Molecular cloning of endopin 1, a novel serpin localized to neurosecretoryvesicles of chromaffin cells - Inhibition of basic residue-cleaving proteases by endopin 1

Serpins represent a diverse class of endogenous protease inhibitors that re gulate important biological functions. In consideration of the importance o f regulated proteolysis within secretory vesicles for the production of pep tide hormones and neurotransmitters, this study revealed the molecular iden tity of a novel serpin, endopin 1, that is localized to neurosecretory vesi cles of neuropeptide-containing chromaffin cells (chromaffin granules). End opin 1 of 68-70 kDa was present within isolated chromaffin granules. Stimul ated cosecretion of endopin 1 with chromaffin granule components, [Met]enke phalin and a cysteine protease known as "pro-hormone thiol protease," demon strated localization of endopin 1 to functional secretory vesicles. Punctat e, discrete immunofluorescence cellular localization of endopin 1 in chroma ffin cells was consistent with its secretory vesicle localization. Endopin 1 contains a unique reactive site loop with Arg as the predicted P1 residue , suggesting inhibition of basic residue-cleaving proteases; indeed, trypsi n was potently inhibited (K7(i(app)) of 5 nM), and plasmin was moderately i nhibited. Although endopin 1 possesses homology with alpha(1)-antichymotryp sin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chro maffin granule prohormone thiol protease (involved in proenkephalin process ing), These results suggest a role for the novel serpin, endopin 1, in regu lating basic residue-cleaving proteases within neurosecretory vesicles of c hromaffin cells.