Elsevier

Neuropeptides

Volume 57, June 2016, Pages 85-94
Neuropeptides

Characterization of endoproteolytic processing of dynorphins by proprotein convertases using mouse spinal cord S9 fractions and mass spectrometry

https://doi.org/10.1016/j.npep.2015.10.008Get rights and content

Abstract

Dynorphins are important neuropeptides with a central role in nociception and pain alleviation. Many mechanisms regulate endogenous dynorphin concentrations, including proteolysis. Proprotein convertases (PCs) are widely expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous big dynorphin (BDyn) and dynorphin A (Dyn A) levels has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild-type (WT), PC1−/+ and PC2−/+ animals and mass spectrometry. Our results clearly demonstrate that both PC1 and PC2 are involved in the proteolysis regulation of BDyn and Dyn A with a more important role for PC1. C-terminal processing of BDyn generates specific peptide fragments dynorphin 1–19, dynorphin 1–13, dynorphin 1–11 and dynorphin 1–7, and C-terminal processing of Dyn A generates dynorphin 1–13, dynorphin 1–11 and dynorphin 1–7, all these peptide fragments are associated with PC1 or PC2 processing. Moreover, the proteolysis of BDyn leads to the formation of Dyn A and Leu-Enk, two important opioid peptides. The rate of formation of both is significantly reduced in cellular fractions of spinal cord mutant mice. As a consequence, even the partial inhibition of PC1 or PC2 may impair the endogenous opioid system.

Introduction

In mammals, the presence of tissue-damaging stimuli is sensed by primary afferent nociceptors. The sensation of pain produced by a noxious stimulus is not always consistent and depends on numerous factors influencing the neurophysiology of pain transmission (Julius and Basbaum, 2001, Gold and Gebhart, 2010). The nervous system has developed very complex mechanisms that control how noxious sensory input is perceived by the organism (Basbaum et al., 2009). It has been shown that extensive modulation of sensory information happens in the dorsal horn of the spinal cord, more specifically during the first synapse (Honore et al., 2000, Levine et al., 1993). There are various molecular events contributing to the transmission of the sensory information during the first synapse and several major neuropeptides were identified including dynorphin, enkephalin and tachykinin peptides (Kuner, 2010, Mika et al., 2011, Felippotti et al., 2012, Pailleux et al., 2013, Ferland et al., 2011).

Neuropeptides are either neurotransmitters or neuromodulators at various levels in the central nervous system and play a critical role in pain transmission (Levine et al., 1993, Seybold, 2009). Many members of the tachykinin family (e.g. Substance P) are mostly pro-nociceptive neuropeptides and have been known to play a fundamental role in central sensitization leading to hyperalgesia and allodynia (Lecci et al., 2000, Teodoro et al., 2013). Opioid peptides (i.e., endomorphins, enkephalins and dynorphins) have potent analgesic effects in the central nervous system (CNS) and play a fundamental role in endogenous pain inhibition (Machelska, 2007, Wahlert et al., 2013, Bali et al., 2014). They are interacting with μ, κ and δ-opioid receptors expressed widely in the brain and in the spinal cord (Carr and Lovering, 2000, Stanojevic et al., 2008, Mulder et al., 1989).

Dynorphin A (Dyn A), one of the major proteolytic fragments of prodynorphin (Civelli et al., 1985, Dores et al., 1985), is an endogenous ligand of the κ and μ-opioid receptors (Beaulieu, 2005, Mika et al., 2011). The agonist activity of Dyn A significantly reduces neuronal activity (Chavkin et al., 1982, Mizoguchi et al., 2006). However, the concentration of Dyn A in the spinal cord can be regulated rapidly by endoproteolysis (Cho and Basbaum, 1989). Neuropeptides are derived from larger protein precursors (i.e., proneuropeptides), and their primary structures include the sequence of the active form of at least one neuropeptide within its full-length (Funkelstein et al., 2010). It has been revealed that many neuropeptides are synthesized by the actions of proprotein convertases (PCs) and endopeptidases within dense core vesicle (Harrison and Geppetti, 2001). Particularly, recent studies outlined the significant contribution of PC1 and PC2 into the proteolytic processing of proneuropeptides (Zheng et al., 1994, Cui et al., 1998, Jin et al., 2005, Miller et al., 2003). Proprotein convertases, particularly PC1 and PC2, are widely expressed in the CNS and specifically cleave at C-terminal of either a pair of basic amino acids (KR, RR, RK and KK), or a single basic residue (R or K). Early prodynorphin (PDYN) endoproteolytic studies suggest that PCs play a significant role in PDYN processing (Berman et al., 2000, Day et al., 1998). These preliminary results generated using indirect methods suggest that the action of PC2 is needed for the formation of Big dynorphin (BDyn), Dyn A and dynorphin B (Dyn B) (Day et al., 1998). The proteolysis control of endogenous BDyn and Dyn A levels has a profound impact on pain perception and the role of PCs remain unclear. As shown in Fig. 1, both peptides contain paired or single basic residues and the action of PCs could be involved in C-terminal processing of BDyn and Dyn A leading to several important N-terminal metabolites. Interestingly, BDyn, Dyn A and all N-terminal metabolites encode one copy of Leu-enkephalin (Leu-Enk), an important opioid peptide. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild-type (WT), PC1−/+ and PC2−/+ animals.

Section snippets

Chemicals and reagents

Big dynorphin (BDyn), dynorphin A (Dyn A), dynorphin 1–13 (Dyn 1–13), dynorphin 1–11 (Dyn 1–11), dynorphin 1–10 (Dyn 1–10), dynorphin 1–9 (Dyn 1–9), dynorphin 1–7 (Dyn 1–7), dynorphin 1–6 (Dyn 1–6) and leu-enkephalin (Leu-Enk) were purchased from Phoenix Pharmaceuticals (Belmont, CA, USA). Dynorphin 1–19 (Dyn 1–19) and deuterium-labeled analog peptides were synthesized de novo (CanPeptide, Inc., Pointe-Claire, QC, Canada). Other chemicals, including acetonitrile, hexane, formic acid,

Mass spectrometry and isotopic dilution method

Full-scan and product ion mass spectra for all peptides and internal standards were obtained in positive ion mode. The full-scan electrospray mass spectrum of targeted peptides displayed the formation of characteristic pseudo molecular ions [M + nH]n +, and the fragment ions observed in MS/MS spectra were annotated based on the Roepstorff and Fohlman nomenclature (Roepstorff and Fohlman, 1984). Details on MS parameters and MRM transitions are reported in Table 1. Full-scan and product ion mass

Conclusion

In neuronal cells, almost all neuropeptides are produced through endoproteolysis specifically at C-terminal pairs of basic residues during their transport along the secretory pathway. Interestingly, it is recognized that proneuropeptides are cleaved using a cell-specific mechanism by members of the family of calcium-dependent subtilisin-like endoproteases (Steiner, 1998, Seidah and Chretien, 1999, Hook et al., 2008). PC1 and PC2 are predominantly expressed in neural cells and our results

Acknowledgements

This project was funded by the National Sciences and Engineering Research Council of Canada (F. Beaudry discovery grant no. 386637-2010). The analyses were performed using analytical instruments acquired with an NSERC Research Tools and Instruments grant (F. Beaudry NSERC-RTI grant no. 439748-2013). Thermo Fisher Scientific provided generous access to a Q-Exactive Orbitrap Mass Spectrometer. A. Ruiz received a scholarship from Agència de Gestió d'Ajuts Universitaris i de Recerca

References (55)

  • S. Harrison et al.

    Substance p

    Int. J. Biochem. Cell Biol.

    (2001)
  • P. Honore et al.

    Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons

    Neuroscience

    (2000)
  • W. Jin et al.

    Proprotein covertases are responsible for proteolysis and inactivation of endothelial lipase

    J. Biol. Chem.

    (2005)
  • A. Lecci et al.

    Peripheral actions of tachykinins

    Neuropeptides

    (2000)
  • H. Machelska

    Targeting of opioid-producing leukocytes for pain control

    Neuropeptides

    (2007)
  • J. Mika et al.

    The role of nociceptin and dynorphin in chronic pain: implications of neuro–glial interaction

    Neuropeptides

    (2011)
  • R. Miller et al.

    Selective roles for the PC2 processing enzyme in the regulation of peptide neurotransmitter levels in brain and peripheral neuroendocrine tissues of PC2 deficient mice

    Neuropeptides

    (2003)
  • A. Minokadeh et al.

    Cathepsin L participates in dynorphin production in brain cortex, illustrated by protease gene knockout and expression

    Mol. Cell. Neurosci.

    (2010)
  • A.H. Mulder et al.

    Selectivity of various opioid peptides towards delta-, kappa; and mu-opioid receptors mediating presynaptic inhibition of neurotransmitter release in the brain

    Neuropeptides

    (1989)
  • F. Pailleux et al.

    Targeted liquid chromatography quadrupole ion trap mass spectrometry analysis of tachykinin related peptides reveals significant expression differences in a rat model of neuropathic pain

    Neuropeptides

    (2013)
  • M. Saidi et al.

    Liquid chromatography-electrospray linear ion trap mass spectrometry analysis of targeted neuropeptides in Tac1 −/− mouse spinal cords reveal significant lower concentration of opioid peptides

    Neuropeptides

    (2015)
  • N.G. Seidah et al.

    Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides

    Brain. Res.

    (1999)
  • S. Stanojevic et al.

    Methionine-enkephalin modulation of hydrogen peroxide (H2O2) release by rat peritoneal macrophages involves different types of opioid receptors

    Neuropeptides

    (2008)
  • F.C. Teodoro et al.

    Peripheral substance P and neurokinin-1 receptors have a role in inflammatory and neuropathic orofacial pain models

    Neuropeptides

    (2013)
  • A. Wahlert et al.

    Spinal astrocytes produce and secrete dynorphin neuropeptides

    Neuropeptides

    (2013)
  • M. Werz et al.

    Dynorphin reduces voltage-dependent calcium conductance of mouse dorsal root ganglion neurons

    Neuropeptides

    (1984)
  • V. Zachariou et al.

    Dynorphin-(1–8) inhibits the release of substance P-like immunoreactivity in the spinal cord of rats following a noxious mechanical stimulus

    Eur. J. Pharmacol.

    (1997)
  • Cited by (0)

    View full text