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PubMed1997

Ontogenic development of secretogranin II and of its processing to secretoneurin in rat brain.

The ontogenic development of secretogranin II was studied by immunochemistry and immunohistochemistry. Extracts of brains from various developmental stages were analyzed by a radioimmunoassay for secretoneurin, a peptide derived from secretogranin II. From gestational day 13 to adulthood the levels increased from 0.1 to 94 fmol/mg wet weight. Characterization of the immunoreactivity by molecular sieve chromatography revealed that throughout all developmental stages the proprotein secretogranin II was fully processed to the free peptide secretoneurin. In immunohistochemistry secretoneurin-IR was first detected at embryonic day 13. Between embryonic days 14 and 18 a strong increase in the number of secretoneurin immunopositive cells was observed in many brain areas, notably in the amygdala, hypothalamus, olfactory bulb and several brainstem nuclei. The pattern of staining during development is quite similar to that in the adult. The present paper demonstrates that secretoneurin immunoreactivity appears early in embryonic life. Processing of the proprotein secretogranin II starts when the protein is first synthesized apparently at about the same time when the prohormone convertase PC1 and PC2 can be demonstrated.

Animals↗
PubMed1995

Molecular cloning and cellular localization of a furin-like prohormone convertase from the atrial gland of Aplysia.

Prohormone convertases (PCs) are Ca(2+)-dependent subtilisin-related endoproteases that have been implicated in the post-translational processing of prohormones and other proproteins. Furin is an ubiquitously expressed PC that has been shown to hydrolyze a wide variety of precursor proteins in secretory pathways. We have screened an Aplysia atrial gland cDNA library using a furin probe prepared by polymerase chain reaction (PCR) and have isolated an Aplysia furin-related 6.7-kb cDNA partial clone that was truncated on the 5' end. The remaining 5' atrial gland furin nucleotide sequence was obtained by two stages of reverse transcription PCR. The final composite nucleotide sequence of the atrial gland furin cDNA was 7,837 bp in length. This sequence encoded a putative preproendoprotease (Afurin2) of 824 amino acid residues that was related to other eukaryotic furins, and showed a high sequence identity with a recently reported Aplysia nervous system furin-like sequence. In situ hybridization demonstrated extensive expression of Afurin2 in atrial gland secretory cells. The cDNA clone contained a relatively long 3' untranslated region of 5,230 nucleotides that included a microsatellite repeat region (TG)n. The characterized Aplysia Afurin2 is a candidate PC that may play an important role in the processing of egg-laying hormone (ELH)-related precursors in the secretory cells of the atrial gland. In addition, comparative structural studies of Afurin2, together with previously reported localization studies, argue for the occurrence of a furin-like convertase within secretory granules.

Amino Acid Sequence↗
PubMed1987

Yeast KEX2 protease has the properties of a human proalbumin converting enzyme.

Several classes of proteolytic enzymes have been proposed to have a role in the processing of precursor forms of proproteins at paired basic amino acid residues. In higher eukaryotes, a single endopeptidase has yet to fulfill the necessary criteria as the physiologically relevant convertase. The observation of proalbumin circulating in a child with a bleeding disorder caused by an unusual alpha 1-antitrypsin mutation led to speculation that the presence of this alpha 1-antitrypsin mutant was inhibitory to the convertase. This provided an additional means of characterizing the processing enzyme. In this study the yeast KEX2 enzyme, a calcium-dependent thiol protease, was found to have all the properties expected for this processing enzyme. KEX2 correctly recognized and cleaved the prosequence in proalbumin. In addition, KEX2 was specifically inhibited by the mutant alpha 1-antitrypsin but not by other serine protease inhibitors.

Cysteine Endopeptidases↗
PubMed1991

Isolation and functional properties of an arginine-selective endoprotease from rat intestinal mucosa. A putative prosomatostatin convertase.

The endoproteolytic activity previously detected in rat intestinal mucosal extracts (Beinfeld M., Bourdais, J., Kuks, P., Morel, A., and Cohen, P. (1989) J. Biol. Chem. 264, 4460-4465), was purified to homogeneity as a 65-kDa molecular species. This putative proprotein-processing enzyme cleaves the peptide bond on the carboxyl side of a single arginine residue in hepta-[Leu62-Gln-Arg-Ser-Ala-Asn-Ser68] or trideca-[Asp56-Glu-Met-Arg-Leu-Glu-Leu-Gln-Arg-Ser-Ala-Asn-+ ++Ser68] peptides, reproducing the prosomatostatin sequence around Arg64, the locus for endoproteolytic release of either somatostatin-28 or its NH2-terminal fragment, somatostatin-28-(1-12), from their common precursor. This enzyme exhibits a strict selectivity for arginyl residues, as demonstrated with related substrates, and did not cleave at lysyl residues. Moreover, only arginyl residues belonging to peptides of the prosomatostatin family were cleaved, since no hydrolysis of peptides from other prohormones was detected. In addition, the arginine residue situated at position -5 on the NH2-terminal side of Arg64 not only did not function as a cleavage locus, but had no effect on the overall cleavage kinetics of the prosomatostatin-(56-68) peptide substrate. This enzyme also cleaved, but with much less efficiency, the peptide bond on the carboxyl side of an arginine in peptides containing either an Arg-Lys or a Lys-Arg doublet corresponding to prohormone cleavage sites. This enzyme was insensitive to divalent cation chelators, was completely inhibited by aprotinin and leupeptin, and was somewhat inhibited by other serine-protease inhibitors. It is concluded that this endoprotease is a serine protease and could be involved in prohormone or proprotein post-translational processing at single arginine cleavage sites.

Amino Acid Sequence↗
PubMed2004

The N-terminal prodomain of sV23 is essential for the assembly of a functional vitelline membrane network in Drosophila.

The vitelline membrane is a specialized extracellular matrix that surrounds and protects the oocyte. Recent studies indicate that it also serves as a storage site for embryonic pattern determinants. sV23, a major vitelline membrane protein, is essential for the morphogenesis of the vitelline membrane as sV23 protein null mutants lay flaccid, infertile eggs. By analyzing a series of sV23 mutant transgenes in the sV23 protein null genetic background, we have shown that sV23 is secreted as a proprotein in functional excess and that C- and N-terminal prodomains are removed successively, following its deposition in the extracellular space. Although a target site for subtilisin-like convertases is essential for N-terminal processing, N-terminal processing is not necessary for the assembly of a functional vitelline membrane layer. While C-terminal truncations were tolerated, the removal of N-terminal sequences lead to the production of flaccid, infertile eggs with a soluble, rather than insoluble, vitelline membrane network. We propose that the hydrophobic N-terminal prodomain plays an early and essential role in aligning molecules within the vitelline membrane network, much like hydrophobic domains within elastin drive the assembly and alignment of molecules within elastin-based extracellular matrices.

Amino Acid Sequence↗
PubMed1991

The processing of human proinsulin and chicken proalbumin by rat hepatic vesicles suggests a convertase specific for X-Y-Arg-Arg or Arg-X-Y-Arg sequences.

Vesicles from rat and chicken livers contain very similar Ca2(+)-dependent proteases that respectively cleave (human) proalbumin at an Arg-Arg site and chicken proalbumin at an Arg-Phe-Ala-Arg site. Similar Ca2(+)-dependent proteases are also present in pancreatic secretory granules and cleave proinsulin at two sites, Arg-Arg and Lys-Arg. The mammalian liver processes a large variety of different proproteins and in order to assess its processing site requirements, we investigated the ability of rat hepatic vesicle extracts to cleave purified chicken proalbumin and human proinsulin. Despite having only a monobasic processing site, chicken proalbumin was cleaved faster than human proalbumin which not only contains a dibasic site, but has an identical propeptide to that of the rat's own proalbumin. Human proinsulin was processed by the rat liver extracts; however, no mature insulin was produced. Cleavage occurred in only one place, presumably the Arg-Arg site at the B-C chain junction. This suggests that the mammalian liver might not contain a Type II Lys-Arg-directed convertase, only a Type I Arg-Arg-specific enzyme. The Type I enzyme that cleaves human proalbumin appears to be the same activity that cleaves chicken proalbumin, suggesting a specificity for either X-Y-Arg-Arg or Arg-X-Y-Arg sequences. This proposal is in keeping with the processing site motif of some 16 different proproteins that are known to be processed in the liver and is entirely consistent with the known in vivo specificity of the enzyme defined by naturally occurring variants of human proproteins.

Amino Acid Sequence↗
PubMed1995

Substrate specificity of rabbit liver metalloendopeptidase and its new fluorogenic peptide substrates.

A metalloendopeptidase (MEP) isolated from rabbit liver microsomes with substrate specificity for peptides containing Arg at the P1 and P4 positions has recently proved to be identical to soluble angiotensin-binding protein present in the cytosol. Here we describe the peptide-degrading specificity of MEP, determined using various bioactive peptides and novel fluorogenic substrates for the enzyme. MEP degraded oligopeptides, including bradykinin, alpha-neoendorphin, bovine adrenal medulla dodecapeptide, substance P, bombesin, neurotensin, and alpha-endorphin, but not polypeptides such as reduced lysozyme and histone H4, hence, MEP probably belongs to the family of endo-oligopeptidases. It cleaved most preferentially at the -Phe-Ser- bond of bradykinin (kcat/Km = 2.8 x 10(4) M-1.S-1) but did not cleave high molecular weight and low molecular weight kininogens, the precursors of bradykinin. MEP did not cleave angiotensin I, dynorphin A 1-13, somatostatin, and luteinizing hormone-releasing hormone, some of which are good substrates for metalloendopeptidase-24.15, metalloendopeptidase-24.16, N-arginine dibasic convertase, and yeast endopeptidase-24.15 related peptidase. An active site-directed inhibitor of metalloendopeptidase-24.15, N-[1-(R,S)-carboxyl-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate also had no effects on the amidolytic activity of MEP. Based on the cleavage sites of bioactive peptides and processing sites of vitamin K-dependent proproteins, intramolecularly quenched fluorogenic peptide substrates were newly synthesized. Among the thirteen substrates used, the most reactive was 2-aminobenzoyl-Ala-Arg-Val-Arg-Arg-Ala- Asn-Ser-2,4-dinitroanilinoethylamide (kcat/Km = 9.3 x 10(5) M-1.S-1). An angiotensin antagonist, [Sar1, Ala8]-angiotensin II, inhibited hydrolysis of the substrate by MEP in a competitive manner (Kl = 7.6 microM). MEP cleaved oligopeptides even on the carboxyl side of proline residue and these peptides are resistant to hydrolysis by the cytosol-derived proteasome, therefore MEP may participate in the catabolism of oligopeptides in the cytosol, together with other endo-oligopeptidases.

Amino Acid Sequence↗
PubMed1997

Ontogeny of prohormone convertases in rat prenatal development.

It has been well established that peptide precursors usually undergo limited proteolysis at pairs or single basic amino acids during their biosynthetic process. This posttranslational modification paradigm is common for numerous membrane-spanning and secreted proteins, neuropeptides, and peptide hormones of physiological significance, in which endoproteolytic cleavage is invariably essential for the accurate biosynthesis and full activity of the mature products. Establishment of an effective peptide profile is dependent on not only the presence of peptide precursor, but also the presence and the enzymatic specificities of cleavage enzymes. We have, therefore, characterized the spatial and temporal patterns of six subtilisin-like serine endoproteases known to be involved in proprotein processing, including furin, PC1, PC2, PC4, PC5, and PACE4, in rat prenatal development and related the results to the expression patterns of several peptide precursors. We have observed largely distinct and sometimes complementary expression patterns of individual PCs in various embryonic structures, suggesting PCs may be functionally distinct in processing different sets of proprotein substrates in development. From these studies, numerous tentative enzyme-substrate relationships in various embryonic structures have been proposed and should encourage more studies to test the in vitro cleavage potentialities of individual PCs toward these precursors. In the future, knowledge gained from these studies, when combined with insights gained from in vivo perturbation and genetic ablation studies, should lead to final comprehensive understanding of specific precursors cleaved by specific enzymes at specific cleavage sites in known spatial and temporal expression patterns during development.

Animals↗
PubMed1989

Identification of a calcium-dependent microsomal proteinase responsible for monobasic cleavage of chicken proalbumin.

The location and nature of the endoproteolytic activity involved in processing of proproteins has been studied in chicken liver microsomes. A membrane-bound, calcium-dependent proteinase was found to cleave chicken proalbumin with a monobasic cleavage site approx. 10-times faster than human proalbumin, which has a dibasic cleavage site. The mutant (human) proalbumin Christchurch (Arg(-1)----Gln), with a potential monobasic site, was not processed. The enzyme, which had a pH optimum of between 5.0 and 7.0, was not inhibited by serine or aspartyl proteinase inhibitors but was affected by some inhibitors of cysteine proteinases. The convertase was specifically inhibited by the reactive centre variant alpha 1-antitrypsin Pittsburgh, but not by normal alpha 1-antitrypsin.

Amino Acid Sequence↗
PubMed1993

Role of beta-turn in proteolytic processing of peptide hormone precursors at dibasic sites.

Proteolytic activation of prohormones and proproteins occurs most frequently at the level of basic amino acids arranged in doublets. Previous predictions by Rholam et al. [Rholam, M., Nicolas, P., & Cohen, P. (1986) FEBS Lett. 207. 1-6] have indicated, on the basis of 20 prohormone sequences containing 53 dibasic potential processing sites, that dibasic sites situated in, or next to, beta-turns were cleaved in vivo, whereas sites included in ordered structures like beta-sheets or alpha-helices were not. We have used peptide analogs of the proocytocin/neurophysin processing domain and a purified preparation of the putative proocytocin convertase from bovine tissues as a model to demonstrate that (1) processing at dibasic sites is associated with a prohormone sequence organized in a beta-turn structure; (2) the beta-turn is an interchangeable motif since the original sequence could be replaced by an heterologous one possessing the ability to organize as a beta-turn; and (3) this particular secondary structure participates in the catalytic reaction, most likely by favoring the interactions of the substrate with the processing endoprotease. It is concluded that, in addition to the dibasic and other amino acids around the cleavage loci, the beta-turn constitutes a key feature in the proteolytic processing reaction in participating as the favorable conformation for optimal substrate-enzyme active site recognition.

Amino Acid Sequence↗
PubMed1997

High-level expression of the prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro prohormone processing.

Prohormone substrates are required for investigation of the proteolytic processing of prohormones and proproteins into active peptide hormones and neurotransmitters. However, the lack of prohormone proteins has been a limiting factor in elucidating proteolytic mechanisms for conversion of prohormones into active peptides. Therefore, in this study, cloned cDNAs encoding the prohormones proenkephalin (PE), pro-neuropeptide Y (pro-NPY), pro-opiomelanocortin (POMC), and beta-protachykinin (beta-PT) were utilized to express recombinant prohormones in Escherichia coli. High-level expression of milligrams of prohormones was achieved with the pET3c expression vector utilizing the T7 promoter for production of PE, pro-NPY, and POMC, as demonstrated by SDS-PAGE gel electrophoresis, Western blots, and 35S-methionine labeling. In addition, beta-PT was expressed at high levels as fusion proteins with the maltose-binding protein and glutathione S-transferase by the pMAL-c and pGEX-2T expression vectors, respectively. Relative rates of processing by the established processing proteases "prohormone thiol protease" (PTP), 70-kDa aspartyl protease, and PC1/ 3 and PC2 (PC, prohormone convertase) were examined with purified PE, pro-NPY, and POMC. Distinct preferences of processing enzymes for different prohormones was demonstrated. PTP preferred PE and pro-NPY substrates, whereas little processing of POMC was detected. In contrast, the 70-kDa aspartyl protease cleaved POMC more readily than pro-NPY or PE. However, PC1/3 and PC2 prefer POMC as substrate. Demonstration of selectivity of processing enzymes for prohormone substrates illustrates the importance of expressing recombinant prohormones for in vitro processing studies.

Animals↗