title: The novel serpin endopin 2 demonstrates cross-class inhibition of papain and elastase: localization of endopin 2 to regulated secretory vesicles of neuroendocrine chromaffin cells.
abstract: This study demonstrates that endopin 2 is a unique secretory vesicle serpin that displays cross-class inhibition of cysteine and serine proteases, indicated by effective inhibition of papain and elastase, respectively. Homology of the reactive site loop (RSL) domain of endopin 2, notably at P1-P1' residues, with other serpins that inhibit cysteine and serine proteases predicted that endopin 2 may inhibit similar proteases. Recombinant N-His-tagged endopin 2 inhibited papain and elastase with second-order rate constants (k(ass)) of 1.4 x 10(6) and 1.7 x 10(5) M(-1) s(-1), respectively. Endopin 2 formed SDS-stable complexes with papain and elastase, a characteristic property of serpins. Interactions of the RSL domain of endopin 2 with papain and elastase were indicated by cleavage of endopin 2 near the predicted P1-P1' residues by these proteases. Endopin 2 did not inhibit the cysteine protease cathepsin B, or the serine proteases chymotrypsin, trypsin, plasmin, and furin. Endopin 2 in neuroendocrine chromaffin cells was colocalized with the secretory vesicle component (Met)enkephalin by confocal immunonfluorescence microscopy, and was present in isolated secretory vesicles (chromaffin granules) from chromaffin cells as a glycoprotein of 72-73 kDa. Moreover, regulated secretion of endopin 2 from chromaffin cells was induced by nicotine and KCl depolarization. Overall, these results demonstrate that the serpin endopin 2 possesses dual specificity for inhibiting both papain-like cysteine and elastase-like serine proteases. These findings demonstrate that endopin 2 inhibitory functions may occur in the regulated secretory pathway.
authors.affiliation: Buck Institute for Age Research, Novato, California 94945, USA.
authors.forename: Shin-Rong
authors.initials: SR
authors.lastname: Hwang
authors.forename: Brent
authors.initials: B
authors.lastname: Steineckert
authors.forename: Thomas
authors.initials: T
authors.lastname: Toneff
authors.forename: Richard
authors.initials: R
authors.lastname: Bundey
authors.forename: Anna V
authors.initials: AV
authors.lastname: Logvinova
authors.forename: Paul
authors.initials: P
authors.lastname: Goldsmith
authors.forename: Vivian Y H
authors.initials: VY
authors.lastname: Hook
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009490:Neurosecretory Systems / Q000201:enzymology / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010206:Papain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D022142:Secretory Vesicles / Q000201:enzymology / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D015843:Serpins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D013379:Substrate Specificity
chemical_list: D015853:Cysteine Proteinase Inhibitors; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C412321:endopin 2; D010196:Pancreatic Elastase; D010206:Papain