Hydrolysis of the synthetic chromophoric hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe catalyzed by bovine pepsin A. Dependence on pH and effect of enzyme phosphorylation level

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu6]substance P6-11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K+ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu6]substance P6-11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe8-Gly9 with minor cleavage sites between Phe7-Phe8 and Gly9-Leu10. In the rat parotid slice system the major cleavage occurs between Gly9-Leu10 with a minor cleavage site between Phe7-Phe8. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH2, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.     

Synthesis and solubility properties of peptide fragments of human hemoglobin alpha-chain (123-136)

The solubility prediction method for protected peptides was successfully applied to relatively small peptide fragments of human hemoglobin alpha-chain (123-136) which contained various polar amino acid residues such as Asp(OBzl), Glu(OBzl), Lys(Z), Ser(Bzl), and Thr(Bzl). As reported previously for hydrophobic peptides and human proinsulin C-peptide fragments, solubility data indicated that the insolubility of protected peptides having a mean value of Pc value below 0.90 appeared to begin at the octa- or nonapeptide sequence level and that beta-sheet structure played an important role in the insolubility of peptides. When a peptide has a beta-sheet structure in the solid state, we can clearly determine the critical chain length for peptide insolubility, the solubility dependence on solvent properties, and the solubility independence of amino acid compositions of peptides.     

The Use of Crown Ethers in Peptide Chemistry – V. Solid-phase Synthesis of Peptides by the Fragment Condensation Approach using Crown Ethers as Non-covalent Protecting Groups

We have previously described the conditions by which peptide synthesis by the solid-phase fragment condensation approach can be carried out using crown ethers as non-covalent protection for the N^α -amino group. Here we demonstrate that the procedure can be extended to large, partially protected peptide fragments possessing free Lys and/or Arg residues. The first step was to ensure that complex formation on the side chain of amino acids was not detrimental to the methodology and exhibited the same solubility and coupling properties as N^α -complexed peptides. Thus, a model hexapeptide was synthesized using Fmoc chemistry containing Lys and Arg residues, which, when complexed with 18-Crown-6, was readily soluble in DCM and coupled quantitatively to a resin-bound tetrapeptide. Two tripeptides were then prepared, one containing a free Ser residue, the other free Tyr, to examine the possible occurrence of side reactions. After coupling using standard conditions only the former tripeptide exhibited the formation of the O-acylation by-product (5%). Another model hexapeptide containing Lys, Tyr, Ser and Asp protected with a TFA-stable adamantyl group was complexed with 18-Crown-6 and coupled to the resin-bound tetrapeptide with near quantative yield. Extending the length of the peptide to 21 and 40 residues, which represent sequences Gly⁵² to Leu⁷² (21-mer) and Pro³³ to Leu⁷² (40-mer) from Rattus norvegicus chaperonin 10 protein, respectively, resulted in partially protected fragments that were readily soluble in water, thus enabling purification by RP-HPLC. Complexation with 18-Crown-6 gave two highly soluble products that coupled to resin-board tetramer with 68% and 50% coupling efficiencies for the 21-mer and 40-mer, respectively. Treatment with 1% DIEA solutions followed by acidolytic cleavage and purification of the major product confirmed that the correct product had been formed, when analysed by amino acid analysis and ESI-MS. These results served to extend the methodology of non-covalent protection of large partially protected peptide fragments for the stepwise fragment condensation of polypeptides.     

Amino acid sequences of hemoglobins I and II from root nodules of the non-leguminous Parasponia rigida-rhizobium symbiosis, and a correction of the sequence of hemoglobin I from Parasponia andersonii

The amino acid sequence of hemoglobins I (pI 6.15 as oxyhemoglobin) and II (pI 5.64 as oxyhemoglobin) from the nitrogen-fixing root nodules of Parasponia rigida have been determined by protein sequencing. The sequence of hemoglobin I (pI 6.16, as oxyhemoglobin) from Parasponia andersonii was re-examined and the corrected primary structure, now in agreement with that predicted from the DNA sequence, is reported. The three Parasponia hemoglobins contain 161 amino acid residues (Mr approximately equal to 18,700 including the heme) with a single cysteine residue and five methionine residues. The N-terminal serine is blocked by an acetyl group. The primary structure of the Parasponia hemoglobins is highly conserved. Hemoglobins I from the two species of Parasponia are identical; both show microheterogeneity at position 30 (Asp/Glu substitution) and hemoglobin I from P. rigida shows microheterogeneity at position 150 (Ala/Val) while hemoglobin I from P. andersonii has only an Ala at 150. P. rigida hemoglobin II shows no microheterogeneity at these positions, having Asp and Val residues respectively, and it contains a single amino acid change of a Gln for an Arg at position 85, which accounts for the 0.5 unit difference in isoelectric point observed between hemoglobins I and II. The sequence data are consistent with allelic heterogeneity at a single locus rather than different genes.     

Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site

Pertussis toxin catalyzes the transfer of ADP-ribose from NAD to the guanine nucleotide-binding regulatory proteins Gi, Go, and transducin. Based on a partial amino acid sequence for a tryptic peptide of ADP-ribosylated transducin, asparagine had been characterized as the site of pertussis toxin-catalyzed ADP-ribosylation. Subsequently, cDNA data for the alpha subunit of transducin indicated that the putative asparagine residue was, in fact, not present in the protein. To determine the amino acid that served as the ADP-ribose acceptor, radiolabel from [adenine-U-14C]NAD was incorporated, in the presence of pertussis toxin, into the alpha subunit of transducin (0.3 mol/mol). An ADP-ribosylated, tryptic peptide was purified and fully sequenced by automated Edman degradation. The amino acid sequence, Glu-Asn 343-Leu-Lys-Asp 346-X-Gly 348-Leu-Phe, corresponds to the cDNA sequence coding the carboxyl-terminal nonapeptide, Glu 342-Phe 350, which includes by cDNA sequence cysteine at position 347. Neither Asn 343 nor Asp 346 appeared to be modified; residue 347 adhered to the sequencing resin. Cysteine, the missing residue, was eluted from the sequencing resin with acetic acid along with 76% of the peptide-associated radioactivity, half of which, presumably ADP-ribosylcysteine, eluted from an anion exchange column between NAD and ADP-ribose; the other half had a retention time corresponding to 5'-AMP. We conclude that Cys 347 and not Asn 343 or Asp 346 is the site of pertusis toxin-catalyzed ADP-ribosylation in transducin.     

Evaluation of peptide libraries: an iterative strategy to analyze the reactivity of peptide mixtures with antibodies.

Peptide libraries corresponding to a presumed mixture of 50,625 tetrapeptides or 16,777,216 hexapeptides were each prepared in a single assembly by standard solid-phase peptide synthesis. By enzyme-linked immunosorbent assay, the tetrapeptide library was shown to inhibit the binding of an antiserum to FMRF amide with an FLRF capture antigen; the hexapeptide library was shown to inhibit the binding of a monoclonal antibody to a 28 amino acid peptide with the corresponding peptide capture antigen. An iterative strategy of variation was used to determine for each position in the tetra- or hexapeptides which amino acid contributed the most to activity. As a result we were able to logically select out of the tetrapeptide library the sequence FLRF and to select out of the hexapeptide library a sequence that differed from the apparent probable epitope but was twice as active. A single amino acid substitution in the logically derived sequence gave a peptide that was 35 times as active as the hexapeptide sequence in the original 28 amino acid peptide.     

The aspartimide problem in Fmoc-based SPPS. Part III.

A newly developed Fmoc-Asp derivative, Fmoc-Asp beta-(2,3,4-trimethyl-pent-3-yl) ester, has been tried in the Fmoc-based SPPS of H-Val-Lys-Asp-Xaa-Tyr-Ile-OH, a well-established peptide model for studying base-catalysed aspartimide formation. When synthesizing the hexapeptide incorporating Gly, Arg(Pbf), Asn(Mtt), Asp(OtBu) or Cys(Acm) for Xaa, considerable amounts of aspartimide-related by-products were to be expected. The Asp(3) beta-carboxy protecting group and the duration of exposure to bases were varied. By-product formation could be reduced by incorporation of the new Asp derivative more efficiently than by introducing the less bulky Asp(OMpe). Significant improvements were observed in cases of prolonged contact with piperidine or DBU. Both beta-carboxy protecting groups were superior to the standard Asp(OtBu) which was also included in this study, but the additional stabilization gained by our new protecting group was valuable especially in syntheses of long peptides or difficult sequences.     

Secondary structure of the Arg-Gly-Asp recognition site in proteins involved in cell-surface adhesion. Evidence for the occurrence of nested beta-bends in the model hexapeptide GRGDSP

The primary sequence Arg-Gly-Asp has been found in a number of proteins which bind to cell surface receptors. Studies with synthetic peptides have shown that the presence of charged side chains alone is not sufficient to confer binding activity. Application of folding algorithms to proteins and peptides having similar sequences indicates that binding activity is strongly correlated with the presence of two or more closely spaced residues that each have a high probability of initiating a beta-bend. Circular dichroic studies on the hexapeptide GRGDSP, whose sequence is contained in fibronectin and which also shows binding activity, demonstrate that it adopts an unusual conformation in aqueous solution. 1H-NMR spectra of the peptide in aqueous solution show that the two amide hydrogens of Asp4 and Ser5 exchange very slowly. Computer-assisted modeling using restrained molecular dynamics and energy minimization results in conformations that include two beta-bends of type III-III or III-I (hydrogen bonds 4----1 and 5----2), fully consistent with constraints imposed by 1H- and 13C-NMR data. It is suggested that this unusual secondary structure provides an additional specificity determinant.     

Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites

Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH2-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe173 and Ser180 and were thus COOH termini of 37K fragments. Two peptides were from the NH2 termini of 57K fragments, starting at Asp218 and Asp222. These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe173-Asp174, Ser180-Asp181, Ser217-Asp218, and Gln221-Asp222, forming eight fragments. The uniformity of cleavages at the NH2-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.     

Purification and characterization of a novel immunomodulatory hexapeptide from alcalase hydrolysate of ultramicro-pretreated silkworm (Bombyx mori) pupa protein

Silkworm (Bombyx mori) pupa protein is one potential source of insect protein for use in food. Immunomodulatory peptides are specific protein fragments that can positively influence human health. Here, we purified a novel immunomodulatory hexapeptide from the alcalase hydrolysate of ultramicro-pretreated silkworm pupa proteinusing Sephadex gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide sequence was determined by liquid chromatography–electrospray ionization– tandem mass spectrometry (LC-ESI-MS/MS). The results showed that the molecular mass of the purified peptide was 656.17 Da, and the amino acid sequence was Pro-Asn-Pro-Asn-Thr-Asn (PNPNTN). Splenocyte proliferation was 87.35% in the presence of 100 μg/ml of purified peptide. The splenocyte proliferation could be promoted upto 248.4% at 100 μg/ml of PNPNTN after induction by Concanavalin A (Con A). PNPNTN was stable in the presence of the gastrointestinal proteases pepsin and trypsin and at temperatures up to120°C. Taken together, these results show that this novel immunomodulatory hexapeptide from silkworm pupae has potential therapeutic value as an immunomodulatory component of functional food.     

Generation of peptides by human erythrocytes: facts and artifacts

Previously reported data on peptide composition of human erythrocyte lysate were obtained under conditions that did not exclude proteolytic degradation of hemoglobin in the process of peptide isolation. Comparative chromatographic analysis of the diluted erythrocyte lysate incubated in acidic conditions with or without proteolytic enzyme inhibitors showed that several peptides earlier identified as intraerythrocyte ones in fact result from hemoglobin degradation by erythrocyte acidic protease(s) during incubation of the lysate. A rational scheme excluding postlysis proteolysis was developed for isolation of peptide fraction. Further analysis resulted in determination of structure and content of about 50 endogenous intraerythrocyte hemoglobin fragments. A primary endopeptidase splitting of alpha- and beta-globin chains followed by consecutive exopeptidase trimming of primary fragments is suggested as a degradation mechanism. The intraerythrocyte peptides were shown to differ from peptides excreted by the erythrocytes to the extracellular medium in the primary culture. It was also found that intraerythrocyte peptides cannot play the role of precursors of hemoglobin fragments present in tissue extracts.     

Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues

Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.     

The structure of a naturally occurring 10K polypeptide derived from the amino terminus of bovine thyroglobulin

A combination of data derived from peptide sequencing and nucleic acid sequencing of cloned cDNA fragments has been used to define the complete amino acid sequence of a 10,000 M.W., thyroxine containing polypeptide derived from bovine thyroglobulin. This fragment, TG-F, which was obtained following reduction and alkylation, has been placed at the amino terminus of the parent protein with hormone located at residue 5 in the primary sequence of the thyroglobulin molecule. The carboxyl terminal sequence of this fragment -Cys-Gln-Leu-Gln is found on the N-terminal side of a lys residue, suggesting that the peptide bond cleavage which occurs to produce this 80 residue fragment from the parent (330K) thyroglobulin chain is a gln-lys. In addition, the amino acid sequence of this 10K fragment contains: No sequence which would be a substrate for glycosylation and no carbohydrate. Several repeated homologous amino acid sequences. A striking number of beta-bends predicted from Chou-Fasman analyses, particularly near its carboxyl terminus.     

Chaperone-like activity of a synthetic peptide toward oxidized gamma-crystallin.

alphaA-Crystallin can function like a molecular chaperone. We recently reported that the alphaA-crystallin sequence, KFVIFLDVKHFSPEDLTVK (peptide-1, residues 70-88) by itself possesses chaperone-like (anti-aggregating) activity during a thermal denaturation assay. Based on the above data we proposed that the peptide-1 sequence was the functional site in alphaA-crystallin. In this study we investigated the specificity of peptide-1 against gamma-crystallin aggregation in the presence of H2O2 and CuSO4. Peptide-1 was able to completely protect against the oxidation-induced aggregation of gamma-crystallin. Removal of N-terminal Lys or the replacement of Lys with Asp (DFVIFLDVKHFSPEDLTVK, peptide-2) did not alter the anti-aggregation property of peptide-1. However, deletion of KF residues from the N-terminus of peptide-1 resulted in a significant loss of its anti-aggregation property. Bio-gel P-30 size-exclusion chromatography of gamma-crystallin incubated with peptide-2 under oxidative conditions revealed that a major portion of the peptide elutes in the void volume region along with gamma-crystallin, suggesting the binding of the peptide to the protein. Peptide-1 and -2 were also able to prevent the UV-induced aggregation of gamma-crystallin. These data indicate that the same amino acid sequence in alphaA-crystallin is likely to be responsible for suppressing the heat-denatured, oxidatively modified and UV-induced aggregation of proteins.     

Isolation and chemical characterization of glicentin C-terminal hexapeptide in porcine pancreas

Using a radioimmunoassay specific for porcine glicentin C-terminal hexapeptide, we isolated a peptide from porcine pancreas and characterized it as the C-terminal 64-69 sequence of glicentin: H-Asn-Lys-Asn-Asn-Ile-Ala-OH. The purification steps included gel filtration, ion-exchange chromatography and HPLC. In each step, the recovery of the desired peptide, radioimmunologically estimated from the respective elution profile, was 71.4-91.7%. The final yield of the hexapeptide was 22 micrograms (4.3%) from 800 g pancreas. The pancreatic content of this peptide was estimated to be approximately equimolar to that of pancreatic glucagon. No hexapeptide-like component was detected in porcine intestinal extracts. The data confirmed that the processing of pancreatic proglucagon liberates the C-terminal hexapeptide of the intramolecular glicentin sequence in a tissue-specific manner during the production of glucagon.     

Substance P degrading systems of rat parotid and hypothalamus

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu⁶]substance P6–11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K⁺ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu⁶]substance P6–11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe⁸-Gly⁹ with minor cleavage sites between Phe⁷-Phe⁸ and Gly⁹-Leu¹⁰. In the rat parotid slice system the major cleavage occurs between Gly⁹-Leu¹⁰ with a minor cleavage site between Phe⁷-Phe⁸. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH₂, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.     

Mechanism of aspartyl proteinase action. VII. Noncovalent complexes of HIV-1 aspartyl proteinase with substrate and substrate-like inhibitors

A computer model of a noncovalent complex of HIV-1 aspartyl protease with substrate-like inhibitor JG-365 was a priori constructed by using the approaches of theoretical conformational analysis and molecular mechanics. The root mean square deviation of the calculated conformation of the inhibitor from the X-ray diffraction analysis data was 0.87 A. These results enabled the a priori calculation of the structure of noncovalent complex of HIV-1 protease with a hexapeptide fragment of its native specific substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val. The only possible orientation of the cleavable peptide bond in this and the nucleophilic water molecule relative to the catalytically active Asp residues of the enzyme (Asp25 and Asp125) was found that provides for the chemical transformation of the substrate to a tetrahedral intermediate. An action mechanism of enzymes of this class was proposed on the basis of the analysis of calculated distances. We showed that neither steric distortion of the cleavable bond nor the formation of unfavorable contacts in molecules of the enzymes and their substrates accompany the optimum orientation of substrate molecules at the active sites of HIV-1 aspartyl proteases and rhizopuspepsin.     

Isolation of a hemoglobin-derived opioid peptide from cerebrospinal fluid of patients with cerebrovascular bleedings.

The hemorphins are peptides with opioid activity, which are enzymatically released from hemoglobin. A decapeptide identical to the sequence 32-41 of the beta-, delta-, gamma- or epsilon-chains of hemoglobin has been isolated from human ventricular cerebrospinal fluid (CSF). The peptide, designated LVV-hemorphin-7, was recovered in relatively high amounts (115-300 pmol per ml) from samples of patients with cerebrovascular bleedings, but was not detectable in control CSF. Its identity with the hemoglobin fragment was confirmed by mass spectrometry and gas-phase sequencing.