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.
Hwang
Shin-Rong
SR
Skaggs School of Pharmacy and Pharmaceutical Sciences and Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093.
Garza
Christina
C
Skaggs School of Pharmacy and Pharmaceutical Sciences and Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093.
Mosier
Charles
C
Skaggs School of Pharmacy and Pharmaceutical Sciences and Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093.
Toneff
Thomas
T
Skaggs School of Pharmacy and Pharmaceutical Sciences and Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093.
Wunderlich
Eric
E
Buck Institute, Novato, California 94545.
Goldsmith
Paul
P
College of Pharmacy, Touro University, Vallejo, California 94592.
Hook
Vivian
V
Skaggs School of Pharmacy and Pharmaceutical Sciences and Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093. Electronic address: vhook@ucsd.edu.
D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D004745:Enkephalins / Q000096:biosynthesis* / Q000378:metabolism*; D004792:Enzyme Precursors / Q000502:physiology; D008247:Lysosomes / Q000201:enzymology; D016716:PC12 Cells; D011506:Proteins / Q000378:metabolism; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
D004745:Enkephalins; D004792:Enzyme Precursors; D011506:Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat