells-Neuroendo-
crine PC12 cells were utilized in this study because they contain the regulated secretory pathway that is utilized for proteolytic processing of proneuropeptides and prohormones (15-24, 28 -30). In addition, PC12 cells lack substantial amounts of prohormone processing enzymes, demonstrated by expression of intact proopiomelanocortin in these cells (29). For these reasons, PC12 cells were an ideal choice for studies of proenkephalin expression and processing during expression of cathepsin L in the regulated secretory pathway.
PC12 cells were transfected with the preproenkephalin (PPE) and preprocathepsin L cDNAs for analyses of (Met)enkephalin production. The production of (Met)enkephalin was quantitated by a specific RIA that specifically detects (Met)enkephalin but not its PE precursor. Cellular (Met)enkephalin was elevated 8-fold in cells expressing cathepsin L and PE when compared with cells expressing PE alone (Fig. 1). Cathepsin L expression in PE-containing cells generated ϳ80-fold greater levels of (Met)enkephalin when compared with vector control. Expression of cathepsin L alone produced a small increase in (Met)enkephalin, suggesting that PC12 cells endogenously express low levels of PE. In addition, expression of PE alone resulted in a small increase in (Met)enkephalin levels, suggesting the presence of low amounts of endogenous PE processing enzyme(s); endogenous enzymes may include low levels of prohormone convertases or cathepsin L itself. Notably, (Met)enkephalin production was increased by nearly 10-fold in cells coexpressing cathepsin L and PE when compared with cells expressing only PE or only  cathepsin L. These results indicate the participation of cathepsin L in the cellular production of (Met)enkephalin.
To assess the cathepsin L-mediated production of cathepsin L with enkephalin in the regulated secretory pathway, nicotinestimulated secretion of (Met)enkephalin was tested. Amounts of (Met)enkephalin secreted during nicotine stimulation of cells were increased by 5-fold in cells expressing cathepsin L when compared with control cells without expression (Fig. 2). These results demonstrate a functional role for cathepsin L in the regulated secretory pathway for the production of (Met)enkephalin.

Cathepsin L Expression in PC12 Cells Generates PE-derived Intermediates That Resemble Those in Vivo in Chromaffin
Granules-Cathepsin L processing of PE in PC12 cells was analyzed by epitope-specific antisera to proenkephalin domains in Western blots. Cells transfected with the PPE cDNA showed the presence of PPE and PE as 33-and 31-kDa bands, respectively, detected by Western blots with anti-enkephalin serum (Fig. 3a). These findings are consistent with the production of PPE with a signal peptide that is removed at the rough endoplasmic reticulum to generate the slightly smaller PE, which is routed to secretory vesicles for proteolytic processing (1).
Importantly, cathepsin L generated PE-derived peptides in transfected PC12 cells that resemble in vivo PE-derived intermediates (Fig. 4). In vivo, the primary PE-derived products in adrenomedullary chromaffin cells consisted of 23-, 18 -19-, 8 -9-, and 4.5-kDa enkephalin-containing peptides (Fig. 4a). PC12 cells expressing PE and cathepsin L showed the same intermediates of 23, 18 -19, 8 -9, and 4.5 kDa (Fig. 4b). These results demonstrate that cathepsin L expression in PC12 cells produces PE-derived intermediates that resemble those in vivo in adrenal medulla. These results support a role for cellular cathepsin L processing of PE.
Secretory Vesicle Localization of Cathepsin L with Enkephalin in Vitro and in Vivo-Support for the cathepsin L production of enkephalin in secretory vesicles was indicated by their cellular  colocalization, examined by immunofluorescence confocal microscopy. During coexpression of cathepsin L and PPE cDNAs, cathepsin L (red immunofluorescence) and (Met)enkephalin (green immunofluorescence) were colocalized, as demonstrated by the merged images indicating their colocalization (Fig. 5, indicated by yellow immunofluorescence). Cathepsin L and enkephalin showed punctate patterns of immunostaining that are consistent with localization to secretory vesicles. Furthermore, in vivo, adrenal medulla chromaffin cells showed a discrete pattern of cathepsin L immunofluorescence that was colocalized with (Met)enkephalin (Fig. 6). These findings illustrate the secretory vesicle localization of cathepsin L in neuronal PC12 cells in vitro and in vivo in adrenal medullary chromaffin cells.

Trafficking of Cathepsin L-DsRed Fusion Protein to Secretory
Vesicles in PC12 Cells-To assess cathepsin L expression and protein trafficking to secretory vesicles, the cathepsin L-DsRed   fusion protein construct was utilized to allow direct fluorescence visualization of the subcellular localization of DsRed fused with cathepsin L, when compared with expression of DsRed alone. Expression of cathepsin L-DsRed resulted in its localization with the endogenous secretory vesicle marker chromogranin A (CgA) (Fig. 7a). Notably, nearly all the cathepsin L-DsRed was colocalized with chromogranin A in a punctate pattern of staining. In contrast, expression of DsRed alone resulted in its presence throughout the cell in nuclei and cytoplasm (Fig. 7b). These results demonstrate that cathepsin L possesses the ability to direct the trafficking of the heterologous DsRed protein to secretory vesicles. Furthermore, neuroendocrine types of cells, represented by PC12 cells, apparently possess mechanisms to route cathepsin L to secretory vesicles. Thus, expression of cathepsin L in PC12 cells that contain the regulated secretory pathway results in trafficking of cathepsin L to secretory vesicles for the production, storage, and secretion of the (Met)enkephalin peptide neurotransmitter.

DISCUSSION
Expression of cathepsin L with PE in neuroendocrine PC12 cells resulted in the conversion of PE to enkephalin-containing intermediates to mature (Met)enkephalin (Fig. 8) in the regulated secretory pathway. Cathepsin L generated high molecular mass PE-derived intermediates of 26 to ϳ4.5 kDa that were similar to those present in vivo in enkephalin-containing secretory vesicles of adrenal medullary chromaffin cells (11,31). Cathepsin L processing of PE resulted in elevated levels of cellular (Met)enkephalin with increased amounts of nicotine-induced secretion of (Met)enkephalin via the regulated secretory pathway. Furthermore, cathepsin L was colocalized to enkephalin-containing secretory vesicles. Expression of cathepsin L-DsRed resulted in its trafficking to secretory vesicles that contain the chromogranin A (a marker for secretory vesicles), whereas DsRed expression alone (not fused to cathepsin L) resulted in the localization of DsRed throughout the cell in nuclei and cytoplasm. Clearly, cathepsin L expression in neuroendocrine PC12 cells allows trafficking of cathepsin L to secretory vesicles. In addition, in vivo colo-calization of cathepsin L with enkephalin in adrenal medulla was demonstrated. These findings indicate that cellular cathepsin L participates in the processing of proenkephalin to produce the active (Met)enkephalin peptide neurotransmitter in the regulated secretory pathway. These results demonstrate that cathepsin L functions as a novel processing enzyme that converts a neuropeptide precursor into (Met)enkephalin that functions as a peptide neurotransmitter. These findings provide significant evidence for cathepsin L as a cysteine protease pathway for the biosynthesis of enkephalins and possibly other neuropeptides including the proopiomelanocortin-derived peptide hormones. 3 Importantly, cellular cathepsin L participates in the proteolytic processing of PE, as illustrated by expression of cathepsin L with PE in neuroendocrine PC12 cells (derived from rat adrenal medullary pheochromocytoma). Immunoblot analyses with anti-(Met)enkephalin and with anti-synenkephalin that recognizes the NH 2 -terminal domain of PE (bovine) showed that cathepsin L generated NH 2 -terminal domain-containing intermediates of 26, 22-23, 18 -19, and 8 -9 kDa (Fig. 8). Moreover, these PE intermediates are present in vivo in chromaffin secretory vesicles (11,31). Clearly, cathepsin L converts PE to intermediates and peptide products in neuroendocrine PC12 cells that resemble those in vivo.
Secretory vesicle cathepsin L has been shown to cleave proenkephalin-and enkephalin-containing intermediates at the NH 2 -terminal side of paired basic residues, and occasionally, between the dibasic residues (32)(33)(34)(35). Such cleavage specificity would result in the production of enkephalin peptide intermediates with basic residue extensions at NH 2 and COOH termini. These basic residues at NH 2 and COOH termini can be removed by the exopeptidases aminopeptidase B and carboxypeptidase E/H that are present in PC12 cells (28,36). Aminopeptidase B removes NH 2 -terminal basic residues from neuropeptides in secretory vesicles (37)(38)(39)(40) and has been demonstrated to be colocalized by electron microscopy with enkephalin in regulated secretory vesicles of adrenal medullary chromaffin cells (37). Furthermore, removal of COOH-terminal basic residues of neuropeptides is accomplished by carboxypeptidase E/H (41,42), which is also present in secretory vesicles with enkephalin and neuropeptides. Thus, expression of proenkephalin with cathepsin L in PC12 cells, combined with endogenous aminopeptidase B and CPE/H, yields mature, processed (Met)enkephalin measured by radioimmunoassay in this study.
Significantly, expression of cathepsin L promotes the production and secretion of (Met)enkephalin in the regulated 3 V. Hook, unpublished observations. secretory pathway, demonstrated by increased nicotinestimulated secretion of (Met)enkephalin. Colocalization of cellular cathepsin L to enkephalin-containing secretory vesicles in transfected PC12 cells, as well as in adrenal medullary chromaffin cells in vivo, supports the hypothesis that cathepsin L functions within secretory vesicles for the production of (Met)enkephalin. These results demonstrate a biological role of cathepsin L for the production of an active peptide neurotransmitter.
The secretory vesicle function of cathepsin L for the production of an active neuropeptide is distinct from its well known lysosomal function for protein degradation. It is notable that nearly all of the expressed cathepsin L in neuroendocrine PC12 cells was localized to neuropeptide-containing secretory vesicles. It was of interest that the cathepsin L-DsRed fusion protein was routed to secretory vesicles, but DsRed alone (without signal peptide) was present throughout the cell in nuclei and cytoplasm. Thus, cathepsin L directs the trafficking of the heterologous DsRed protein to secretory vesicles. It is notable that cathepsin L in PC12 cells is routed almost entirely to secretory vesicles, based on its colocalization with the secretory vesicle markers enkephalin and chromogranin A. These results suggest that PC12 cells route the majority of cathepsin L to secretory vesicles rather than to lysosomes. It is apparent that in neuroendocrine PC12 cells, cathepsin L is routed to secretory vesicles that produce, store, and secrete neuropeptides.
These studies of cathepsin L expression in PC12 cells for (Met)enkephalin production complement cathepsin L gene knock-out studies that demonstrate reduction of (Met)enkephalin in brain by ϳ50% (14). The direct expression experiments of this study demonstrate the active participation of cathepsin L in the proteolytic processing of PE that is required for the biosynthesis of (Met)enkephalin. Thus, the cellular expression studies of cathepsin L, combined with the cathepsin L gene knock-out data, together provide support for a role of cathepsin L in producing (Met)enkephalin.
The role of cathepsin proteases in secretory vesicles has been extended in several studies, demonstrating biological roles for cathepsins in these vesicles. For example, cathepsin B has been found to be present in granules of rat islets of Langerhans where proinsulin is converted to insulin (43,44). In the pancreatic secretory pathway, cathepsin B is involved in trypsinogen activation during hereditary pancreatitis (45). In juxtaglomerular secretory granules, cathepsin B participates in processing prorenin (46,47). With respect to neuronal functions, cathepsin B has been identified in neurosecretory vesicles of adrenal medullary chromaffin cells as a candidate ␤-secretase for converting amyloid precursor protein into ␤-amyloid peptides that are secreted (48); extracellular ␤-amyloid peptides accumulate in aged brains as notable amyloid deposits in Alzheimer disease (49 -51). Moreover, the cysteine cathepsins B, L, and H are localized in Golgi and regulated secretory pathways of several neuroendocrine tissues (52)(53)(54). These secretory vesicle functions of cysteine cathepsins contrast with the well known functions of cathepsin proteases in lysosomes for protein degradation. Clearly, cathepsins participate in biological functions of neuroendocrine systems.
Overall, this study demonstrates that cathepsin L functions as a proneuropeptide processing enzyme for the conversion of proenkephalin to mature (Met)enkephalin, an opioid peptide neurotransmitter and hormone. The cathepsin L processing pathway represents an alternative route for the biosynthesis of active neuropeptides, in addition to the well known proprotein convertase family of processing proteases. Among the neuroendocrine members of the proprotein convertase family, PC2, has been shown to be involved in proneuropeptide processing for the production of (Met)enkephalin (55,56) and other peptide neurotransmitters and hormones. Findings from this study lead to the hypothesis for distinct protease pathways for processing proneuropeptide and related prohormones, the cysteine protease cathepsin L and the subtilisin-like proprotein convertases. It will be of interest in future studies to assess the coordinate regulation of these protease pathways for the biosynthesis of active peptide neurotransmitters.