Notch1 antiapoptotic activity is abrogated by caspase cleavage in dying T lymphocytes.

Excessive signaling via the Notch1 receptor inhibits apoptosis in T lymphocytes. Since several antiapoptotic proteins are cleaved by caspases during cell death, we investigated whether Notch1 was a caspase substrate. Results demonstrate that the intracellular domain of Notch1 (NICD) is cleaved into six fragments during apoptosis in Jurkat cells or peripheral T lymphocytes. Notch1 cleavage is prevented by the caspase inhibitors DEVD-fmk and VEID-fmk or by Bcl-2 expression. Caspase-3 and caspase-6 cleave the NICD into six fragments using sites located within the NF-kappaB binding domain, the ankyrin repeats and the transactivation domain. Notch1 cleavage correlates with the loss of HES-1 expression in apoptotic T cells. Notch1 fragments cannot inhibit activation-induced cell death in a T-cell hybridoma, confirming the abrogation of Notch1 antiapoptotic activity by caspases. The ability of the NICD but not the fragments to antagonize Nur77 activity supports a role for this factor in Notch1 antiapoptotic function.

Characterization of ostrich (Struthio camelus) beta-microseminoprotein (MSP): identification of homologous sequences in EST databases and analysis of their evolution during speciation.

Beta-microseminoprotein, alternatively called prostatic secretory protein of 94 amino acids, is a hydrophilic, unglycosylated, small protein rich in conserved half-cystine residues. Originally found in human seminal plasma and prostatic fluids, its presence was later shown in numerous secretions and its homologs were described in many vertebrate species. These studies showed that this protein had rapidly evolved, but they failed to unambiguously identify its biological role. Here, we show that a protein isolated from ostrich pituitary gland is closely related to a similar one isolated from chicken serum and that the two are structurally related to the mammalian beta-microseminoprotein. The complete 90-amino acid sequence of the ostrich molecule was established through a combination of automated Edman degradation and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric procedures, including postsource decay (PSD) and ladder sequencing analyses. This study documents for the first time that beta-microseminoprotein is present in aves. It is also the first report of a C-terminal amidated form for a member of this protein family and the first in which the disulfide linkages are established. Database searches using the herein-described amino acid sequence allowed identification of related proteins in numerous species such as cow, African clawed frog, zebrafish, and Japanese flounder. These small proteins show a strikingly high rate of amino acid substitutions, especially across phyla boundaries. Noticeably, no beta-microseminoprotein-related gene could be found in the recently completed fruit fly genome, indicating that if such a gene exists in arthropods, it must have extensively diverged from the vertebrate ones.

Purification and characterization of active recombinant rat kallikrein rK9.

The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.

Comparative characterization of two forms of recombinant human SPC1 secreted from Schneider 2 cells.

SPC1 (furin/PACE), an enzyme belonging to the S8 group of serine endoproteases, is a type I integral membrane protein that catalyzes the processing of a multitude of precursor proteins. We report here the use of transfected Drosophila melanogaster Schneider 2 cells to produce milligram amounts of two forms of recombinant human SPC1. In order to investigate the role of the cysteine-rich region (CRR) of SPC1, we compared the biochemical and enzymatic properties of hSPC1/714 that has the C-terminal tail and transmembrane region of the native enzyme removed with that of hSPC1/585 which had, in addition, the CRR deleted. Two stable cell lines were established. The S2-hSPC1/714 line secreted a major form of apparent molecular weight of 83 kDa and a minor form of 80 kDa whereas the S2-hSPC1/585 line secreted a single 59-kDa protein. PNGase F treatment of the different forms demonstrated that the enzymes were glycosylated. Automated NH(2)-terminal sequencing revealed that all purified forms resulted from processing at the expected zymogen activation site. Removal of the CRR resulted in a broadening of the enzyme's pH range, a shift of K(0.5) for Ca(2+), and a shorter enzymatic half-life when compared to the longer form, which suggest that the CRR of hSPC1 may help in stabilizing the enzyme's proteolytic activity. The use of this high-level expression system will meet the demand for material necessary to perform biochemical and structural studies that are needed to further our understanding of this and other SPCs at the molecular level.

Enzymic characterization in vitro of recombinant proprotein convertase PC4.

Proprotein convertase PC4A, a member of the subtilisin/kexin family of serine proteases, was obtained in enzymically active form following expression of vaccinia virus recombinant rat (r)PC4A in GH4C1 cells. It displayed maximal activity at pH 7.0 and a Ca(2+) concentration of 2.0 mM. Using PC4-specific antibodies, Western blot analysis of the medium revealed a major band at approximately 54 kDa, corresponding to the molecular size of mature rPC4A. Among the various peptidyl-[4-methylcoumarin 7-amide (MCA)] substrates tested, the one that was preferred the most by rPC4A was acetyl (Ac)-Arg-Lys-Lys-Arg-MCA, which is cleaved 9 times faster (as judged from V(max)/K(m) measurements) than the best furin and PC1 substrate, pGlu-Arg-Thr-Lys-Arg-MCA. Recombinant rPC4A, along with human (h)furin and hPC1, cleaved a 17-amino-acid synthetic peptide, YQTLRRRVKR downward arrowSLVVPTD (where downward arrow denotes site of cleavage, and the important basic residues are shown in bold), encompassing the junction between the putative pro-segment of rPC4A and the active enzyme, suggesting a possible auto-activation of the enzyme. In an effort to identify potential physiological substrates for PC4, studies were performed with pro-[insulin-growth-factor (IGF)]-derived synthetic peptides, namely Ac-PAKSAR downward arrowSVRA (IGF-I(66-75)) and Ac-PAKSER downward arrowDVST (IGF-II(63-72)), as well as two lysine mutants [(IGF-I(66-75)Lys(70)) and (IGF-II(63-72)Lys(67))]. Unlike PC1 and furin, rPC4A cleaved efficiently both IGF-I(66-75) and IGF-II(63-72), suggesting a possible role of PC4 in the maturation of IGF-I and -II. In contrast, the peptides with a position 2 (P2) lysine mutation, IGF-I(66-75)Lys(70) and IGF-II(63-72)Lys(67), were cleaved more efficiently by PC1 and furin compared with rPC4A. Furthermore, using synthetic peptides containing the processing sites of pituitary adenylate-cyclase-activating polypeptide (PACAP)-38, we were able to confirm that, of the two testicular enzymes PC4 and PC7, PC4 is the best candidate enzyme for maturation of PACAP. Our data suggest that rPC4A is a functionally active convertase, with a substrate specificity somewhat different from that of other convertases, namely KXXR downward arrow (where X denotes any other residue). As expected, p-chloromercuribenzoic acid and metal chelators such as EDTA, EGTA and trans-1,2-diaminocyclohexane-N,N,N', N'-tetraacetic acid inhibit the proteolytic activity of rPC4A, whereas it is activated by dithiothreitol. PC4A was also inhibited by transition-metal ions (Cu(2+)>Hg(2+)>Zn(2+) Ni(2+)>Co(2+)), as well as by small peptide semicarbazones (SCs), such as Arg-Lys-Lys-Arg-SC (K(i) 0.75 microM) and Arg-Ser-Lys-Arg-SC (K(i) 11.4 microM).

High concentrations of the macrophage migration inhibitory factor in human seminal plasma and prostatic tissues.

During purification procedures to isolate kallikrein hK2 from human seminal plasma, kallikrein hK2 was found to be associated with another protein after several chromatographic steps. This study was conducted to identify the hK2 companion protein and characterize its properties and distribution. The protein was identified as macrophage migration inhibitory factor (MIF) by its NH2-terminal amino acid sequence. It had an enzymatic activity identical to that of recombinant MIF. Its concentration varied between 1 and 10 micrograms/mL in various seminal plasma. By immunohistochemical analysis, MIF was found to be localized mainly in the epithelial cells of normal and cancerous prostates. Since MIF is a well-known proinflammatory mediator, these results suggest that it may have important functions in both human reproduction and prostatic physiology.

The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis.

Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.

Prostatic kallikrein hK2, but not prostate-specific antigen (hK3), activates single-chain urokinase-type plasminogen activator.

Our work was undertaken to compare the relative efficiency of 2 purified prostatic kallikreins, namely, hK2 and prostate-specific antigen (PSA or hK3), in the activation of single-chain urokinase (scuPA). We found that hK2 converts scuPA into an active enzyme with an efficiency equal to approximately 1/50 that of plasmin. During the activation of scuPA by hK2, two fragments of 33 and 22 kDa were generated. The NH2-terminal amino acid sequence of the 33 kDa fragment showed that hK2 cleaved scuPA between Lys158 and Ile159. In contrast to a previous report by another group, our purified hK3 preparation containing no trypsin-like contaminants was totally unable to activate scuPA. Our results show that kallikrein hK2 has plasmin-like activity and suggest that it could be the initiator of a proteolytic cascade leading to prostatic cancer invasion.

Purification of enzymatically active kallikrein hK2 from human seminal plasma.

Kallikrein hK2 is a member of the human glandular kallikrein family which includes prostate-specific antigen (PSA) and pancreatic-renal kallikrein. The purpose of this work was to isolate and characterize for the first time the enzymatically active form of the hK2 protein starting from the PCI-hK2 complex isolated from human seminal plasma (Deperthes, D., Chapdelaine, P., Tremblay, R.R., Brunet, C., Berton, J., Hébert, J., Lazure, C. and Dubé, J.Y. (1995) Biochim. Biophys. Acta 1245, 311-316). That complex was dissociated by an incubation at alkaline pH and final purification was achieved by C-18 reverse phase HPLC. The purified material contained a 27 kDa band by SDS gel electrophoresis and had the expected NH2-terminal amino acid sequence of hK2. It hydrolyzed synthetic chromogenic substrates containing esters of lysine and arginine but not of phenylalanine. Furthermore, hK2 formed molecular complexes with alpha 2 -antiplasmin, alpha 1-antichymotrypsin, antithrombin III and alpha 2-macroglobulin but not with alpha 1-antitrypsin. In conlusion, the new findings of the present paper are that the PCI-hK2 complex can be dissociated by mild procedures, that the free hK2 protein can be purified thereafter by standard HPLC procedures, that the recovered free hK2 is a trypsin-like enzyme and that it can form molecular complexes with many of the major serum proteinase inhibitors.

Secretion of a type II integral membrane protein induced by mutation of the transmembrane segment.

Signal peptide/membrane anchor (SA) domains of type II membrane proteins initiate the translocation of downstream polypeptides across the endoplasmic reticulum (ER) membrane. In contrast with signal peptides, however, SA domains are not cleaved by signal peptidase and thus anchor the protein in the membrane. In the present study we have introduced mutations in the SA domain of neprilysin (neutral endopeptidase-24.11; NEP) to identify structural elements that would favour the processing of SA domains by signal peptidase. Mutants of full-length and truncated (without cytoplasmic domain) protein were constructed by substitution of the sequences SQNS, QQTT or YPGY for VTMI starting at position 15 of the NEP SA domain. In addition, a Pro residue was substituted for Thr at position 16 of the SA domain. The rationale for the use of these sequences was decided from our previous observation that substitution in the NEP SA domain of the sequence SQNS, which is polar and has alpha-helix-breaking potential, could promote SA domain processing under certain conditions (Roy, Chatellard, Lemay, Crine and Boileau (1993) J. Biol. Chem. 268. 2699-2704; Yang. Chatellard, Lazure, Crine and Boileau (1994) Arch. Biochem. Biophys. 315, 382-386). The QQTT sequence is polar but, according to secondary structure predictions, is compatible with the alpha-helix structure of the NEP SA domain. The YPGY sequence and single Pro residue are less polar and have alpha-helix-breaking potential. The predicted effects of these mutations on the structure of the NEP SA domain were confirmed by CD analysis of 42-residue peptides encompassing the hydrophobic segment and flanking regions. Wild-type and mutated proteins were expressed in COS-I cells and their fate (membrane-bound or secreted) was determined by immunoblotting and by endoglycosidase digestions. Our biochemical and structural data indicate that: (I) the cytosolic domain of NEP restricts the conformation of the SA domain because mutants not secreted in their full-length form are secreted in their truncated form; (2) alpha-helix-breaking residues are not a prerequisite for cleavage; (3) the presence, in close proximity to a putative signal peptidase cleavage site, of a polar sequence that maintains the alpha-helical structure of the SA domain is sufficient to promote cleavage. Furthermore pulse chase studies suggest that cleavage is performed in the ER by signal peptidase and indicate that cleavage is not a limiting step in the biosynthesis of the soluble form of the protein.

Human kallikrein hK2 has low kininogenase activity while prostate-specific antigen (hK3) has none.

In the present paper, we determined the kinin-releasing activity of human prostatic kallikrein hK2 and compared it to one of the kallikreins hK1 and prostate specific antigen (hK3). Kinin-like substances active on the rabbit jugular vein were progressively produced when nanomolar concentrations of hK2 were incubated with heated plasma. However in these experiments, hK1 appeared much more potent than hK2 while hK3 was totally inactive. When hK2 was incubated with purified high molecular weight kininogen, several peptides were generated as shown by the analysis on C18 reverse-phase HPLC. Kinin activity was localized exclusively in a small peak having an elution time identical to that of bradykinin while the only important peak obtained with hK1 corresponded to Lys-bradykinin. Finally, the rate of kinin production of hK2 was found to be more than a thousandfold lower than that of hK1. These experiments show that kallikreins hK2 has only a low kininogenase activity. However, it is not excluded that some of the peptides produced by hK2 action could have other types of biological activity.

The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments.

The proprotein convertase PC5 is encoded by multiple mRNAs, two of which give rise to the COOH-terminal variant isoforms PC5-A (915 amino acids [aa]) and PC5-B (1877 aa). To investigate the differences in biosynthesis and sorting between these two proteins, we generated stably transfected AtT-20 cell lines expressing each enzyme individually and examined their respective processing pattern and subcellular localization. Biosynthetic analyses coupled to immunofluorescence studies demonstrated that the shorter and soluble PC5-A is sorted to regulated secretory granules. In contrast, the COOH-terminally extended and membrane-bound PC5-B is located in the Golgi. The presence of a sorting signal in the COOH-terminal 38 amino acids unique to PC5-A was demonstrated by the inefficient entry into the regulated secretory pathway of a mutant lacking this segment. EM of pancreatic cells established the presence of immunoreactive PC5 in glucagon-containing granules, demonstrating the sorting of this protein to dense core secretory granules in endocrine cells. Thus, a single PC5 gene generates COOH-terminally modified isoforms with different sorting signals directing these proteins to distinct subcellular localization, thereby allowing them to process their appropriate substrates.

Comparative cellular processing of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases.

We present here the pulse and pulse-chase analysis of the biosynthesis of the envelope glycoprotein gp160 and its intracellular processing by the subtilisin/kexin-like convertases furin, PACE4, PC1, PC5 and its isoform PC5/6-B. We demonstrate that furin and to a much lesser extent PACE4, PC5/6-B and PC1 are candidate enzymes capable of processing gp160 intracellularly. Furthermore we show that furin can also process gp160/gp120 into gp77/gp53 products by cleavage at the sequence RIQR/GPGR just preceding the conserved GPGR structure found at the tip of the hypervariable V3 loop. The results show that processing into gp120 could occur at or before the trans-Golgi network (TGN) where sulphation of the oligosaccharide moieties of gp160 was detected. In contrast, the formation of gp77/gp53 by furin is a late event occurring after exit from the TGN. Our data also revealed that the alpha glucosidase I inhibitor N-butyldeoxynojirimycin, although affecting the oligosaccharide composition of gp160, does not impair the processing of either gp160 or gp120 by either furin or PACE4. Finally, the co-expression of the [Arg355, Arg358]-alpha-1-antitrypsin Portland variant was shown to potently inhibit the processing of both gp160 and gp120 by these convertases.

Peptidyl substrates containing unnatural amino acid at the P'1 position are potent inhibitors of prohormone convertases.

In order to study further the importance of the P'1 residue upon the activity of human PC1 and human furin, two important members of subtilisin/kexin family of enzymes, we have prepared by solid-phase Fmoc or recently introduced FastMoc chemistry a series of 10 peptidyl substrate analogs. The structures of these analogs are based upon the core sequence of pro-mPC1(83-93) namely, D-Tyr-Lys-Glu-Arg-Ser-Lys-Arg-Xaa-Val-Gln-Lys-Asp, where D-Tyr replaces the native L-Tyr residue and Xaa, representing the P'1 position, corresponds to L-Ser or to nonproteinacous amino acids such as Tle, Sarc, MLeu, Aib, D-Tic or L-Tic. Two more analogs with L-Tic at P'1 position but with one amino acid less, namely P5 Glu or P'3 Gln, and one with a Cit residue in place of Arg at P1 site of the dodecapeptide were also obtained. These peptides were all fully characterized by a combination of MS, 1H-NMR and amino acid analysis. In contrast to the Ser analog, which is an excellent substrate for both hPC1 and hfurin, these analogs displayed moderate to strong inhibition of both hPC1 and hfurin activity in a reversible competitive manner. They all exhibited higher potency for hfurin than for hPC1, with an inhibition constant (Ki) ranging from 0.8 to 10 microM and from 1.0 to 170 microM, respectively. Incorporation of L-Tic yielded an analog with a two to four-fold increased inhibition of either enzymes when compared to its D-Tic counterpart, the effect being more pronounced for hPC1 than for hfurin. Comparison of these data with those for the corresponding N-terminal Fmoc protected peptides revealed that the highly hydrophobic N-terminal Fmoc function occupying the P8 position can contribute positively or negatively towards proteinase inhibition depending on the nature of the unnatural amino acid at P'1 and the enzyme used. Finally, none of the analogs was significantly cleaved by either enzyme. FTIR data on these analogs revealed some important structural differences between the substrate and inhibitor analogs, as there appears to be a conformational shift from a more beta-sheet-like structure for the substrates to a more alpha-helical-like structure for the inhibitors.

N-terminal truncated forms of insulin-like growth factor binding protein-3 in the peritoneal fluid of women without laparoscopic evidence of endometriosis. Le groupe d'investigation en gynécologie.

OBJECTIVE: To characterize and purify peritoneal mitogens able to stimulate the proliferation of human endometrial cells in vitro. DESIGN: Peritoneal fluids (PFs) from 50 patients were collected at laparoscopy and pooled (270 mL) for purification of mitogenic activity. SETTING: University infertility clinic and endocrinology of reproduction and molecular endocrinology laboratories. PATIENTS: Fifty subjects presenting for tubal ligation, pelvic pain, mass, or infertility but otherwise having no evidence of endometriosis inflammation, infection, or tumor. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Assessment of mitogenic activity by 3H-thymidine incorporation into mouse embryo fibroblasts and into primary cultures of isolated epithelial and stromal cells of human endometrium. RESULTS: The PF mitogens were purified successively on carboxymethyl-sepharose and heparin-sepharose columns followed by fractionation on cartridges of C18 silica and reverse-phase high-performance liquid chromatography (HPLC). Four distinct bands were eluted from Sep-Pak, (Mississauga, Ontario, Canada) with molecular weights of 17 to 18, 20, 25, and 29 to 30 kd. The eluted fractions of Sep-Pak exerted preferential mitogenic activity on epithelial-derived human endometrial cells at an equimolar ratio with epidermal growth factor. Microsequencing of the 17 to 18, 20, 25, and 29 to 30 kd bands showed a homologous sequence with N-terminal amino acid sequences of insulin-like growth factor binding protein-3 (IGFBP-3). CONCLUSION: These data indicate that the PF of normal women without evidence of endometriosis contains N-terminal truncated forms of IGFBP-3 that mediate an apparent preferential mitogenic action on epithelial-derived endometrial cells. Therefore, they could play a role in the ectopic growth of endometrial cells.

Processing specificity and biosynthesis of the Drosophila melanogaster convertases dfurin1, dfurin1-CRR, dfurin1-X, and dfurin2.

Pro-protein and pro-hormone convertases are subtilisin/kexin-like enzymes implicated in the activation of numerous precursors by cleavage at sites mostly composed of pairs of basic amino acids. Six members of this family of enzymes have been identified in mammals and named furin (also called PACE), PC1 (also called PC3), PC2, PACE4, PC4, and PC5 (also called PC6). Multiple transcripts are produced for all the mammalian convertases, but only in the cases of PC4, PACE4, and PC5 does differential splicing result in the modification of the C-terminal sequence of these enzymes. A similar molecular diversity is also observed for the convertases of Hydra vulgaris, Caenorhabditis elegans, and Drosophila melanogaster. In the third species, two genes homologous to human furin called Dfur1 and Dfur2 have been identified. The Dfur1 gene undergoes differential splicing to generate three type I membrane-bound proteins called dfurin1, dfurin1-CRR, and dfurin1-X, which differ only in their C-terminal sequence. By using recombinant vaccinia viruses that express each of the dfurin proteins, we investigated the potential effect of the C-terminal domain on their catalytic specificities. For this purpose, these enzymes were coexpressed with the precursors pro-7B2, pro-opiomelanocortin, and pro-dynorphin in a number of cell lines, and the processed products obtained were characterized. Our studies demonstrate that these proteases display cleavage specificities similar to that of mammalian furin but not to that of PC2. In contrast, we noted significant differences in the biosynthetic fates of these convertases. All dfurins undergo rapid removal of their transmembrane domain within the endoplasmic reticulum, resulting in the release of several truncated soluble forms. However, in the media of cells containing secretory granules, such as GH4C1 and AtT-20, dfurin1-CRR and dfurin2 predominate over dfurin1, whereas dfurin1-X is never detected. While pro-segment removal occurs predominantly in the trans-Golgi network for all the dfurins, in the presence of brefeldin A, only dfurin1-CRR and dfurin2 can undergo partial zymogen cleavage. The conclusions drawn from the results of this study may well be applicable to the mammalian convertases PC4, PACE4, and PC5, which also display C-terminal sequence heterogeneity.

Insertion of hydrophilic amino acid residues in the signal peptide/membrane anchor domain of neprilysin (neutral endopeptidase-24.11) results in its cleavage: role of the position of insertion.

We have expressed in COS-1 cells mutants of neprilysin (neutral endopeptidase-24.11; NEP) in which the hydrophilic sequence S-Q-N-S was either substituted for V42-T-M-I or inserted after T38 in the signal peptide/membrane anchor (SA) domain. These mutations were introduced in full-length NEP (mutants NEP(H1) and NEP(H2), respectively) and a form of NEP lacking its cytosolic tail (mutants NEP delta cyto(H1) and NEP delta cyto(H2), respectively). Immunoblotting showed that NEP(H1) was membrane-bound while NEP delta cyto(H1), NEP(H2), and NEP delta cyto(H2) were secreted. Furthermore, carbonate treatment of isolated intracellular membranes suggested that cleavage of the SA domain was performed in the endoplasmic reticulum, presumably by signal peptidase. Sequencing of the secreted proteins indicated that cleavage of the SA domain mostly occurred at the carboxy side of Ala46 but also at the carboxy side of Ala41 in NEP(H2) and NEP delta cyto(H2). We conclude that the position of the S-Q-N-S sequence influences the accessibility of the cleavage site and, in the case of NEP(H1) and NEP(H2), the efficiency of cleavage of the SA domain.

Design and synthesis of novel inhibitors of prohormone convertases.

Prohormone convertase-1 (PC1) and furin are subtilisin-like endopeptidases involved in the biosynthesis of peptide hormones. Five decapeptides representing the junction between the pro-region and the catalytic region of PC1 were prepared. The core sequence corresponded to D-Tyr-Arg-Ser-Lys-Arg- Xaa-Val-Gln-Lys-Asp where D-Tyr replaces the native Glu residue and Xaa, representing the P1' position, corresponds to L-Ser, L-Leu or the unnatural amino acids, D-Ser, beta-Ala, gamma-Abu, beta-Cha or gamma-Hyp. Another analog incorporating an Orn residue in place of the Arg at the P1 site was also prepared. These peptides, synthesized by solid-phase Fmoc chemistry, were fully characterized by FAB-MS, 1H-NMR and amino acid composition. Except for Orn, gamma-Hyp, L/D-Ser and L-Leu containing analogs, the others were found to be moderate to potent competitive inhibitors of hPC1 activity in the following order: gamma-Abu > beta-Cha > beta-Ala, with Ki values ranging from 1 to 8.6 microM. Both L-Ser and L-Leu analogs were correctly cleaved at the acyl carbon COOH-terminal to the Lys-Arg pair by human PC1, whereas beta-Cha, gamma-Abu, beta-Ala and D-Ser analogs proved to be very poor substrates. The Orn and gamma-Hyp derivatives were not cleaved by the enzyme at all. The three analogs containing beta-Cha, gamma-Abu and beta-Ala also proved to be potent inhibitors of the human furin activity in the following order: beta-Ala > beta-Cha > gamma-Abu, with Ki ranging from 0.8 to 2.2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotinylation of an enkephalin-containing heptapeptide via various spacer arms. Synthesis, comparative binding studies toward avidin, and application as substrates in enzymatic reactions.

The preparation of an enkephalin-containing heptapeptide of the sequence Tyr-Gly-Gly-Phe-Leu-Arg-Arg-OH with a biotinyl moiety linked to the carboxy terminus is described. A series of biotinylated derivatives, each containing a different linker (LC) moiety between the biotin function and the carboxyterminal Arg residue, were synthesized by solution-phase chemistry following the coupling of the side chain protected peptide with previously prepared appropriate biotinylamine derivative. Both linear and flexible spacer arms of variable chain lengths [LC = (CH2)x, x = 2, 4, or 6] as well as semirigid cyclohexyl spacers (racemic 1,2-cyclohexane, cis or trans) were incorporated. The relative binding aptitudes of these molecules toward the glycoprotein, avidin, either in immobilized form or in solution were compared using both 125I-labeled and unlabeled peptide derivatives and were found to be in the following order, trans > or = 6C > 4C > 2C > cis. The potential application of these materials as substrates for enzymatic analysis is illustrated for one of the derivatives, namely the LC-2C analogue.

Processing of prodynorphin by the prohormone convertase PC1 results in high molecular weight intermediate forms. Cleavage at a single arginine residue.

Processing of rat prodynorphin (proDyn) by the mouse prohormone convertase PC1 was investigated. Recombinant vaccinia virus vectors were used to coexpress proDyn and PC1 in rat PC12 pheochromocytoma and mouse AtT-20 corticotroph cells. In vitro experiments were also conducted by co-incubating purified proDyn and PC1. The results demonstrate that PC1 cleaves proDyn at pairs of basic residues to yield 10 and 16 kDa high molecular weight (HMW) intermediates. Additionally, PC1 cleaves proDyn at a single arginine residue to yield an 8 kDa product and the C-peptide. This demonstrates that PC1 cleaves proDyn at single and pairs of basic residues.