The diapause hormone-pheromone biosynthesis activating neuropeptide gene of Helicoverpa armigera encodes multiple peptides that break, rather than induce, diapause

FXPRLamide peptides encoded by the DH-PBAN (diapause hormone-pheromone biosynthesis activating neuropeptide) gene induce embryonic diapause in Bombyx mori, but terminate pupal diapause in Helicoverpa armigera (Har). Here, we explore the mechanisms of terminating pupal diapause by the FXPRLamide peptides. Using quantitative RT-PCR, we observed that expression of Har-DH-PBAN mRNA in the SG of nondiapause-type pupae was significantly higher than in diapause-type pupae. Immunocytochemical results indicated that the level of FXPRLamide peptides and axonal release are related to the diapause decision. Ecdysteroidogenesis in prothoracic glands (PGs) was stimulated by synthetic Har-DH in vivo and in vitro, and labeled Har-DH bound to the membrane of the PG, thus suggesting that DH breaks diapause by activating the PG to synthesize ecdysone. Furthermore, the response of DH in terminating diapause was temperature dependent. Decerebration experiments showed that the brain can control pupal development through the regulation of DH, and DH can terminate diapause and promote development without the brain. This result suggests a possible mechanism of response for the signals of DH and other FXPRLamide peptides in H. armigera.     

Facile SPS of peptides having C-terminal Asn and Gln

Attachment of Fmoc-asparagine or glutamine to p-alkoxybenzyl alcohol type resins has always been difficult and not very effective. A very simple and effective method for the preparation of peptides terminating in asparagine or glutamine is described. The method involves quantitative attachment of Fmoc-Asp-OtBu or Fmoc-Glu-OtBu via their side-chain carboxyl group to a resin functionalized with our TMBPA linker for peptide amides. Peptide synthesis is performed using our standard Fmoc chemistry, and treatment with acid, e.g. TFA/DCM/scavenger mixtures, releases the Asn or Gln peptides.     

The amidating enzyme in pituitary will accept a peptide with C-terminal D-alanine as substrate

A series of tripeptides which terminated in d-alanine, d-serine, d-leucine or l-alanine was synthesized and the peptides tested for their ability to act as substrates for an amidating enzyme present in porcine pituitary. The peptides were allowed to compete with a radiolabelled substrate 125I d-Tyr Phe Gly in the presence of a rate limiting concentration of amidating enzyme and the degree of conversion to 125I d-Tyr Phe amide was determined by ion exchange chromatography. An accelerated procedure was developed for investigating the rates of reaction. The results showed that d-Tyr Phe d-Ala has a significant affinity for the amidating enzyme; no affinity could be demonstrated with d-Tyr Phe 1-Ala, d-Tyr Phe d-Ser or d-Tyr Phe d-Leu. Direct evidence that d-Tyr Phe d-Ala can undergo amidation was obtained by incubating the 125I labelled tripeptide with the pituitary enzyme. Amidation took place readily with d-Tyr Phe d-Ala but not with the other tripeptides; thus, while the enzyme is unable to catalyse the conversion of a peptide terminating in 1-alanine, it can accept a peptide terminating in d-alanine. The results indicate that the amidating enzyme has a highly compact substrate binding site.     

Characterization of beta-endorphin peptides in the spinal cord of the rat

The objective of the present studies was to estimate the total content of beta-endorphin-like immunoreactivity (beta-EPLIR) and to characterize the beta-endorphin-like peptides in distinct regions of the spinal cord using gel filtration and reverse phase high performance liquid chromatography. The concentration of beta-EPLIR expressed as pg per mg tissue was similar in the four regions of the spinal cord. Sephadex G-75 chromatography demonstrated the presence, in all four regions of the spinal cord, of beta-endorphin (beta-EP) immunoreactive peptides eluting at the positions of standard beta-EP and beta-lipotropin (beta-LPH) peptides as well as a high molecular weight form eluting prior to beta-LPH. High performance liquid chromatography of the beta-EP-sized peptides indicated some differences in the relative proportions of the various beta-EP-sized peptides among the four regions of the spinal cord, which suggest a different origin of the beta-EP fibers terminating in different regions of the spinal cord as well as different physiological importance of the beta-endorphin peptides in the various spinal cord regions.     

Identification by radioimmunoassay and HPLC of morphine modulatory peptide-immunoreactivity in rat spinal cord and brain

Two so-called morphine modulatory peptides, an octapeptide and an octadecapeptide, have recently been isolated from bovine spinal cord. We have raised antibodies to the octapeptide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH₂: FF-8), which in radioimmunoassay react with peptides terminating in Arg-Phe-NH₂. This dipeptide is common to both the morphine modulatory peptides and the molluscan neuropeptide FMRF amide. The distribution and molecular forms of immunoreactive peptides were examined in the rat central nervous system and gastrointestinal tract. Highest concentrations of FF-8-like immunoreactivity were found in the dorsal spinal cord, brain stem and hypothalamus. The immunoreactive material in central nervous system extracts was resolved by reversed phase HPLC into three peaks of activity, the two largest peaks eluted in similar positions to the standard octapeptide and octadecapeptide. It appears that previously observed FMRF amide-like immunoreactivity in the rat central nervous system corresponds to peptides immunochemically and chromatographically similar to the two bovine spinal cord peptides.     

Characterisation of N-terminally extended met-enkephalin Arg6Gly7Leu8 variants in the porcine upper digestive tract

Proenkephalin A-derived peptides are known to occur in the gut, but their precise identity is uncertain. We report here the isolation of N-terminally extended forms of Met-enkephalin Arg6Gly7Leu8 from porcine upper digestive tract monitored by radioimmunoassay. A single major form was identified in pyloric antral muscle and mucosa, but in the duodenum two major forms were detected. Microsequence analysis together with immunological data revealed that the antral mucosal peptide and the most acidic duodenal peptide had identical amino-acid sequences, corresponding to a 5.3 kDa peptide terminating in Met-enkephalin Arg6Gly7Leu8. The data indicate that high-molecular-weight peptides may constitute a major proportion of gut opioid peptide immunoactivity.     

Immunological Monitoring of Fenton Fragmentation of Fibrinogen

Fibrinogen is transformed into insoluble “neofibe” by reaction with up to IOOpM Cu(II) and 1.5 mM ascorbate. The soluble peptides which are released during the reaction can be monitored by amino acid analysis and by measuring released keto-carbonyl (with DNPH). Immunologic characterization of the soluble peptides. with anibodies directed against fibrino-peptide A (FPA) clearly show the release of this epitope. optimally at 50 pM Cu(II). Anti-FPB gives no evidence for the release of that epitope. However, N-terminal amino acid analyses reveals the presence of 3 peptides terminating in ALA (alpha chain FPA). GLU (beta chain FPB) and SER/ASP (unknown). The release of fibrinopeptides is interpreted within the context of a general mechanism for OH'-induced peptide chain cleavage via intermediate Schiff-base hydrolysis.     

Immunological Monitoring of Fenton Fragmentation of Fibrinogen

Fibrinogen is transformed into insoluble “neofibe” by reaction with up to IOOpM Cu(II) and 1.5 mM ascorbate. The soluble peptides which are released during the reaction can be monitored by amino acid analysis and by measuring released keto-carbonyl (with DNPH). Immunologic characterization of the soluble peptides. with anibodies directed against fibrino-peptide A (FPA) clearly show the release of this epitope. optimally at 50 pM Cu(II). Anti-FPB gives no evidence for the release of that epitope. However, N-terminal amino acid analyses reveals the presence of 3 peptides terminating in ALA (alpha chain FPA). GLU (beta chain FPB) and SER/ASP (unknown). The release of fibrinopeptides is interpreted within the context of a general mechanism for OH'-induced peptide chain cleavage via intermediate Schiff-base hydrolysis.     

A novel peptide designated PYLa and its precursor as predicted from cloned mRNA of Xenopus laevis skin

A variety of peptides closely related to mammalian hormones and neurotransmitters are secreted from amphibian skin. Using cDNA clones of mRNA isolated from skin of Xenopus laevis, we have been searching for precursors of some of these constituents. Here we present the sequences of parts of cloned mRNAs which code for precursors of a novel peptide. In the predicted polypeptides, pairs of basic residues flank a sequence of 25 amino acids terminating with glycine, the signal for the formation of a terminal amide. The predicted final product liberated from these precursors would be a peptide comprised of 24 amino acids starting with tyrosine and ending with leucine amide, which has therefore been designated PYLa. This peptide can form an amphipathic helix similar to that found in peptides with cytotoxic, bacteriostatic and/or lytic properties.     

Authentic FMRFamide is present in the polychaete Nereis virens

The tetrapeptide FMRFamide is but one member of a large family of invertebrate neuropeptides which includes another tetrapeptide, FLRFamide, and several longer peptides terminating in one or the other of these tetrapeptide sequences. These peptides have been isolated from both molluscs and arthropods, but so far not one has been isolated from an annelid. Since the annelid worms are believed to share a common ancestor with molluscs and arthropods, they should contain FMRFamide-like peptides. We found two immunoreactive peaks in Nereis virens, but microsequencing and fast atom bombardment mass spectrometry revealed that they represent only one native peptide, FMRFamide. (The other peak is its methionyl sulfoxide derivative.) Each worm contained only 100 to 600 fmols of peptide, which is at least 10-100 times less than the levels in molluscs. Our identification of a tetrapeptide, and only a tetrapeptide, in this worm suggests that the tetrapeptides are the more ancient members of the family, and were probably present in the common ancestors of the annelids, arthropods, and molluscs.     

Proteolytic excision and in situ cyclization of a bioactive loop from an REI-VL presentation scaffold.

Covalent cyclization of peptides is an important tool in structure-function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor-bound conformation. Previously we described a 2-step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N-terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res 39:533-539). We show here that the cyclization reaction scheme can be extended to peptides excised from proteins by endo-LysC proteolysis, which generates fragments terminating with Lys. To illustrate the method, we used an immunoglobulin VL domain (REI-VL) with an RGD-containing sequence engineered into its CDR3 and flanked by Lys residues. This REI-VL/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor alpha IIb beta 3. The RGD-containing peptide excised by endo-LysC from the REI-VL presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, and IC50 of about 10 nM. Significantly, both the N alpha-acylation and the cyclization reactions occur efficiently even in the context of the other endo-LysC fragments of REI-VL, which suggests that the reaction may prove useful in converting mixtures of endo-LysC products of many proteins into the corresponding cyclic peptides in situ.     

Sequence requirements for the activity of membrane-active peptides.

Synthetic peptides were constructed with the sequence of the first 20 residues of melittin and terminating with a range of different amino acid amides. These were found to have haemolytic and cytolytic activity similar to that of melittin, provided that certain charge constraints were observed. The nature of the 21st residue was not critical except when the residue introduced a negative charge. The presence of at least two positive charges in the molecule was found to be essential for activity. One of these charges could be the amino-terminal amine. Peptides could be inactivated by the addition of a non-acidic presequence which was acetylated at the N-terminus. Introducing a protease cleavable sequence into an N-terminal extension of the peptides produced analogues with low haemolytic activity that could be activated by proteolytic action. A peptide with extra positive charges introduced on the hydrophilic face of the helix possessed a haemolytic activity that was greater than that of melittin.     

Medium-chain fatty acid synthesis by goat mammary-gland fatty acid synthetase. The effect of limited proteolysis.

Fatty acid synthetase from goat mammary gland was subjected to limited proteolysis by trypsin and elastase. Both proteolytic enzymes selectively cleaved the chain-terminating thioester hydrolase component from the enzyme complex, leaving all other partial activities intact in the core peptides. Trypsin, but not elastase, caused extensive degradation of the released thioester hydrolase. The released thioester hydrolase could be purified to homogeneity by gel filtration. The molecular weight was estimated as 29 000 and the enzyme showed only significant hydrolytic activity toward long-chain acyl-CoA esters. The core peptides retained the ability to synthesize medium-chain acyl-CoA esters in the presence of 2,6-di-O-methyl-alpha-cyclodextrin. The results conclusively show that the terminating thioester hydrolase of goat mammary-gland fatty acid synthetase is not involved in termination of medium-chain-length fatty acid synthesis by this enzyme.     

Analysis of the site-specific N-glycosylation of beta1,6 N-acetylglucosaminyltransferase V

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a beta1,6-linked GlcNAc to the alpha1,6 mannose of the trimannosyl core to form tri- and tetraantennary N-glycans and contains six putative N-linked sites. We used mass spectrometry techniques combined with exoglycosidase digestions of recombinant human GnT-V expressed in CHO cells, to identify its N-glycan structures and their sites of expression. Release of N-glycans by PNGase F treatment, followed by analysis of the permethylated glycans using MALDI-TOF MS, indicated a range of complex glycans from bi- to tetraantennary species. Mapping of the glycosylation sites was performed by enriching for trypsin-digested glycopeptides, followed by analysis of each fraction with Q-TOF MS. Predicted tryptic glycopeptides were identified by comparisons of theoretical masses of peptides with various glycan masses to the masses of the glycopeptides determined experimentally. Of the three putative glycosylation sites in the catalytic region, peptides containing sites Asn 334, 433, and 447 were identified as being N-glycosylated. Asn 334 is glycosylated with only a biantennary structure with one or two terminating sialic acids. Sites Asn 433 and 447 both contain structures that range from biantennary with two sialic acids to tetraantennary terminating with four sialic acids. The predominant glycan species found on both of these sites is a triantennary with three sialic acids. The appearance of only biantennary glycans at site Asn 433, coupled with the appearance of more highly branched structures at Asn 334 and 447, demonstrates that biantennary acceptors present at different sites on the same protein during biosynthesis can differ in their accessibility for branching by GnT-V.     

Cyclic Peptides Arising by Evolutionary Parallelism via Asparaginyl-Endopeptidase-Mediated Biosynthesis

The cyclic miniprotein Momordica cochinchinensis Trypsin Inhibitor II (MCoTI-II) (34 amino acids) is a potent trypsin inhibitor (TI) and a favored scaffold for drug design. We have cloned the corresponding genes and determined that each precursor protein contains a tandem series of cyclic TIs terminating with the more commonly known, and potentially ancestral, acyclic TI. Expression of the precursor protein in Arabidopsis thaliana showed that production of the cyclic TIs, but not the terminal acyclic TI, depends on asparaginyl endopeptidase (AEP) for maturation. The nature of their repetitive sequences and the almost identical structures of emerging TIs suggest these cyclic peptides evolved by internal gene amplification associated with recruitment of AEP for processing between domain repeats. This is the third example of similar AEP-mediated processing of a class of cyclic peptides from unrelated precursor proteins in phylogenetically distant plant families. This suggests that production of cyclic peptides in angiosperms has evolved in parallel using AEP as a constraining evolutionary channel. We believe this is evolutionary evidence that, in addition to its known roles in proteolysis, AEP is especially suited to performing protein cyclization.     

Structure-based design, synthesis, and pharmacologic evaluation of peptide RGS4 inhibitors.

Regulators of G-protein signaling (RGS) proteins form a multifunctional signaling family. A key role of RGS proteins is binding to the G-protein Galpha-subunit and acting as GTPase-activating proteins (GAPs), thereby rapidly terminating G protein-coupled receptor (GPCR) signaling. Using the published RGS4-Gialpha1 X-ray structure we have designed and synthesized a series of cyclic peptides, modeled on the Gialpha Switch I region, that inhibit RGS4 GAP activity. These compounds should prove useful for elucidating RGS-mediated activity and serve as a starting point for the development of a novel class of therapeutic agent.     

Modulators of the activation of the proteasome by PA28 (11S Reg)

The degradation of chromogenic substrates and oligopeptides by the 20S proteasome is markedly enhanced and the generation of antigens for presentation by the MHC class-I system is facilitated by combination with an activator protein known as PA28 or 11S reg. We have described the properties of a PA28-proteasome modulator, N-benzyloxycarbonyl-Ile-Glu(O-t-Bu)-Ala-leucinol which shifts the pathway of peptide hydrolysis by the activated proteasome to products terminating in an acidic amino acid at the expense of products terminating in a hydrophobic amino acid. We now report that piperazinyl phenothiazines and several other antipsychotic drugs modulate the PA28-20S activated proteasome in an opposite manner. Fluphenazine, trifluoperazine and prochlorperazine antagonize the peptidylglutamyl peptide bond hydrolyzing activity of the activated proteasome much more strongly than the chymotrypsinlike activity. The chicken ovalbumin immunodominant epitope SIINFEKL is degraded by the activated proteasome to SIINFE and SIINF in approximately equimolar amounts. Piperazinyl phenothiazines promote formation of SIINF whereas Psi-ol promotes formation of SIINFE. PA28- proteasome modulators by modifying the profile of peptides produced by the activated proteasome, may either enhance or suppress the immune response.     

Peptidergic modulation of the hippocampus synaptic activity

Effects of two newly synthesized nootropic and anxiolytic dipeptides: Noopept and Selank on inhibitory synaptic transmission in hippocampal CA1 pyramidal cells were investigated using patch-clamp technique in whole-cell configuration. Bath application of Noopept (1 microM) or Selank (2 microM) significantly increased the frequency of spike-dependent spontaneous m1PSCs, whereas spike-independent mlPSCs remained unchanged. It was suggested that both peptides mediated their effect sue to activation of inhibitory interneurons terminating on CA1 pyramidal cells. Results of current clamp recording of inhibitory interneurons residing in stratum radiatum confirmed this suggestion, at least for Noonent.     

Affinity labelling of proteinases with tryptic specificity by peptides with C-terminal lysine chloromethyl ketone

Methods are described for the synthesis of peptides terminating in Lys-CH(2)Cl. The products were examined as affinity labels for several enzymes of trypsin-like specificity which are resistant to Tos-Lys-CH(2)Cl. In part, the inertness of the latter may be due to the sulphonamide group, since Z-Lys-CH(2)Cl was more effective. However, a number of tripeptides with C-terminal Lys-CH(2)Cl were superior in their ability to inactivate subtilisin, thrombin and plasma kallikrein. The possibility of developing enzyme-specific reagents selective for members within the trypsin-like group is demonstrated by Ala-Phe-Lys-CH(2)Cl, which readily inactivates plasma kallikrein but not thrombin.