Cathepsin L Colocalizes with Chromogranin A in Chromaffin Vesicles to Generate Active Peptides

Abstract

Chromogranin A (CgA), the major soluble protein in chromaffin granules, is proteolytically processed to generate biologically active peptides including the catecholamine release inhibitory peptide catestatin. Here we sought to determine whether cysteine protease cathepsin L (CTSL), a novel enzyme for proteolytic processing of neuropeptides, acts like the well-established serine proteases [prohormone convertase (PC)1/3 or PC2] to generate catestatin by proteolytic processing of CgA. We found that endogenous CTSL colocalizes with CgA in the secretory vesicles of primary rat chromaffin cells. Transfection of PC12 cells with an expression plasmid encoding CTSL directed expression of CTSL toward secretory vesicles. Deconvolution fluorescence microscopy suggested greater colocalization of CTSL with CgA than the lysosomal marker LGP110. The overexpression of CTSL in PC12 cells caused cleavage of full-length CgA. CTSL also cleaved CgA in vitro, in time- and dose-dependent fashion, and specificity of the process was documented through E64 (thiol reagent) inhibition. Mass spectrometry on CTSL-digested recombinant CgA identified a catestatin-region peptide, corresponding to CgA(360-373). The pool of peptides generated from the CTSL cleavage of CgA inhibited nicotine-induced catecholamine secretion from PC12 cells. CTSL processing in the catestatin region was diminished by naturally occurring catestatin variants, especially Pro370Leu and Gly364Ser. Among the CTSL-generated peptides, a subset matched those found in the catestatin region in vivo. These findings indicate that CgA can be a substrate for the cysteine protease CTSL both in vitro and in cella, and their colocalization within chromaffin granules in cella suggests the likelihood of an enzyme/substrate relationship in vivo.

Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters

“…Results here indicate the presence of cathepsin V in neuronal tissue regions that is compatible with its unique role in the production of enkephalin and NPY peptide neurotransmitters. The findings from this study of human cathepsin V and from prior studies of cathepsin L (mouse, bovine, and human) indicate their role in processing dibasic residue sites of proprotein precursors in secretory vesicles for the production of neuropeptides (1)(2)(3)(4)(5)(6)(7)36). Both cathepsin V and cathepsin L show cleavage of proneuropeptides at the N-terminal side of dibasic processing sites (i.e.…”

“…The unique nature of cathepsin V in the human genome raises the issue of the biological function of human cathepsin V with that of the highly homologous human cathepsin L. Notably, results of this study with others (7,36) in the field indicate the joint roles of human cathepsin V and human cathepsin L for proneuropeptide processing. However, in contrast to the human genome, the genomes of mouse and other mammals (bovine, sheep, and others) encode only cathepsin L but not orthologues of cathepsin V (8 -11).…”

“…Studies of transgenic mice with knock-out of the cathepsin L gene indicated the important role of this protease in the production of numerous peptide neurotransmitters and hormones, including enkephalin, ␤-endorphin, dynorphins, NPY, ACTH, ␣-MSH (␣-melanocyte stimulating hormone), cholecystokinin, and catestatin (1)(2)(3)(4)(5)(6)(7)36). Expression of cathepsin V in the cathepsin L knock-out mouse rescues defects in T cell function (37) and keratinocyte proliferation (38,39).…”

Molecular mechanism of interactions of the physiological anti-hypertensive peptide catestatin with the neuronal nicotinic acetylcholine receptor

“…It is generated from chromogranin A (CHGA) (Biswas et al, 2009;Helle, 2010), a major protein costored and co-released with catecholamines from secretory granules of chromaffin cells and adrenergic neurons (Sahu et al, 2010;Taupenot et al, 2003). Although CHGA is overexpressed in genetic hypertension (Friese et al, 2005;O'Connor et al, 1999;Takiyyuddin et al, 1995), the plasma CST level is diminished not only in established hypertensive patients but also in their normotensive offspring .…”

The Natural Antimicrobial Chromogranins/Secretogranins- Derived Peptides – Production, Lytic Activity and Processing by Bacterial Proteases

“…More recently, it was shown that cathepsin L colocalizes with CGA in chromaffin granules. In vitro it is able to generate after digestion of recombinant hCGA, a catestatin (CAT)-derived fragment hCGA360-373 (Biswas et al, 2009). In addition to the inhibitory effect of CAT on catecholamine release from chromaffin cells (Mahata et al, 1997), we have shown for this peptide and its shorter active sequence bCGA344-358 (cateslytin, CTL), (Figure 3) a potent antimicrobial activity with a MIC in the lowmicromolar range against Gram-positive bacteria (Micrococcus luteus, Bacillus megaterium at concentration of 0.8 µM), Gram-negative bacteria (Escherichia coli D22 at concentration of 8 µM), filamentous fungi (Neurospora crassa, Aspergillus fumigatus, Nectria haematococca at concentration of 0.2-10 µM) and yeasts (Candida albicans, Candida tropicalis, Candida glabrata, Candida neoformans at concentration of 1.2-8 µM).…”