Human growth hormone releasing factor and somatostatin from two pancreatic tumors: isolation and characterization

Peptides with high intrinsic activity to release growth hormone from pituitary cells in tissue cultures were isolated from two different human pancreatic tumors that had caused acromegaly. Homogeneous peptides were obtained after gel filtration and two steps of reverse-phase high-performance liquid chromatography. From one tumor a 44-residue peptide (human pancreas growth hormone releasing factor, hpGRF-44) was isolated, together with two shorter fragments of reduced bioactivity having 40 and 37 amino acid residues (hpGRF-40, hpGRF-37). In contrast, the other tumor contained only one form of GRF which proved to be identical to hpGRF-40. These hpGRFs are indistinguishable from partially purified preparations of hypothalamic growth hormone releasing factor of human, porcine and murine origins with respect to biological activity and are very similar in their physicochemical properties (molecular weight, retention behavior on reverse-phase HPLC, absence of sulfhydryl groups). One of the pancreatic tumors also contained two forms of immunoreactive somatostatin. One form, after isolation and partial microsequencing, was identified as somatostatin-14 with a structure identical to that of the peptide found in other species. The second form has tentatively been identified as somatostatin-28 on the basis of chromatographic behavior.     

Isolation and chemical characterization of somatostatin-28 from rat hypothalamus

A peptide with somatostatin-like immuno- and bioactivity has been isolated from an aqueous extract of 96 800 rat hypothalamic by means of immunoaffinity chromatography, gel filtration and reverse-phase high-performance liquid chromatography (HPLC). Chemical characterization by amino acid analysis, tryptic peptide mapping and retention behavior in two HPLC system showed that the peptide was indistinguishable from somatostatin-28 previously characterized from several species. This evidence suggests that rat hypothalamic somatostatin-28 is identical in structure to porcine and ovine somatostatin-28.     

Post-translational processing of preprosomatostatin-II examined using fast atom bombardment mass spectrometry

The products and an intermediate of preprosomatostatin-II processing in the anglerfish islet were purified and subjected to structural analysis. The peptides isolated identify the site of signal cleavage (between Ser-24 and Gln-25). The prohormone is further processed at Arg-97 and, to a lesser extent, at the two adjacent basic amino acid residues Lys-61 and Arg-62. A 28-residue somatostatin is also generated which can be hydroxylated at Lys-23. A proteolytic processing site which would form the 14-residue somatostatin does not appear to be used to a significant degree. Fast atom bombardment mass spectrometry (FABMS) was used to demonstrate that the amino-terminal residues of peptides 25-60, and 25-90 are pyroglutamic acid, a modification which precludes Edman degradation of these peptides. Analysis of the peptides and tryptic peptide maps by FABMS allowed confirmation of the sites of prohormone conversion and indicated that terminal basic residues were removed during processing. Three amino acid residues were also found to differ from the amino acid sequence deduced from the cDNA and were localized to specific regions by FABMS analysis. Residues found to differ from the cDNA (cDNA in parentheses) were: Asp-77 (Thr), Val-78 (Phe), and Gly-90 (Glu). Mass assignments were confirmed by running a single cycle of Edman degradation prior to FABMS. The peptides noted above were also examined by Edman sequence analysis. The sequence of a cDNA clone to preprosomatostatin-II was re-examined in light of the observed differences at the protein level. This study emphasizes the utility of FABMS in prohormone processing studies and in identification of post-translational processing events.     

Solid phase synthesis of somatostatin-28 II. A new biologically active octacosapeptide from anglerfish pancreatic islets

Somatostatin-28 II, an octacosapeptide recently isolated from anglerfish pancreatic islets, was synthetized by the solid phase method along with its somatostatin-14 II and somatostatin-28 II-(1-12) corresponding domains. Homogeneity of the synthetic peptides was demonstrated by analytical RP-HPLC, thin layer chromatography and electrophoresis. The peptides were further characterized by amino acids analysis, fast atomic bombarding mass spectrometry and/or 252Cf plasma desorption mass spectrometry. Synthetic somatostatin-28 II and somatostatin-14 II displace equally well the potent agonist (Tyr0,D-Trp8)-somatostatin-14 from its specific binding sites on anterior pituitary cells membranes. Both peptides activate adenylate cyclase from dispersed rat anterior pituitary cells.     

Catfish somatostatin is unique to piscine tissues

It has been previously shown that catfish islets contain two distinct forms of somatostatin: somatostatin-14 and the predominant form, catfish somatostatin, which is a 22-residue peptide structurally related to somatostatin-14. Using antisera against this catfish somatostatin and somatostatin-14, other tissues of the catfish and pancreatic tissue of various animals were examined for the presence of these two peptides. Catfish intestine also contained large amounts of catfish somatostatin in comparison to that of somatostatin-14, but the predominant form in catfish brain tissues was somatostatin-14. Relatively small quantities of catfish somatostatin were found in extracts of anglerfish islet, but none were detected in pancreatic tissues of frog, chicken, or rat. Somatostatin-14 was found in relatively large amounts in these other pancreata. These results suggest that catfish somatostatin is found only in piscine tissues and that there may be a differential expression of the two somatostatin genes in those tissues.     

The somatostatin-28 convertase of rat brain cortex generates both somatostatin-14 and somatostatin-28

The products generated after addition of the ARG-LYS esteropeptidase activity purified from rat brain to synthetic somatostatin-28 were analyzed using radioimmunoassay, HPLC and amino acid analysis. In addition to somatostatin-14, both free arginine and free Lysine were identified together with somatostatin-28. The dipeptide ARG-LYS was not present, which indicates that three peptide bonds were hydrolyzed in order to achieve excision of the doublet. Since it is likely that the octacosapeptide is a precursor for both somatostatin-14 and somatostatin-28, these observations add further support to the hypothesis that the convertase is also involved in the in vivo processing of endogenous somatostatin-28.     

Purification and cDNA cloning of a cecropin-like peptide from the great wax moth, Galleria mellonella

A cecropin-like antimicrobial peptide, Gm cecropin, was purified from hemolymph of larvae of the wax moth, Galleria mellonella, immunized against E. coli, and its antibacterial activity was examined in a radial diffusion assay. The molecular mass of Gm cecropin was 4,160.69 Da by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis. The full-length cDNA of the Gm cecropin precursor was cloned by a combination of RT-PCR, based on the N-terminal sequence obtained by Edman degradation, and 5'-RACE-PCR. Analysis of the cDNA showed that cecropin is synthesized as a prepropeptide, with a putative 22-residue signal peptide, a 4-residue propeptide and a 39-residue mature peptide with a calculated mass of 4,344.18 Da the difference between the calculated and measured masses suggests that Gm cecropin is a 37-residue peptide generated by removal of the C-terminal residue and amidation.     

The use of plasma desorption time-of-flight mass spectrometry to screen for products of prohormone processing in crude tissue extracts

Californium-252 plasma desorption mass spectrometry (252Cf PDMS) of a crude, desalted, extract of piscine endocrine pancreas provided mass information for the major biologically active peptide hormones present in this tissue. An extraction procedure compatible with 252Cf PDMS analysis was developed. In extracts of catfish pancreas, strong molecular ions were identified in the positive mode for somatostatin-14 (1638 amu), O-glycosylated somatostatin-22 (2944 amu), glucagon (3512 amu), glucagon-like peptide (3785 amu), insulin (ca. 5550 amu), and other prohormone-derived peptides. Both protonated species and sodium adducts were apparent in the mass spectrum. A number of other molecular ions were observed including somatostatin-26, 1-10 (1014 amu) and the entire portion of prosomatostatin-22 remaining after removal of somatostatin-22 (6465 amu). The data obtained by this method also resulted in the identification of the third major product of proglucagon processing in catfish pancreas, glicentin-related polypeptide. Subtractive Edman degradation analyzed by 252Cf PDMS was also used to confirm a mass assignment.     

A novel conotoxin from Conus betulinus,kappa-BtX,unique in cysteine pattern and in function as a specific BK channel modulator

A novel conotoxin, kappa-conotoxin (kappa-BtX), has been purified and characterized from the venom of a worm-hunting cone snail, Conus betulinus. The toxin, with four disulfide bonds, shares no sequence homology with any other conotoxins. Based on a partial amino acid sequence, its cDNA was cloned and sequenced. The deduced sequence consists of a 26-residue putative signal peptide, a 31-residue mature toxin, and a 13-residue extra peptide at the C terminus. The extra peptide is cleaved off by proteinase post-processing. All three Glu residues are gamma-carboxylated, one of the two Pro residues is hydroxylated at position 27, and its C-terminal residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that: 1) among voltage-gated channels, kappa-BtX is a specific modulator of K(+) channels; 2) among the K channels, kappa-BtX specifically up-modulates the Ca(2+)- and voltage-sensitive BK channels (252 +/- 47%); 3) its EC(50) is 0.7 nm with a single binding site (Hill = 0.88); 4) the time constant of wash-out is 8.3 s; and 5) kappa-BtX has no effect on single channel conductance, but increases the open probability of BK channels. It is concluded that kappa-BtX is a novel specific biotoxin against BK channels.     

Isolation and characterization of S. cerevisiae mutants defective in somatostatin expression: cloning and functional role of a yeast gene encoding an aspartyl protease in precursor processing at monobasic cleavage sites.

The peptide somatostatin exists as two different molecular species. In addition to the most common form, somatostatin-14, there is also a fourteen amino acid N-terminally extended form of the tetradecapeptide, somatostatin-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which upon cleavage generate either somatostatin-14 or -28, respectively. In some species of fish two distinct, but homologous, precursors (prosomatostatin-I and -II) give rise to somatostatin-14 and -28, respectively. Whereas anglerfish prosomatostatin-II was previously shown to release exclusively somatostatin-28, the yeast Saccharomyces cerevisiae proteolytically matures the homologous prosomatostatin-I precursor to somatostatin-28 and -14 as well as to a lysine-extended form of somatostatin-14. The Kex2 endoprotease appears to be essential for the formation of lysine somatostatin-14 and is involved either directly or indirectly in the release of mature somatostatin-14. The isolation of yeast mutants defective in somatostatin-28 expression (sex mutant) allowed the cloning of a non-essential gene, which encodes an aspartyl protease, whose disruption severely affects the cleavage of mature somatostatin-28 from both somatostatin precursors. We conclude that two distinct endoproteases, which demonstrate some cross specificity in vivo, are involved in the proteolytic maturation of prosomatostatin at mono- and dibasic processing sites in yeast.     

Prosomatostatin processing in anglerfish brain, gut and pancreas

The distribution of somatostatin immunoreactive forms in three tissues of the anglerfish (Lophius piscatorius L.) was analyzed by a combination of gel permeation, High Pressure Liquid Chromatography and amino acid analysis. The data indicate that prosomatostatins I and II are expressed in both neural and gastro-intestinal tissues and that their post-translational processing gives rise to somatostatin-14 I, somatostatin-28 II and to some of its hydroxylysine23-derivative, respectively. It is concluded that, in contrast to the mammals, production of two somatostatins in the Teleostean fish requires two structurally distinct precursors whose processing operates in a fixed pattern rather than in a tissue-specific manner.     

Direct evidence for two distinct prosomatostatin converting enzymes. Detection using a rapid, sensitive, and specific assay for propeptide converting enzymes

Many bioactive peptides are initially synthesized via larger precursors from which they are released by proteolytic cleavage at basic amino acids. Some precursors contain more than one final product peptide, multiple copies of a single peptide, or both. Different product peptides can be produced from a common precursor in different tissues. It is not currently known whether this cell-type specific production of bioactive peptides is mediated by different, specific propeptide converting enzymes (PCEs) or by a small number of similar PCEs. To resolve this issue for the conversion of prosomatostatin, the processing of prosomatostatin-I (aPSS-I) and prosomatostatin-II (aPSS-II) to either somatostatin-14 (SS-14) or somatostatin-28 (aSS-28), respectively, was examined in anglerfish islets. Two distinct forms of PSS PCE activity were detected using a rapid, sensitive, and specific assay. Examination of the specificity of these two enzyme activities showed that one proteolytic activity performs the aPSS-I to SS-14 conversion, while the other protease liberates aSS-28 from aPSS-II. The SS-14-generating PCE also cleaves aPSS-II to produce [Tyr7,Gly10]SS-14 (a tetra-decapeptide analog of SS-14) and converts proinsulin to insulin. The aSS-28-generating PCE does not process proinsulin. These results provide direct evidence that different, specific PCEs are required for liberation of SS-14 and aSS-28 from their precursors.     

Analysis of the primary structure and post-translational modifications of the Schistosoma mansoni antigen Smp28 by electrospray mass spectrometry

The Schistosoma mansoni glutathione-S-transferase with an apparent molecular mass of 28 kDa, Smp28, has a blocked N-terminus which has been elucidated with the aid of the cDNA sequence combined with mass spectrometry and amino acid composition analysis of the N-terminal tryptic peptide. The blocked N-terminal tryptic peptide (m/z 695.8) contained an equimolar ratio of E, G, H, A, I and K³ upon amino acid composition analysis in agreement with its expected sequence AGEHIK, and showed a Δm = +41.7 Da compared to the predicted mass, which is consistent with the N-terminal alanine being acetylated (Δm = +42.0 Da). The mass of the complete molecule (23 744.5 ± 3.3 Da) determined by electrospray mass spectrometry showed a further mass increase of 14 Da with respect to Smp28 containing an N-acetylated alanine. This result is consistent with one of the seven methionines being present as a methionine sulfoxide in ca. 90% of the Smp28 molecules in this preparation. Tryptic mapping of Smp28 showed five of the seven methionines to be partially oxidized by mass spectrometry. This is indicative of the ease with which this modification occurs. Two minor components were detected along with the intact molecule, corresponding to modified forms of the molecule, originating from reaction of the only cysteine residue either with itself forming a covalent dimer or with glutathione. On-line liquid chromatography—mass spectrometry has been compared with the off-line complete tryptic map of Smp28 confirming 97% of the primary structure in less than 2 h.     

Piscidin 4, a novel member of the piscidin family of antimicrobial peptides

Piscidins are linear, amphipathic, antimicrobial peptides (AMPs) with broad, potent, activity spectrum. Piscidins and other members of the piscidin family appear to comprise the most common group of AMPs in teleost fish. All piscidins and related members of the piscidin family described to date are 18–26 amino acids long. We report here the isolation of a novel 5329.25 Da, 44-residue (FFRHLFRGAKAIFRGARQGXRAHKVVSRYRNRDVPETDNNQEEP) antimicrobial peptide from hybrid striped bass (Morone chrysops female x M. saxatilis male). We have named this peptide “piscidin 4” since it has considerable (to > 65%) N-terminal sequence homology to piscidins 1–3 and this distinctive, 10 to 11-residue, N-terminus is characteristic of piscidins. The native peptide has a modified amino acid at position 20 that, based upon mass spectrometry data, is probably a hydroxylated tryptophan. Synthetic piscidin 4 (with an unmodified tryptophan at position 20) has similar antibacterial activity to that of the native peptide. Piscidin 4 demonstrates potent, broad-spectrum, antibacterial activity against a number of fish and human pathogens, including multi-drug resistant bacteria. Its potent antimicrobial activity suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass.     

Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor

Entactin (nidogen), a 150-kD sulfated glycoprotein, is a major component of basement membranes and forms a highly stable noncovalent complex with laminin. The complete amino acid sequence of mouse entactin has been derived from sequencing of cDNA clones. The 5.9-kb cDNA contains a 3,735-bp open reading frame followed by a 3'- untranslated region of 2.2 kb. The open reading frame encodes a 1,245- residue polypeptide with an unglycosylated Mr of 136,500, a 28-residue signal peptide, two Asn-linked glycosylation sites, and two potential Ca2+-binding sites. Analysis of the deduced amino acid sequence predicts that the molecule consists of two globular domains of 70 and 36 kD separated by a cysteine-rich domain of 28 kD. The COOH-terminal globular domain shows homology to the EGF precursor and the low density lipoprotein receptor. Entactin contains six EGF-type cysteine-rich repeat units and one copy of a cysteine-repeat motif found in thyroglobulin. The Arg-Gly-Asp cell recognition sequence is present in one of the EGF-type repeats, and a synthetic peptide from the putative cell-binding site of entactin was found to promote the attachment of mouse mammary tumor cells.     

Synthesis of a biological active tumor growth factor from the predicted DNA sequence of Shope fibroma virus

A 55-residue peptide comprising the carboxyl portion (residues 26-80) of the Shope fibroma virus growth factor (SFGF), a predicted 80-residue DNA virus gene product that encoded a homologous sequence with the epidermal growth factor transforming growth factor alpha family, was synthesized by a stepwise solid-phase method. The synthetic SFGF (26-80) purified to homogeneity by reverse-phase HPLC was characterized by fission ionization mass spectrometry and amino acid analysis. The disulfide pairings were established by enzymatic digestion and mass spectrometry and were found to be similar to those of EGF and TGF alpha. Synthetic SFGF (26-80) was found to share about 10% of the activities as EGF in the radioreceptor binding to A431 cells, stimulation of [3H]thymidine uptake in NRK cells, and induction of colony formation in soft-agar assay. Our results therefore confirmed that SFGF contained the putative biological activities of the EGF-TGF alpha family and that production of SFGF by Shope fibroma virus infected cells may account for the proliferative diseases associated with this particular virus.     

Somatostatin receptor type 5 modulates somatostatin receptor type 2 regulation of adrenocorticotropin secretion

Somatostatin inhibits adrenocorticotropin (ACTH) secretion from pituitary tumor cells. To assess the contribution of somatostatin receptor subtype 5 (SST5) to somatostatin receptor subtype 2 (SST2) action in these cells, we assessed multipathway responses to novel highly monoreceptor-selective peptide agonists and multireceptor agonists, including octreotide and somatostatin-28. Octreotide and somatostatin-28 cell membrane binding affinities correlated with their respective SST2-selective peptide ligand. Although octreotide had similar inhibiting potency (picomolar) for cAMP accumulation and ACTH secretion as an SST2-selective agonist, somatostatin-28 exhibited a higher potency (femtomolar). Baseline spontaneous calcium oscillations assessed by fluorescent confocal microscopy revealed two distinct effects: SST2 activation reduced oscillations at femtomolar concentrations reflected by high inhibiting potency of averaged normalized oscillation amplitude, whereas SST5 activation induces brief oscillation pauses and increased oscillation amplitude. Octreotide exhibits an integrated effect of both receptors; however, somatostatin-28 exhibited a complex response with two separate inhibitory potencies. SST2 internalization was visualized with SST2-selective agonist at lower concentrations than for octreotide or somatostatin-28, whereas SST5 did not internalize. Using monoreceptor-selective peptide agonists, the results indicate that, in AtT-20 cells, SST5 regulates the dominant SST2 action, attenuating SST2 effects on intracellular calcium oscillation and internalization. This may explain superior somatostatin-28 potency and provides a rationale for somatostatin ligand design to treat ACTH-secreting pituitary tumors.     

Biosynthesis of the lantibiotic Pep5. Isolation and characterization of a prepeptide containing dehydroamino acids

Pep5 is a tricyclic peptide antibiotic which contains the unusual amino acids dehydrobutyrine, lanthionine and 3-methyllanthionine. It is matured from a 60-amino-acid precursor peptide (pre-Pep5) deduced from the sequence of the structural gene pepA. To study the biosynthesis of Pep5 we tried to isolate the primary translation product. We identified a peptide in crude extracts of the Pep5-producing Staphylococcus epidermidis strain using antibodies raised against a synthetic 26-residue peptide representing the leader peptide region of pre-Pep5. The putative precursor was purified by reversed-phase HPLC. The isolated peptide did not react with antibodies directed against a C-terminal fragment of mature Pep5 containing two sulfide bridges. Neither lanthionine nor 3-methyllanthionine was detected in amino acid analysis of the isolated precursor. Its amino acid sequence was identical with the sequence predicted from pepA, but Edman degradation stopped at the first threonine residue of the prolantibiotic region indicating a posttranslational modification at this position. The molecular mass of the isolated peptide was 6575.4 +/- 1.7 Da, determined by ion-spray mass spectrometry. This is in agreement with a molecule being dehydrated at the four threonine and the two serine residues in the propeptide region; such a peptide has a calculated molecular mass of 6576.7 Da. The results strongly suggest that maturation of the lantibiotic Pep5 is initiated by selective dehydration of hydroxyamino acids in the propeptide region of the primary translation product and that thioether ring formation is not closely linked to dehydration.     

Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides.

We recently isolated from pig intestine and characterized a 31-residue antibacterial peptide named cecropin-P1 with activity against Escherichia coli and several other Gram-negative bacteria. The isolation involved a number of batch-wise steps followed by several chromatography steps. The continued investigation of these antibacterial peptides has now yielded another antibacterial peptide with high activity against both E. coli and Bacillus megaterium. Amino acid analysis showed a very high content of proline (49 mol%) and arginine (26 mol%), an intermediate level of phenylalanine and low levels of leucine, tyrosine, isoleucine, and glycine. The primary structure was determined by a combination of Edman degradation, plasma desorption mass spectrometry and C-terminal sequence analysis by carboxypeptidase Y degradation using capillary zone electrophoresis for detection of liberated residues. The calculated molecular mass was 4719.7 Da, which is in excellent agreement with 4719 Da obtained by plasma desorption mass spectrometry. The peptide was named PR-39 (proline-arginine-rich with a size of 39 residues). The lethal concentration of the peptide was determined against six Gram-negative and four Gram-positive strains of bacteria.     

Seven peptides derived from pro-somatostatin in rat brain

Acid extracts of murine hypothalamic and extra-hypothalamic rat brains were analyzed by specific radioimmunoassays for the presence of somatostatin-14, somatostatin-28 and somatostatin-28(1–12)-like immunoreactivity. Seven molecular forms were observed after gel permeation chromatography. In addition to somatostatin-14, somatostatin-28 and somatostatin-28(1–12), there were two peptides of 4,400 and 7,500 mol. wt. which contained the somatostatin-28(1–12) sequence with an extension towards the NH₂-terminus of pro-somatostatin. Moreover, two other peptides of 6,000 and 9,500 mol. wt. were detected containing the whole somatostatin-28 structure. These results imply that the processing of brain pro-somatostatin involves a minimum of four cleavage sites and yields at least seven peptides.