The role of EC 3.4.24.15 in the post-secretory regulation of peptide signals.

In the present studies, we characterized the degradation of gonadotropin-releasing hormone (GnRH) by tissues of the ovine hypothalamo-pituitary axis. Membrane and soluble fractions of the medial basal hypothalamus, the pre-optic area, the median eminence and the anterior pituitary demonstrated greater GnRH-degrading activity than either hypophysial-portal or jugular plasma. The primary stable product of the membrane fractions was GnRH1-3, while the major product of the soluble fractions was GnRH1-5, both fragments were generated by plasma. Of all tissue fractions, the highest specific activity was observed in the soluble median eminence. Partial purification and characterization of soluble hypothalamic peptidase activity suggested that GnRH degradation by this tissue occurs via a two-step mechanism involving both post-proline cleaving enzyme and the metalloendopeptidase 3.4.24.15.

Characterization of membrane-associated peptidase activities expressed by endothelial cells of the ovine median eminence.

The capillary endothelial cells of the median eminence represent a potential site for the degradation/modification of both circulating and hypothalamic peptides passing through the hypophysial portal system toward the pituitary. This study examines endothelial cell peptidase expression in vitro by monitoring the metabolism of gonadotropin-releasing hormone (GnRH) by cultured endothelial cells from sheep median eminence. Cleavage of GnRH by median eminence endothelial cell membranes generated GnRH1-5 as the primary stable product, which was then degraded to GnRH1-3 and free amino acids. Degradation of GnRH was completely inhibited by TPCK, ZnCl2 and N-ethylmaleimide, and partially inhibited by EDTA and by a specific inhibitor of the metalloendopeptidase EC 3.4.24.15, CFP-AAY-pAB. Interestingly, an increase in GnRH1-9 production was seen with the latter inhibitors, suggesting a two-step mechanism of GnRH degradation involving a primary cleavage at the Pro9-Gly10-NH2 bond, inhibitable by TPCK, ZnCl2, and NEM, followed by cleavage by EC 3.4.24.15 to generate GnRH1-5. Phosphoramidon and angiotensin converting enzyme inhibitors (as well as other non-specific inhibitors) were without effect, indicating that endopeptidase EC 3.4.24.11 and angiotensin converting enzyme are not involved. Neither bovine aortic endothelial cell nor AtT-20 cell membranes exhibited this pattern of peptidase activity. Degradation of GnRH by intact median eminence endothelial cells in culture was also observed, suggesting an extracellular orientation for these enzymes; the potential role of such peptidases in the fine regulation of both pituitary function and local blood flow is currently under investigation.

Binding of the von Willebrand factor A1 domain to histone.

Activation of the von Willebrand Factor (vWF) A1 domain is a critical factor in regulating the interaction of vWF with its platelet membrane receptor, the glycoprotein (GP) Ib-IX-V complex. This activation controls vWF-dependent platelet adhesion at high shear. The vWF-GP Ib-IX-V interaction is induced in vivo by exposure of platelet-rich plasma to high shear force, or by association of vWF with one or more unidentified components of the subendothelial matrix. In vitro, soluble vWF is activated to bind to platelets by nonphysiological modulators, such as the bacterial glycopeptide, ristocetin, or the snake venom protein, botrocetin, or by removal of negatively-charged sialic acid residues. Analysis of vWF modulators and the very marked charge asymmetry of amino acid sequences within the A1 domain has led to an electrostatic model for vWF modulation. Endothelial membrane/matrix and detergent-soluble fractions of human placenta were screened for the ability to bind vWF by electrophoresis of extracts on SDS-polyacrylamide gels, electrotransferring to nitrocellulose and probing with fluid-phase 125I-labeled vWF or a 39/34-kDa vWF fragment (Leu-480-Gly-718) that encompasses the A1 domain. In the course of these studies, it was found that both vWF and the 39/34-kDa vWF fragment bound strongly to histone. Purified soluble histone also bound vWF since, like ristocetin, it induced vWF flocculation. Histone binding to vWF did not activate or inhibit vWF binding to platelets. While the vWF-histone interaction has no conceivable physiological role, it suggests that binding to the A1 domain of vWF alone is insufficient to modulate vWF adhesive activity. This implies that specific interactions of the vWF A1 domain with either ristocetin or botrocetin are required for GP Ib-IX-V recognition to occur.

Purification, characterisation and distribution of ovine neuronal nitric oxide synthase.

Nitric oxide (NO) is an ubiquitous intercellular messenger molecule synthesised from the amino acid L-arginine by the enzyme nitric oxide synthase (NOS). A number of NOS iso-enzymes have been identified, varying in molecular size, tissue distribution and possible biological role. To further understand the role of NO in the regulation of neuroendocrine function in the sheep, we have purified and characterised ovine neuronal NOS (nNOS) using anion exchange, affinity and size-exclusion chromatography. SDS-PAGE reveals that ovine nNOS has an apparent denatured molecular weight of 150 kDa which correlates well with the other purified nNOS forms such as rat, bovine and porcine. The native molecular weight predicted by size-exclusion chromatography was 200 kD which is in close agreement with that found for porcine and rat nNOS. Internal amino acid sequences generated from tryptic digests of the purified ovine nNOS are highly homologous to rat nNOS. There was no significant difference in the cofactor dependence and kinetic characteristics of ovine nNOS when compared to rat and bovine nNOS, (K(m) for L-arginine 2.8, 2.0 and 2.3 microM respectively). A polyclonal anti-peptide antibody directed toward the C-terminal end of the rat nNOS sequence showed full cross-reactivity with the purified ovine nNOS. Immunohistochemical and Western analysis using this antiserum demonstrate the expression of nNOS in the cortex, cerebellum, hypothalamus and pituitary of the sheep. The lack of staining in the neural and anterior lobes of the pituitary seems to suggest that NOS plays a varied role in the control of endocrine systems between species.

Cleavage of incoming Semliki Forest virus capsid protein within the endocytotic pathway: a feature common to both invertebrate and mammalian cells.

During the initial stages of Semliki Forest virus (SFV) infection in mammalian (baby hamster kidney, BHK) cells, the cleavage of SFV capsid protein could be detected. Analysis of subcellular fractions from SFV-infected BHK cells showed that (a) cleavage of the capsid protein occurred within a prelysosomal compartment of the endocytotic pathway, and (b) following release of the nucleocapsid into the cytoplasm, a 17.5 kD capsid protein fragment could be detected in the subcellular fraction which contained ribosomes. We have previously reported the cleavage of incoming SFV capsid protein in mosquito cells, too. Thus, the proteolytic cleavage of incoming SFV capsid protein is a feature which is common to both invertebrate and mammalian cells. These data further support our hypothesis that the cleavage of incoming capsid protein might provide the conformational change which primes the SFV nucleocapsid for uncoating.

Plasmid-controlled resistance to copper in Escherichia coli.

The copper resistance of a strain of Escherichia coli isolated from the effluent of a piggery where pigs were fed a diet supplemented with copper sulfate was controlled by a conjugative 78-megadalton plasmid designated pRJ1004. Plasmid pRJ1004 exhibited surface exclusion and incompatibility with standard plasmids belonging to incompatibility groups I1 and K. Sensitive strains of E. coli K-12 were unable to form colonies on nutrient agar containing more than 4 mM copper, whereas transconjugants which harbored pRJ1004 were able to form colonies on medium containing up to 20 mM copper.

Epitope mapping of apolipoprotein A-I using endoproteinase cleavage and monoclonal antibodies in an enzyme-linked immunosorbent assay.

The epitopes for two monoclonal antibodies (MAbs) directed towards human apolipoprotein A-I (apoA-I), designated AI-1 and AI-3, have been more precisely defined. Previous work in our laboratory demonstrated that AI-1 and AI-3 recognize antigenic determinants located within cyanogen bromide (CNBr) fragments 1 (CF1) and 3 (CF3), respectively. Using peptides generated from endoproteinase cleavage of CF1 and CF3, we now report that both MAbs are specific for two previously unreported epitopes along the apoA-I molecule. The ability of whole endoproteinase digest mixtures to bind the MAbs, as determined by means of a competitive enzyme-linked immunosorbent assay (ELISA), indicated regions of CF1 and CF3 that were likely to form the epitopes. Purified peptides derived from the digests were then used to localize the epitopes recognized by MAbs AI-1 and AI-3 to within residues 28-47 and 140-147 of apoA-I, respectively. We have previously reported that the epitopes for both MAbs are exposed on HDL2, HDL3, and free apoA-I. Thus, the precise mapping of the binding sites recognized by AI-1 and AI-3 has enabled the identification of regions along apoA-I that are exposed on the surface of lipoprotein particles.

Identification and purification of a novel serine proteinase inhibitor.

We report the identification, purification, and partial amino acid sequence of a novel serine proteinase inhibitor which is present in extracts from human placentas and in the cytosolic fraction of the leukemic cell line K562. Extracts from these tissues exhibited time-dependent inhibition of the serine proteinase thrombin. This activity was not accelerated by heparin and corresponded to a protein which formed a 67-kDa complex with 125I-thrombin. The complex was stable on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cleaved and functionally inactive form of the protein was purified from placental tissue by chromatography on DEAE-Sepharose, followed by affinity chromatography on thrombin-Sepharose. Antibodies raised against the placental protein recognized the inhibitor from K562 cells and placental extract. Western blotting experiments using the antibody showed that the uncleaved inhibitor has a molecular mass of 38 kDa. Amino acid sequencing was performed on the purified protein. Sequences of peptides resulting from digestion with cyanogen bromide followed by Endoproteinase Lys-c confirmed that this is a novel inhibitor with significant homology to the serpin family.

Separation of human apolipoproteins A-IV, A-I and E by reversed-phase high-performance liquid chromatography on a TSK Phenyl-5PW column.

A method has been developed for the rapid separation of the medium-molecular-weight apolipoproteins A-IV, A-I and E by high-performance liquid chromatography. Separations were achieved using a commercially available column of very low hydrophobicity (TSK Phenyl-5PW) in the reversed-phase mode rather than the conventional mode of hydrophobic interaction. Delipidated apolipoproteins were dissolved in 20 mM orthophosphoric acid (pH 2.3), applied to the column which was pre-equilibrated with the same buffer, and eluted with an increasing gradient of acetonitrile. Purified apolipoproteins were identified by a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, amino acid analysis and N-terminal sequence analysis. In one step the method can be used to separate the major human chylomicron apolipoproteins A-IV, A-I and E, following preliminary removal of apolipoprotein A-II and the C apolipoproteins by size-exclusion chromatography.

Application of reversed-phase high-performance liquid chromatography to the separation of apolipoproteins A-IV, A-I and E from rat high-density lipoprotein.

Apolipoproteins A-IV, A-I and E from rat high-density lipoprotein (HDL) were successfully purified by reversed-phase high-performance liquid chromatography (RP-HPLC), using a method which we have previously developed for the separation of apolipoproteins A-IV, A-I and E from human lymph chylomicrons [T. Tetaz, E. Kecorius, B. Grego and N. Fidge, J. Chromatogr., 511 (1990) 147]. Since analytical-scale RP-HPLC indicated that the C apolipoproteins from rat HDL coeluted with both apo A-IV and apo A-I, delipidated rat HDL was first subjected to preparative-scale size-exclusion HPLC (HPSEC) on a Serva Si300 column, which effectively separated the C apolipoproteins from all but apolipoprotein E. Fractions from HPSEC which were enriched for apolipoproteins A-IV, A-I or E were directly applied to RP-HPLC on a TSK Phenyl-5PW column. This procedure yielded fractions containing apolipoproteins A-IV, A-I or E which were pure as assessed by N-terminal sequencing and silver staining of sodium dodecyl sulphate-polyacrylamide gels.

Characterization of novel hsp70 in mammalian cells.

A new protein detected in heat resistant mutants of a murine fibrosarcoma has been identified as a member of the hsp70 family. The protein is similar to the constitutive hsp70 of the parent cells with regard to its antibody cross-reactivity, its ability to bind to an ATP affinity column and partial amino acid sequencing. It is present in addition to, and in similar amounts to, the constitutive isoform of the heat resistant mutant cells and the parent cell line. The lack of either of two post-translational modifications common to other hsps, phosphorylation and ADP-ribosylation, and the demonstration of mRNA for the novel protein suggest that it is a separate gene product and not a post-translational modification of the constitutive hsp70. To our knowledge, this protein has not been described in other systems and may be important in the expression of the heat resistant phenotype of these cells. The in vivo phosphorylation patterns also show that hsp90 and hsp28 are heavily phosphorylated in the heat resistant mutant, but that two other stress proteins reported to be phosphorylated under some conditions are not phosphorylated in these cells.

Association of a phospholipase A2 (14-3-3 protein) with the platelet glycoprotein Ib-IX complex.

Platelet adhesion to subendothelial von Willebrand factor involves receptor recognition by the platelet glycoprotein (GP) Ib-IX and initiates activation signals that contribute to primary hemostasis. We show here that GPIb-IX is specifically associated with an intracellular 29-kDa protein. The physicochemical characteristics and amino acid sequence of this protein indicate that it is identical to the human zeta-isoform 14-3-3 protein, previously characterized as a platelet phospholipase A2 (PLA2). As activation of PLA2 is an early event in GPIb-IX-mediated signaling, this result suggests that ligand occupancy of GPIb-IX may directly activate PLA2, leading to platelet activation.

Characterization of a cDNA encoding the 43-kDa membrane-associated inositol-polyphosphate 5-phosphatase.

Agonist stimulation of cells results in phosphatidylinositol turnover and the generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular calcium. The inositol-polyphosphate 5-phosphatase (5-phosphatase) enzymes hydrolyze Ins(1,4,5)P3 in a signal-terminating reaction. We have isolated a 2.7-kilobase (kb) composite cDNA, encoding the 43-kDa membrane-associated 5-phosphatase, by screening a human placental lambda gt11 library, using degenerate oligonucleotides. The 2.7-kb cDNA contains a 1.1-kb open reading frame, comprising 363 amino acids, which encodes a protein of a predicted molecular mass of 42 kDa. Amino acid sequence analysis demonstrates a number of potential sites for phosphorylation by protein kinase C and a CAAX motif in the COOH terminus, which may mediate membrane localization. The recombinant enzyme was expressed in COS-7 cells, resulting in a 50-fold increase in enzyme activity in the detergent-soluble membrane fraction of the cell (nanomole of Ins(1,4,5)P3 hydrolyzed per min/mg), but only a 2.5-fold increase in 5-phosphatase activity in the total cell homogenate. Sequence analysis demonstrated a 73-amino acid domain in the COOH terminus of the 43-kDa membrane-associated 5-phosphatase, which had 30% sequence identity and 67% similarity to a region in the 75-kDa 5-phosphatase and 34% identity and 70% similarity to a sequence in the protein that is encoded by the gene, defective in Lowe's oculocerebrorenal syndrome. As shown by RNA analysis the 43-kDa membrane-associated 5-phosphatase appears to be predominantly expressed in heart, brain, and skeletal muscle.

Purification and characterization of two forms of soluble thrombomodulin from human urine.

We have isolated and characterized two forms of soluble thrombomodulin from human urine. The purification procedure consisted of ultrafiltration, chromatography on DEAE-Sepharose, affinity chromatography on diisopropyl-phosphate-thrombin and/or monoclonal anti-thrombomodulin IgG affigel followed by reverse-phase HPLC. An active soluble form of thrombomodulin was purified 1600-fold from 34-l urine. The purified protein migrated as a doublet, with molecular mass 76/72 kDa under reducing conditions and 63/57 kDa under non-reducing conditions as determined by SDS/PAGE. Amino acid analysis of the 63/57-kDa soluble thrombomodulin confirmed sequence identity with human thrombomodulin and demonstrated N-terminal heterogeneity. Compared to membrane-type thrombomodulin, the purified 63/57-kDa soluble thrombomodulin was more active as a cofactor for protein-C activation. The second major thrombomodulin fragment urine is an inactive 35-kDa thrombomodulin polypeptide derived from the N-terminal extracellular region of thrombomodulin.

Molecular cloning of cDNA encoding a novel platelet-endothelial cell tetra-span antigen, PETA-3.

Platelet-endothelial cell tetra-span antigen (PETA-3) was originally identified as a novel human platelet surface glycoprotein, gp27, which was detected by a monoclonal antibody (MoAb), 14A2.H1. Although this glycoprotein is present in low abundance on the platelet surface, MoAb 14A2.H1 stimulates platelet aggregation and mediator release. We now report isolation of a cDNA clone encoding PETA-3 from a library derived from the megakaryoblastic leukemia cell line MO7e. The clone encodes an open reading frame of 253 amino acids that displays 25% to 30% amino acid sequence identity with several members of the newly defined Tetraspan, or Transmembrane 4 superfamily. These proteins consist of four conserved putative transmembrane domains with a large divergent extracellular loop between the third and fourth membrane-spanning regions. PETA-3 has a single consensus sequence for N-linked glycosylation located in this extracellular loop. A single PETA-3 RNA transcript (1.6 kb) was detected in RNA isolated from MO7e cells, bone marrow stromal cells, the C11 endothelial cell line, and several myeloid leukemia cell lines. No transcript was detected in the lymphoblastoid cell lines MOLT-4 and BALM-1. This pattern correlates well with previous protein expression data. Northern blot analysis of RNA from a range of human tissues indicated that the transcript was present in most tissues, the notable exception being brain.

Development of methods for purification of membrane associated gonadotropin-releasing hormone binding proteins.

Gonadotropin-releasing hormone (GnRH) is the primary regulator of mammalian reproduction. It stimulates the release of luteinizing hormone and follicle stimulating hormone via receptors on the cell membranes of pituitary gonadotrope cells. This paper describes the development of a protocol for purification of GnRH binding proteins from sheep pituitary membranes. Membranes were best solubilized using a zwitterionic detergent. Solubilized membranes were applied to an affinity column prepared with a GnRH analogue. The most effective analogue was the agonist [D-Lys6,Pro9-NHEt]-GnRH. The column was washed with a gradient of sodium chloride up to 0.4 M and GnRH binding activity was eluted from the column using the acidic buffer. Eluted fractions bound labelled GnRH agonist after neutralization of the buffer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis revealed a major protein band with a relative molecular weight of 67 kD. Amino acid sequence analysis showed that the protein is different from the cloned GnRH receptor, but homologous with a similar protein recently purified from bovine pituitary. This protein may have a function which is modulated by binding of GnRH, GnRH fragments or GnRH-related peptides.

Relaxed specificity of endoproteinase Asp-N: this enzyme cleaves at peptide bonds N-terminal to glutamate as well as aspartate and cysteic acid residues.

Asp-N, an endoproteinase specific for cleavage of protein or polypeptide bonds N-terminal to aspartate or cysteic acid residues, has been shown to possess a similar affinity for certain glutamate residues. Of 18 glutamate residues present in 2 cyanogen bromide fragments of apolipoprotein A-I, 5 residues were cleaved at rates comparable to that of cleavage at the 12 internal aspartate residues present in these polypeptides (all of which were cleaved). Cleavage of these 5 glutamate residues was obtained under standard enzyme digestion conditions, and the identities of all peptides obtained by Asp-N digestion were determined by amino acid sequencing of peaks obtained from reversed-phase high performance liquid chromatography.

Evidence for a two-step mechanism of gonadotropin-releasing hormone metabolism by prolyl endopeptidase and metalloendopeptidase EC 3.4.24.15 in ovine hypothalamic extracts.

The metalloendopeptidase EC 3.4.24.15 is believed to degrade gonadotropin-releasing hormone (GnRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) by cleavage at the Tyr5-Gly6 bond. We compared the ability of crude and partially purified endopeptidase 24.15 from ovine hypothalamus with recombinant rat testicular endopeptidase 24.15 to degrade synthetic GnRH. Both soluble and membrane hypothalamic fractions degraded GnRH to GnRH1-5, with some production of GnRH1-9 and GnRH1-3. Generation of the smaller fragments was blocked by a specific endopeptidase 24.15 inhibitor (CFP-AAY-pAB), but production of GnRH1-9 was reciprocally enhanced, suggesting this peptide may be an intermediate generated by prolyl endopeptidase. Indeed, both bacitracin and Z-Pro-prolinal, inhibitors of this enzyme, markedly reduced GnRH degradation to any product. Degradation of synthetic GnRH1-9 was more rapid than that of GnRH and was inhibited by CFP-AAY-pAB but not bacitracin. Activity against either substrate was greater in the soluble fraction. Repeated washing of the membrane fraction followed by extraction with Triton X-114 suggested that both endopeptidase 24.15 and prolyl endopeptidase, although predominantly soluble, can be peripherally associated with membranes. When fractionated by hydrophobic interaction chromatography, soluble endopeptidase 24.15 degraded GnRH only in fractions that also exhibited prolyl endopeptidase activity. In contrast, maximal degradation of GnRH1-9 was observed in adjacent fractions, which also contained the highest levels of immunoreactive endopeptidase 24.15. The affinity of recombinant endopeptidase 24.15 for GnRH was low (Km = 1.35 mM), was improved 10-15-fold by removal of the COOH-terminal amide or glycinamide (Km = 90 and 119 microM, respectively), and could be inhibited by CFP-AAY-pAB but not bacitracin. Taken together, these results suggest that GnRH metabolism in the hypothalamus may occur via a two-step process involving first removal of Gly10-NH2 by prolyl endopeptidase, followed by cleavage by endopeptidase 24.15 at the Tyr5-Gly6 bond.

Substrate specificity differences between recombinant rat testes endopeptidase EC 3.4.24.15 and the native brain enzyme.

We have characterized and compared the substrate specificity of affinity-purified recombinant rat testes endopeptidase EC 3.4.24.15 (EP 24.15) with that reported for the isolated brain enzyme. Of the peptides tested, only bradykinin, dynorphin A1-8, and neurotensin were efficiently cleaved by the recombinant enzyme (kcat/Km = 3.0, 2.8 and 0.5 x 10(5) M-1sec-1, respectively); other peptides considered substrates of EP 24.15 (gonadotropin-releasing hormone, substance P, somatostatin and angiotensin) were not metabolized. The enzyme was inhibited by metal ion chelators and thiol-reactive agents, as well as a specific EP 24.15 inhibitor (N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate), thus confirming the enzyme as a thiol-dependent metalloendopeptidase. The observed discrepancies in substrate specificity of the recombinant testicular and the isolated brain enzymes may result from tissue-specific forms and/or post-translational modifications of EP 24.15.

The globin fragment LVV-hemorphin-7 is an endogenous ligand for the AT4 receptor in the brain.

Angiotensin IV (Val-Tyr-Ile-His-Pro-Phe) has been reported to interact with specific high-affinity receptors to increase memory retrieval, enhance dopamine-induced stereotypy behavior, and induce c-fos expression in several brain nuclei. We have isolated a decapeptide (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) from sheep brain that binds with high affinity to the angiotensin IV receptor. The peptide was isolated using 125I-angiotensin IV binding to bovine adrenal membranes to assay receptor binding activity. This peptide is identical to the amino acid sequence 30-39 of sheep betaA- and betaB-globins and has previously been named LVV-hemorphin-7. Pharmacological studies demonstrated that LVV-hemorphin-7 and angiotensin IV were equipotent in competing for 125I-angiotensin IV binding to sheep cerebellar membranes and displayed full cross-displacement. Using in vitro receptor autoradiography, 125I-LVV-hemorphin-7 binding to sheep brain sections was identical to 125I-angiotensin IV binding in its pattern of distribution and binding specificity. This study reveals the presence of a globin fragment in the sheep brain that exhibits a high affinity for, and displays an identical receptor distribution with, the angiotensin IV receptor. This globin fragment, LVV-hemorphin-7, may therefore represent an endogenous ligand for the angiotensin IV receptor in the CNS.