Molecular cloning demonstrates structural features of homologous bovine prohormone convertases 1 and 2.

PC1 and PC2 (prohormone convertase) represent neuroendocrine members of the mammalian subtilisin-like family of proprotein convertases. The goal of this study was to compare the primary sequence motifs of bovine PC1 and PC2 with those of homologs from other mammalian species to establish the structural basis for PC1 and PC2 activities in bovine that resemble other mammalian homologs. Molecular cloning from bovine adrenal medulla resulted in the isolation of cDNAs for bovine PC1 and PC2 with highly conserved primary sequences with respect to signal sequence, prosegment, catalytic domain, and P domain. Bovine PC1 and PC2 contained the catalytic triad residues Asp, His, Ser, which are identical to the triads in PC1 and PC2 from other mammalian species. Bovine PCl contained Asn as the oxyanion hole residue; in contrast, bovine PC2 contained Asp as the oxyanion hole residue, which is identical to PC2 in other mammalian species. Bovine PC1 and PC2 possessed the P domain that contains the functional RRGDL motif. The cloned cDNAs detected expression of PC1 and PC2 mRNAs in bovine adrenal medulla. These results establish the defined structural domains of bovine PC1 and PC2 that are known to be essential for the activities of these enzymes in various species.

Expression and mutagenesis of the novel serpin endopin 2 demonstrates a requirement for cysteine-374 for dithiothreitol-sensitive inhibition of elastase.

The primary sequence of the serpin endopin 2 predicts a reactive site loop (RSL) region that possesses high homology to bovine elastase inhibitor, suggesting inhibition of elastase. Moreover, endopin 2 possesses two cysteine residues that implicate roles for reduced Cys residue(s) for inhibitory activity. To test these predicted properties, mutagenesis and chemical modification of recombinant endopin 2 were performed to examine the influence of dithiothreitol (DTT), a reducing agent, on endopin 2 activity. Endopin 2 inhibited elastase in a DTT-dependent manner, with enhanced inhibition in the presence of DTT. The stoichiometry of inhibition in the presence of DTT occurred at a molar ratio of endopin 2 to elastase of 8/1, resulting in complete inhibition of elastase. However, a higher molar ratio (25/1) was required in the absence of DTT. DTT enhanced the formation of SDS-stable complexes of endopin 2 and elastase, a characteristic property of serpins. Site-directed mutagenesis of endopin 2, with substitution of Ala for Cys-232 or Cys-374, demonstrated that Cys-374 (but not Cys-232) was required for the DTT-sensitive nature of endopin 2. Chemical modification of Cys-374 by bis(maleimido)ethane also reduced inhibitory activity. Modified electrophoretic mobilities of mutant endopin 2 suggested the presence of intramolecular disulfide bonds; in addition, chemical modification suggested that Cys-374 influences the electrophoretic and conformational properties of endopin 2. Moreover, the reducing agent glutathione enhanced endopin 2 activity, suggesting that glutathione can function as an endogenous reducing agent for endopin 2 in vivo. These findings demonstrate the importance of Cys-374 for DTT-sensitive inhibition of elastase by endopin 2.

Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

An alpha1-antichymotrypsin-like serpin has been implicated in Alzheimer's disease (AD) based on immunochemical detection of alpha1-antichymotrypsin (ACT) in amyloid plaques from the hippocampus of AD brains. The presence of neuroendocrine isoforms of ACTs and reported variations in human liver ACT cDNA sequences raise the question of the molecular identity of ACT in brain. In this study, direct reverse transcription-polymerase chain reaction and cDNA sequencing indicate that the hippocampus ACT possesses the reactive site loop that is characteristic of serpins, with Leu as the predicted P1 residue interacting with putative chymotrypsin-like target proteases. The deduced primary sequence of the human hippocampus ACT possesses more than 90% homology with reported primary sequences for the human liver ACT. Moreover, identical ACT primary sequences deduced from the cDNAs were demonstrated in the hippocampus of control and AD brains. Northern blots showed that ACT mRNA expression in hippocampus was 900 times lower than that in liver. Also, hippocampus and liver ACT proteins demonstrated differential sensitivities to deglycosylation. Overall, reverse transcription-polymerase chain reaction combined with cDNA and primary sequence analyses have defined the molecular identity of human hippocampus ACT in control and AD brains. The determined reactive site loop domain of hippocampus ACT will allow prediction of potential target proteases inhibited by ACT in AD.

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

Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68-70 kDa was present within isolated chromaffin granules. Stimulated cosecretion of endopin 1 with chromaffin granule components, [Met]enkephalin and a cysteine protease known as "prohormone thiol protease," demonstrated localization of endopin 1 to functional secretory vesicles. Punctate, discrete immunofluorescence cellular localization of endopin 1 in chromaffin 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, trypsin was potently inhibited (K(i(app)) of 5 nM), and plasmin was moderately inhibited. Although endopin 1 possesses homology with alpha(1)-antichymotrypsin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chromaffin granule prohormone thiol protease (involved in proenkephalin processing). These results suggest a role for the novel serpin, endopin 1, in regulating basic residue-cleaving proteases within neurosecretory vesicles of chromaffin cells.