Differential accessibilities of dibasic prohormone processing sites of proenkephalin to the aqueous environment revealed by H-D exchange mass spectrometry.

Proenkephalin and prohormones are inactive protein precursors that must undergo proteolytic processing to generate the smaller, active peptide neurotransmitters and hormones. The mature enkephalin neuropeptide functions in brain regulation of analgesia and behavior (1-3). Proenkephalin (PE) contains multiple copies of (Met)enkephalin and related peptides (4,5), which are produced by proteolytic processing of PE at multiple dibasic processing sites (6-9) that flank the enkephalin peptide sequences within PE. The processed, mature enkephalin peptides are stored within secretory vesicles and then undergo regulated secretion induced by cellular stimuli, allowing it to function as an extracellular peptide neurotransmitter. Proteolysis of proenkephalin in the regulated secretory pathway has been extensively studied and involves the cysteine protease cathepsin L combined with the subtilisin-like prohormone convertases (6-9). The prohormone convertases known as PC1/3 and PC2 represent the primary subtilisin-like proteases (6-9) that cleave at the COOH-terminal side of the dibasic residue prohormone processing sites; their actions are followed by carboxypeptidase E (CPE) that removes COOH-terminal basic residues to generate active enkephalin and related neuropeptides (6,10). The more recently discovered cathepsin L protease pathway for PE processing involves preferential cleavage at the NH2-terminal side of dibasic residue sites (6,11), followed by aminopeptidase B to remove NH2-terminal basic residues (12) in the formation of mature enkephalin. Studies of protease gene knockout mice (6-9, 11, 13-16) have demonstrated the significant roles of secretory vesicle cathepsin L, combined with the well-known prohormone convertases, in the production of enkephalin and numerous neuropeptides functioning as key neurotransmitters and hormones. The dibasic processing sites of PE are recognized and cleaved by the prohormone processing proteases. However, little is known about the conformational orientation and structural features of proenkephalin and prohormones at their proteolytic processing sites. For this reason, this study sought to gain knowledge of the relative accessibility of the proteolytic processing sites of proenkephalin by hydrogen-deuterium exchange mass spectrometry (DXMS) (17-19). DXMS allows evaluation of the relative rates of exchange of hydrogens of the polypeptide backbone of PE with deuterium of D2O (heavy water), and can compare protein subdomains with respect to their relative accessibility to the aqueous solvent environment. DXMS experiments demonstrated differences in the relative accessibilities of the dibasic KR, KK, and RR cleavage sites to the aqueous environment. The mid-domain processing sites of PE showed higher accessibility to the aqueous solvent compared to other NH2- and COOH-domains of PE. However, the NH2- and COOH-terminal dibasic sites showed relative high DXMS exchange rates. In the presence of the organic solvent trifluoroethanol (TFE), PE displayed differences in DXMS properties, combined with differences in secondary structural features determined by CD (circular dichroism) (20-22). These findings suggest that in more hydrophobic conditions, such as association of PE with membranes in cells, the orientation of the dibasic processing sites to the aqueous environment may differ. The observed differences in relative accessibility of dibasic sites within PE suggest differential conformational features among these proteolytic processing sites. These findings suggest that dibasic prohormone processing sites with differences in accessibility to the aqueous solvent undergo proteolytic processing to generate mature active enkephalin and related neuropeptides.