Type A allatostatins from Drosophila melanogaster and Diplotera puncata activate two Drosophila allatostatin receptors, DAR-1 and DAR-2, expressed in CHO cells.

The type-A allatostatins A (AST-A) are a group of insect peptides with a common C-terminal motif Y/FXFGL-NH(2). The existence of at least four putative type A Drosophila melanogaster ASTs (called type A drostatins or DST-As) has been predicted from the sequence of a recently cloned DST-A preprohormone [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 126-1131]. SRPYSFGL-NH(2), (DST-3A), the only DST isolated from Drosophila so far, activated the first cloned DST-A GPCR (DAR-1) [N. Birgül et al. (1999) EMBO J. 18, 5892-5900]. A newly cloned orphan Dm GPCR, which shares 47% overall and 60% transmembrane region sequence identity with DAR-1, was classified as a second putative Dm DST-A receptor (DAR-2) [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 571-577]. Although activation of DAR-2 by DSTs has been postulated, no experimental evidence for that has been presented to date. In this study, we expressed both DAR-1 and DAR-2 in CHO cells and used a GTPgammaS and a Ca(2+) mobilization assay for pharmacological evaluation of the receptors. Synthetically prepared DST-As, as well as selected Diplotera punctata (cockroach) ASTs, activated DAR-1 and DAR-2 in both functional assays indicating ligand redundancy and cross species activity. Cell pretreatment with pertussis toxin led to some differences in the nature and magnitude of signaling pathways at the DAR-1 and DAR-2 receptors, suggesting possible differential coupling to cellular effector system(s) and distinct biological functions of each receptor in vivo.

Pharmacology of FMRFamide-related peptides in helminths.

Nervous systems of helminths are highly peptidergic. Species in the phylum Nematoda (roundworms) possess at least 50 FMRFamide-related peptides (FaRPs), with more yet to be identified. To date, few non-FaRP neuropeptides have been identified in these organisms, though evidence suggests that other families are present. FaRPergic systems have important functions in nematode neuromuscular control. In contrast, species in the phylum Platyhelminthes (flatworms) apparently utilize fewer FaRPs than do nematodes; those species examined possess one or two FaRPs. Other neuropeptides, such as neuropeptide F (NPF), play key roles in flatworm physiology. Although progress has been made in the characterization of FaRP pharmacology in helminths, much remains to be learned. Most studies on nematodes have been done with Ascaris suum because of its large size. However, thanks to the Caenorhabditis elegans genome project, we know most about the FaRP complement of this free-living animal. That essentially all C. elegans FaRPs are active on at least one A. suum neuromuscular system argues for conservation of ligand-receptor recognition features among the Nematoda. Structure-activity studies on nematode FaRPs have revealed that structure-activity relationship (SAR) "rules" differ considerably among the FaRPs. Second messenger studies, along with experiments on ionic dependence and anatomical requirements for activity, reveal that FaRPs act through many different mechanisms. Platyhelminth FaRPs are myoexcitatory, and no evidence exists of multiple FaRP receptors in flatworms. Interestingly, there are examples of cross-phylum activity, with some nematode FaRPs being active on flatworm muscle. The extent to which other invertebrate FaRPs show cross-phylum activity remains to be determined. How FaRPergic nerves contribute to the control of behavior in helminths, and are integrated with non-neuropeptidergic systems, also remains to be elucidated.

Use of the HIV-1 protease for excision of growth-hormone-releasing factor from synthetic and recombinant peptide precursors.

An autolysis-resistant mutant of the HIV-I protease was employed for removal of metabolically stabilized and highly bioactive analogues of bovine growth-hormone-releasing factor (bGRF) from their larger either synthetic or recombinant precursors. The N-terminal four amino acids in two selected model GRF analogues, Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQVF32-OH (I; GRF32) and Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQ30-OH (IA; GRF30), conform well to the specificity of the HIV-I protease for residues in the P1' to P4' positions of its peptide substrates. A variety of amino acids were tried in the N-terminal extension (positions P4-P1) to fit the protease substrate specificity for the 8 amino acids in positions P4-P4'. A synthetic precursor of I, extended N-terminally with RQVF-, a sequence representing the four C-terminal residues in I, was effectively cleaved by the protease at the Phe-1-Tyr1 bond (... RQVF-decreases-YIDA ...) to release GRF32. However, when several soluble fusion proteins linked to GRF32 by the RQVF sequence were expressed in Escherichia coli, attempts to cleave out the core GRF32 met with variable, and only limited, success. By random mutagenesis in a propeptide segment, [MGQSVAQVF]-decreases-GRF30, (II) was identified as a construct that showed reasonably high-level expression in E. coli and was effectively processed by the HIV-I protease. A yield of 5 mg of pure GRF30 was obtained/litre of culture medium after a single HPLC purification step.

Importance of the proline residue to the functional activity and metabolic stability of the nematode FMRFamide-related peptide, KPNFIRFamide (PF4).

PF4 has previously been shown to have potent inhibitory effects on myoactivity of somatic muscle strips from the nematode. Ascaris suum. This study examined the bioactivity and metabolic stability of position 2- and position 5-modified analogues of PF4. Although the analogues [Leu5]PF4,[Ala2]PF4, [Gly2]PF4, [Ala2,Leu5]PF4, and [Gly2,Leu5]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishable and had the order of potency: PF4 = [Leu5]PF4 > > [Ala2]PF4 = [Ala2,Leu5]PF4 > > [Gly2]PF4 = [Gly2,Leu5]PF4, Leu5 for Ile5 substitutions in PF4 did not alter the activity of this peptide: however, Gly2/Ala2 for Pro2 substitutions reduced, but did not abolish, peptide activity. Peptide stability studies revealed that [Gly2]PF4(2-7) and -(3-7) and [Ala2]PF4(2-7), -(3-7), and -(4-7) fragments were generated following exposure to A. suum somatic muscle strips. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases. Observed analogue metabolism appeared to be due to the activity of released aminopeptidases as identical fragments were generated by incubation in medium that had been exposed to somatic muscle strips and from which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of the tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reveal that Pro2 in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the actions of native nematode aminopeptidases.

Isolation and preliminary biological characterization of KPNFIRFamide, a novel FMRFamide-related peptide from the free-living nematode, Panagrellus redivivus.

A novel FMRFamide-related heptapeptide, Lys-Pro-Asn-Phe-Ile-Arg-Phe-NH2 (KPNFIRFamide), was isolated and characterized from acid ethanol extracts of the free-living nematode, Panagrellus redivivus. Whole-worm extracts contained > or = 9 pmol KPNFIRFamide/g wet weight. A synthetic replicate of this peptide induced a rapid relaxation of tone and inhibited spontaneous contractility in isolated innervated and denervated body-wall muscle strips of the parasitic nematode, Ascaris suum. KPNFIRFamide (0.1 nM) induced measurable relaxations in 50% of the muscle preparations examined. Concentrations > or = 0.3 nM induced relaxation in 100% of muscle preparations examined. The relaxation was short-lived at concentrations of peptide > or = 1 microM and displayed a profile typical of receptor desensitization. These data suggest the occurrence of a closely related peptide in A. suum and add further evidence to the concept of primary structural conservation of FaRPs within the nematodes.

Metabolism of mouse growth hormone-releasing factor, mGRF(1-42)OH, and selected analogs from the bovine GRF series in mouse and bovine plasma in vitro.

The presence of Val2 in mGRF(1-42)OH is unique and, as shown in this study, renders this GRF resistant to plasma DPP-IV, the main enzyme responsible for rapid hydrolysis and inactivation of Ala2-containing GRFs from other species via cleavages between Ala2-Asp3. The presence of DPP-IV activity in mouse serum, and mouse and bovine plasma has been demonstrated with Gly-Pro-p-nitroanilide and/or with two DPP-IV-sensitive bGRF analogs, [Leu27]bGRF(1-29)NH2 and [Ala15,Leu27]bGRF(1-29)NH2, which were effectively converted to their respective (3-29) fragments. During incubations of mGRF(1-42)OH in mouse serum or plasma, as well as in bovine plasma in vitro, no major fragments were detectable, except for small amounts of metabolites with HPLC retention times corresponding to those of mGRF(12-42)OH and mGRF(21-42)OH, indicative of possible trypsin-like cleavages between Arg11-Lys12 and Arg20-Lys21. Both mGRF(1-42)OH (t1/2 52-78.5 min) and [Val2,Ala15,Leu27]-bGRF(1-29)NH2 (t1/2 78.5 min) disappeared 5 to 7 times faster in mouse than in bovine plasma, indicating much higher activity of various degrading enzymes in mouse plasma. In summary, our data provide evidence that mGRF(1-42)OH, despite its resistance to plasma DPP-IV, is degraded relatively fast in mouse plasma or serum because of trypsin-like and other, non-DPP-IV-related, proteolytic cleavages.

Application of thermally assisted electrospray ionization mass spectrometry for detection of noncovalent complexes of bovine serum albumin with growth hormone releasing factor and other biologically active peptides.

Ion-spray ionization mass spectrometry with gentle conditions for solvent removal has been reported as a useful tool for detection of high-affinity noncovalent complexes of biological relevance formed in solution. Two main objectives of this study were (i) to find whether other types of electrospray ionization (ESI) sources, e.g. where the solvent is removed with the help of heat (thermally assisted electrospray), could be utilized for detection of noncovalent biological complexes of high and low affinity and (ii) to find whether ESI-MS can be used for detection of the association of bovine serum albumin (BSA) with biologically active peptides. Using a well-defined high-affinity association of FK506 with its binding protein (FKBP) as model system we proved that ESI-MS with thermally assisted interphase can be used for detection of the FK506-FKBP complexes in a similar way as was previously shown for electrospray mass spectrometry (Ganem et al., J. Am. Chem. Soc. 113, 6294 (1991)). In mixtures of BSA with a 9-10 molar excess of biologically active peptides, such as growth hormone releasing factor (GRF), glucagon, bradykinin or insulin in ammonium acetate at pH 7.5, complexes with a ratio of 1:1, 1:2 and in some cases 1:3 were detected. On the other hand, these complexes disappeared upon acidification, pointing to their noncovalent nature.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of secondary structure on the rate of deamidation of several growth hormone releasing factor analogs.

The objective of this study was to determine whether the rates of deamidation of Asn8 in selected growth hormone releasing factor (GRF) analogs were related to the peptide's secondary structures in solution. Bovine or human [Leu27]GRF(1-32)NH2 (both having Gly at position 15), [Ala15Leu27]bGRF(1-32)NH2 and [Pro15Leu27]bGRF(1-32)NH2 were used as model peptides. The peptide helical content (assessed by CD) increased with the increasing methanol concentration and was as follows: 7, 12 and 18% in 0% MeOH; 24, 48 and 52% in 40% MeOH; and 41, 77 and 81% in 80% MeOH for Pro15Leu27 bGRF(1-32)NH2, [Leu27]hGRF(1-32)NH2 and Ala15Leu27 bGRF(1-32)NH2, respectively. 2D NMR studies done in the presence of 40% CD3OH indicated more helical structure for the Ala15 analog as compared to [Leu27]hGRF(1-32)NH2. In both these peptides Asn8 was included in the helical region. In contrast, the lack of conformational information for the Pro15 analog indicated little helical structure around Asn8. The peptides' deamidation rates decreased and their half-lives increased with increasing MeOH concentrations. At 40% MeOH, the least helical Pro15 bGRF analog (t1/2 = 10.78 h) deamidated 1.5 and 2 times faster than its Gly15 (t1/2 = 15.74 h) and Ala15 (t1/2 = 21.53 h) counterparts, respectively. This study indicates that helical environment around Asn8 in GRF makes this residue less prone to deamidation.

Dipeptidyl peptidase IV (DPP-IV) from pig kidney cleaves analogs of bovine growth hormone-releasing factor (bGRF) modified at position 2 with Ser, Thr or Val. Extended DPP-IV substrate specificity?

The literature reported DPP-IV substrate specificity includes oligopeptides with a penultimate Pro, Hyp or Ala residue. Bovine GRF is a substrate for DPP-IV and is rapidly degraded by the enzyme via removal of its N-terminal Tyr-Ala. Incubation of selected GRF analogs from the [X2,Ala15,Leu27]bGRF(1-29)NH2 series with a porcine-kidney-derived DPP-IV in PBS (pH 7.4) resulted in cleavage at the X2-Asp3 bond. The extent of enzymatic hydrolysis varied with X2 as reflected in the following relative cleavage rates: Ala2 (100%), Ser2 (4%), Thr2 (2.5%), Val2 (0.53%), Ile2 (0%). These cleavages were sequestered when similar experiments were performed in the presence of the DPP-IV-specific inhibitor N-epsilon-(p-NO2-benzyloxycarbonyl)-Lys-Pro-OH. A side reaction, buffer-induced deamidation of Asn8, contributed less than 5% of the total substrate degradation. Although our finding qualitatively extends the DPP-IV substrate specificity to also include N-terminal X-Ser, X-Thr and X-Val sequences, quantitatively, relatively fast cleavages of the GRFs with Ala2 make the latter preferred substrates for DPP-IV. The data presented here indicates that the observed GRF(3-29) fragment formation upon incubation of Ser2- and Thr2-substituted bGRF analogs in bovine plasma could have been DPP-IV-related.

Position 2 and position 2/Ala15-substituted analogs of bovine growth hormone-releasing factor (bGRF) with enhanced metabolic stability and improved in vivo bioactivity.

In order to prepare GRF analogs with high activity in vivo, a strategy was undertaken to stabilize the peptide to dipeptidylpeptidase IV (DPP-IV), a protease found in plasma which inactivates native human and bovine GRF by cleavage of the Ala2-Asp3 bond. Replacement of the Ala2 residue with Ser, Thr, or Gly in [Leu27]bGRF(1-29)NH2 resulted in peptides greatly stabilized against proteolysis in plasma, but having low inherent GH-releasing activity when tested in bovine pituitary cell cultures. Replacement of Gly15 with Ala15 was marginally effective in improving the in vitro bioactivity of this group of peptides. When tested for GH-hormone release in steers, however, the Thr2,Ala15 analog was four times more potent than bGRF(1-44)NH2. Eleven additional analogs from the [X2,Ala15,Leu27]bGRF(1-29)NH2 series were synthesized and evaluated for metabolic stability in bovine plasma and for GH releasing activity in steers in vivo and in rat pituitary cells in vitro. Two compounds, [Val2,Ala15,Leu27]dGRF(1-29)NH2 and [Ile2,Ala15,Leu27]-bGRF(1-29)NH2, had increased GH-releasing activity in steers over that of [Thr2,Ala15,Leu27]-bGRF(1-29)NH2 and over a previously reported super-potent analog, [desNH2Tyr1,D-Ala2,Ala15]-hGRF(1-29)NH2.

1H NMR analysis and in vitro bioactivity of Leu27-bGRF(1-29)NH2 and its D-Ala2 and des-(Tyr1-Ala2)-analogs.

Relative growth hormone-releasing potencies of bovine growth hormone-releasing factor (bGRF) analogs bGRF(1-44)NH2 (I), Leu27-bGRF(1-29)NH2 (II) and D-Ala2, Leu27-bGRF(1-29)NH2 (III) in in vitro bovine anterior pituitary cell cultures were determined to be 100%, 48% and 77%, respectively. The potencies of II and III, although numerically different, were not statistically different. Leu27-bGRF(3-29)NH2 (IV) was approximately 10,000 times less potent than 1. 1H NMR studies of peptides II, III and IV in 35% d3-2,2,2-trifluorethanol (TFE)/65% phosphate buffer at pH 4 revealed very similar, highly helical secondary structures in the 8-29 region, with only subtle differences at the N-termini. This lack of correlation between secondary structure in solution and in vitro bioactivity suggests that either 1) the biological conformations induced at the GRF receptor for II and III vs. IV are different from those generated in TFE/buffer, 2) similar secondary structures may be necessary but not sufficient for the observed bioactivity or 3) residues 1 and 2 of analogs II and III are important contact residues crucial for effective GRF-receptor interaction.

In vitro metabolic degradation of a bovine growth hormone-releasing factor analog Leu27-bGRF(1-29)NH2 in bovine and porcine plasma. Correlation with plasma dipeptidylpeptidase activity.

A bovine growth hormone-releasing factor analog, Leu27-bGRF(1-29)NH2, was rapidly hydrolyzed to Leu27-bGRF(3-29)NH2 when incubated at 0.03 mM with porcine and bovine plasma at 37 degrees C in vitro (t1/2 = 8.4 min and 22.1 min, respectively). The site of cleavage was the same as that reported by Frohman et al. (J. Clin. Invest. 78, 906-913, 1986) for the GRF/human plasma system and was suggested by the authors to be due to the presence of dipeptidylpeptidase IV (DPP-IV) in human plasma. The DPP-IV-like activity of porcine plasma, determined with Gly-Pro-p-nitroanilide as substrate at pH 7.6 was about 2- to 3-fold higher than that of bovine plasma and seems to correlate well with the more rapid degradation of the GRF analog in porcine plasma. The hormone half-life was extended to 83.3 min when Leu27-bGRF(1-29)NH2 was incubated in vitro with bovine plasma in the presence of an equimolar amount of diprotin A (a competitive DPP-IV inhibitor). Dipeptidylpeptidase II-like activity of porcine and bovine plasma (which may overlap with substrate specificity of DPP-IV) was measured with Lys-Ala-beta-naphthylamide and at pH 7.6 was found to be relatively low (3% and 21% of the corresponding plasma DPP-IV activities). Tyr-beta-naphthylamide was hydrolyzed slowly by porcine plasma and not degraded at all by bovine plasma, which suggests that the sequential cleavage from the GRF N-terminus starting with Tyr at position 1 is not dominant.(ABSTRACT TRUNCATED AT 250 WORDS)