Identification of a novel recognition sequence for the integrin alpha 4 beta 1 in the COOH-terminal heparin-binding domain of fibronectin.

The type III connecting segment of fibronectin contains two cell binding sites, represented by the peptides CS1 and CS5, that are recognized by the integrin receptor alpha 4 beta 1. Using assays measuring the spreading of A375-SM human melanoma cells, we now report that the adhesion promoting activity of a 29 kDa protease fragment of fibronectin containing the COOH-terminal heparin-binding domain (HepII), but lacking CS1 and CS5, is completely sensitive to anti-alpha 4 and anti-beta 1 antibodies, suggesting that HepII contains a third alpha 4 beta 1-binding sequence. Examination of the primary structure of HepII revealed a sequence with homology to CS1. A 19mer peptide spanning this region (designated H1) was found to support cell spreading to the same level as the 29 kDa fragment. H1-dependent adhesion was completely sensitive to anti-alpha 4 and anti-beta 1 antibodies. When soluble peptides were tested for their ability to block cell spreading on the 29 kDa fragment, a 13mer peptide comprising the central core of H1 was found to be completely inhibitory. The active region of H1 was localized to the pentapeptide IDAPS, which is homologous to LDVPS from the active site of CS1. Taken together, these results identify a novel peptide sequence in the HepII region of fibronectin that supports alpha 4 beta 1-dependent cell adhesion.     

Molecular association of lectin and beta-glucosidase in corn coleoptile

Corn coleoptile lectin is present with beta-glucosidase (EC. 3.2.1.2.1) in a single tightly bound molecular association complex (88.7 kDa). SDS-PAGE of the molecular complex dissociates into two main components. Of these, at a concentration of 75%, the corn coleoptile beta-glucosidase (60 kDa) is identified by enzymatic activity, with two 16-amino acid tryptic peptides displaying close homology with the primary structure of the enzyme. In separate experiments, we isolated homogenous monomeric enzyme of corn coleoptile. This allowed us to conclude that lectin properties like erythrocyte agglutination, found in the (88.7 kDa) molecular complex, is not due to the beta-glucosidase bound in it. Another protein (30 kDa) dissociated from the same SDS-PAGE gels rendered several tryptic peptides, including a 20-amino acid sequence V(L)GP(Q)W(A)GGSGGSPVDITAEPQR closely homologous to the putative beta-glucosidase aggregating factor (BGAF) precursor described recently. Tryptic peptide SAFTE(A)WN(V)ELK(V) was also present in the BGAF precursor. KFHEQR peptide was not present in BGAF precursor or any other protein sequence examined. Tryptic peptide TYGPFGA showed good homology with the BGAF precursor protein, FEGLYLFHTPLGSGAN peptide displayed identity with the BGAF precursor sequence. Thus, the 30 kDa protein does not appear to be identical to BGAF, but is rather a similar molecule which could be endowed with the lectin properties of the 88.7 kDa molecular complex.     

Identification and characterization of a novel molecular-recognition and self-assembly domain within the islet amyloid polypeptide

The islet amyloid polypeptide (hIAPP) is a 37 amino acid residue polypeptide that was found to accumulate as amyloid fibrils in the pancreas of individuals with type II diabetes. Previous studies identified various fragments of hIAPP that can form amyloid fibrils in vitro (e.g. hIAPP(8-20), hIAPP(23-27), and hIAPP(30-37)). However, no comparative and systematic information was available on the role of these structural domains (or others) in the process of molecular recognition that mediates fibrillization, in the context of the full-length polypeptide. To systematically map and compare potential recognition domains, we studied the ability of hIAPP to interact with an array of 28 membrane-spotted overlapping peptides that span the entire sequence of hIAPP (i.e. hIAPP(1-10), hIAPP(2-11...), hIAPP(28-37)). Our study clearly identified a major domain of molecular recognition within hIAPP, as the polypeptide was found to bind with high affinity to a defined linear group of peptides ranging from hIAPP(7-16) to hIAPP(12-21). The maximal binding of the full-length polypeptide was to the hIAPP(11-20) peptide fragment (with the sequence RLANFLVHSS). In order to define the minimal fragment, within this apparent recognition motif, that is capable of self-association and thus may serve as the core molecular recognition motif, we examined the ability of truncated analogs of the recognition sequence to self-assemble into amyloid fibrils. The shortest active fragments capable of self-assembly were found to be the pentapeptides FLVHS and NFLVH. The apparent role of this motif in the process of hIAPP self-assembly is consistent with the profile of the hIAAP-binding distribution to the peptide array. The identification of such short recognition motifs is extremely useful in the attempts to develop means to block amyloid fibril formation by hIAPP. It is worth mentioning that this is only the second time in which peptides as short as a pentapeptide were shown to form amyloid fibrils (the other pentapeptide is FGAIL).     

Mass spectrometric evaluation of synthetic peptides as primary structure models for peptide and protein deamidation.

Solid-phase peptide synthesis and deamidation measurements using a novel mass spectrometric technique were carried out for 94 model asparaginyl peptides from 3 to 13 residues in length. Deamidation rates of these peptides in pH 7.4, 37.0 degrees C, 0.15 M Tris-HCl buffer were measured and evaluated. It was found that they validate the use of pentapeptide models as surrogates for the primary sequence dependence of peptide and protein deamidation rates and the discovery by difference of secondary, tertiary and quaternary structure effects. Deamidation of the pentapeptide models, compared with that of longer peptides of more intricate structure, is discussed, and the application of this technique to deamidation measurement of intact proteins is demonstrated.     

Inhibition of peptide amidation by disulfiram and diethyldithiocarbamate

Peptidylglycine alpha-amidating monooxygenase is a copper- and ascorbate-dependent enzyme that converts peptides with COOH-terminal glycine residues into the corresponding alpha-amidated product peptides. The relatively selective copper chelator N,N-diethyldithiocarbamate (DDC) and its disulfide dimer, disulfiram (Antabuse), were used to determine whether the availability of copper affects the production of two alpha-amidated pro-ACTH/endorphin-derived peptides, alpha-melanotropin (alpha MSH) and joining peptide. When mouse pituitary corticotropic tumor cells (AtT-20) were grown in medium containing micromolar concentrations of disulfiram or DDC, alpha-amidation of newly synthesized joining peptide was specifically inhibited in a dose-dependent manner. In rats injected twice with disulfiram or DDC, the ability of the intermediate pituitary to alpha-amidate newly synthesized alpha MSH and joining peptide was inhibited in a dose-dependent manner; at disulfiram doses equivalent to those used in alcohol abuse therapy (4 mg/kg/day), only about 10% of the newly synthesized peptides were correctly alpha-amidated. Chronic treatment of rats with DDC or disulfiram produced a dose-dependent increase in the pituitary content of glycine-extended alpha MSH and joining peptide; the total amount of pro-ACTH/endorphin-related material was unaltered. After 11 days of treatment with 4 mg/kg/day disulfiram, about one-third of the pituitary alpha MSH and joining peptide were present in the glycine-extended rather than the alpha-amidated form; pituitary extracts normally contain almost entirely alpha-amidated peptides.     

Isolation and primary structure of a novel pheromonotropic neuropeptide structurally related to leucopyrokinin from the armyworm larvae, Pseudaletia separata.

A novel pheromonotropic neuropeptide has been isolated from a head extract of the armyworm larvae, Pseudaletia separata, by a seven step purification procedure using an in vivo assay with decapitated female moths of Bombyx mori. Amino acid sequence analysis and comparison with synthetic peptides established the primary structure of the peptide, termed Pseudaletia pheromonotropin (Pss PT), as H-Lys-Leu-Ser-Tyr-Asp-Asp-Lys-Val-Phe-Glu-Asn-Val-Glu-Phe-Thr-Pro-Arg-Le u-NH2. Pss PT is structurally related to leucopyrokinin, an insect myotropic neuropeptide, and possesses the C-terminal pentapeptide, Phe-Thr-Pro-Arg-Leu-NH2, responsible for the biological activity.     

Characterization of photosystem 1 chlorophyll a/b-binding apoprotein accumulation in developing soybean using type-specific antibodies.

The structure and supramolecular assembly of the soybean photosystem 1 (PS 1) chlorophyll a/b-binding antenna (LHC 1) was examined. We identified the subunit composition of LHC 1 in soybean and followed the accumulation of individual subunits during light-induced assembly. We observed four LHC 1 subunits, at 23, 22, 21 and 20.5 kDa, obtained partial sequence information by amino-terminal sequence analysis, and classified the 20.5, 22, and 21 kDa subunits as being encoded by type I, II, and IV chlorophyll a/b binding protein genes, respectively. Antisera against LHC 1 subunits were used to follow the accumulation of individual subunits during the light-initiated transition from etioplast to chloroplast. Several points are noteworthy. First, monospecific antibody against the 22 kDa subunit decorated a 25 kDa peptide in etiolated tissue, which declined during maturation. This decline correlated with the light-induced appearance of mature 22 kDa peptide, suggesting a precursor/product relationship. Second, the same antibody identified a 22 kDa protein in mature corn, but not a larger band in etiolated corn, suggesting that LHC 1 accumulation is regulated differently between species before the onset of chlorophyll biosynthesis. Third, the mature 22 kDa subunit appeared somewhat later than the other LHC 1 peptides during greening, implying that this subunit is less intimately associated with the PS1 core than are the subunits appearing earlier in development.     

A heme- and metal-binding hexapeptide from the sequence of rabbit plasma histidine-rich glycoprotein

Rabbit histidine-rich glycoprotein (HRG) binds low-spin heme and metals tightly at several sites that contain histidine. As part of an on-going effort to define and locate the binding sites for these and the other ligands of HRG, the sequence: NH2-Gly-His-Phe-Pro-Phe-His-Trp-... was found in a 16 kDa heme-binding peptide isolated from HRG. The spacing of the histidyl residues in this peptide, which contains the C-terminal 79 residues of HRG, together with molecular modeling suggested that this sequence might constitute one heme binding site of HRG by accommodating heme in a bis-histidyl linkage. Three peptides based on this sequence (I, HFPFHW; II, WHFPFH; and III, HFGFHW) were synthesized, and their ability to bind heme and metals examined. All three peptides bind heme as demonstrated by the changes produced in the absorbance of heme when mixed with the peptides. Substituting glycine for proline in the central position or moving the location of the tryptophan did not affect heme binding. The apparent Kd's of the mesoheme/peptide I, II and III complexes are 75 +/- 25 microM, indicative of heme binding approximately 100 times less avid than the mesoheme/HRG complex (Kd ca. 1 microM), but nearly 1000 times tighter than that of the mesoheme/histidine complex (Kd ca. 60 mM). The absorbance spectra of the mesoheme/peptide complexes, the loss of binding caused by modification of histidine residues, and the pH dependence of heme binding, all indicate that heme forms a low spin, bis-histidyl type of complex with these peptides, like that formed with HRG itself. Copper, but not cadmium or nickel, was an effective inhibitor of heme binding by the peptides. The sequence of HRG congruent with the sequence of peptide I is proposed to be one heme- and metal-binding site of rabbit HRG.     

Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.     

Optimal designing of beta-conglycinin to genetically incorporate RPLKPW, a potent anti-hypertensive peptide

Previously, we introduced the RPLKPW sequence, a highly potent hypotensive peptide designed based on ovokinin (2-7), into three homologous sites in the soybean beta-conglycinin alpha' subunit by site-directed mutagenesis. The modified protein expressed in Escherichia coli reduced blood pressure of spontaneously hypertensive rats (SHRs) after oral administration at a dose of 10 mg/kg, which suggested about 30% of the introduced peptide was released in vivo. In this study amino acid residues around the RPLKPW sequence were optimized with a use of synthetic peptides to facilitate release of RPLKPW by gastrointestinal proteases. Then, fourth RPLKPW was also introduced into the extension domain of the protein. The newly modified protein, which was produced in E. coli, significantly lowered blood pressure in SHRs at a dose of 2.5 mg/kg 4 h after oral administration. Furthermore, we produced an extension domain that corresponds to residues 1-143 of the modified alpha' subunit containing four RPLKPW sequences by introducing a termination codon. The minimum effective dose of the modified extension domain was 1.0 mg/kg, which is 1/2000 that of ovalbumin.     

PEGylation enhances the therapeutic potential of peptide antagonists of the neonatal Fc receptor, FcRn

Peptides targeting the human neonatal Fc receptor (FcRn) were conjugated to poly(ethylene glycol) (PEG) polymers to study their effect on inhibition of the IgG:FcRn protein-protein interaction both in vitro and in mice. Both linear (5-40kDa) and branched (20, 40kDa) PEG aldehydes were conjugated to an amine-containing linker of a homodimeric anti-FcRn peptide using reductive alkylation chemistry. It was found that conjugation of PEG to the peptide compromised the in vitro activity, with larger and branched PEGs causing the most dramatic losses in activity. The conjugates were evaluated in transgenic mice for their ability to accelerate the catabolism of human IgG. Optimal pharmacodynamic properties were observed with PEG-peptide conjugates that contained 20-40kDa linear PEGs and a 20kDa branched PEG. The optimal PEG-peptide conjugates were more effective in vivo than the unconjugated peptide control on a mole:mole and mg/kg basis, and represent potential new longer-acting peptide therapeutics for the treatment of humorally-mediated autoimmune disease.     

Beneficial effect of nitric oxide synthase inhibitor on hepatotoxicity induced by allyl alcohol

The effect of aminoguanidine (a selective inhibitor of inducible nitric oxide synthase) on allyl alcohol-induced liver injury was assessed by the measurement of serum ALT and AST activities and histopathological examination. When aminoguanidine (50-300 mg/kg, i.p.) was administered to mice 30 min before a toxic dose of allyl alcohol (75 microL/kg, i.p.), significant changes related to liver injury were observed. In the presence of aminoguanidine the level of ALT and AST enzymes were significantly decreased. All symptoms of liver necrosis produced by allyl alcohol toxicity almost completely disappeared when animals were pretreated with aminoguanidine at 300 mg/kg. Depletion of hepatic glutathione as a consequence of allyl alcohol metabolism was minimal in mice pretreated with aminoguanidine at 300 mg/kg. It was found that the inhibition of toxicity was not due to alteration in allyl alcohol metabolism since aminoguanidine did not effect alcohol dehydrogenase activity both in vivo and in vitro.     

Evenly tritium-labeled peptides and their in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50-150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium-labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a tg scale of biological samples both in vitro and in vivo.     

Fast atom bombardment mass spectrometric characterization of peptides

Structural characterization of peptides in the range of 500–5000 Da, using fast atom bombardment (FAB) and Cs⁺ ion liquid secondary ion mass spectrometry (SIMS), is reviewed. These include syntheitc peptides Kemptamide (mol wt 1516); GIF-C15 (mol wt 1875), an isolated natural product as an acylated pentapeptide; and polypeptides generated from enzymatic digests of proteins. MS data is shown to reveal molecular weight and sequence information as well as determine disulfide bonds between cysteine residues and glycosylation sites in the case of a glycopeptide. The complementarity of MS technique to classical biochemical methods for peptide characterization is highlighted. The reader is briefly acquainted with two newer ionization techniques namely, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Synthetic chemists and biochemists can refer to the in-depth review articles that are cited throughout this article.     

Identification of cross-reactive peptides using combinatorial libraries circumvents tolerance against Her-2/neu-immunodominant epitope

The majority of the currently defined tumor-associated Ags are often overexpressed products of normal cellular genes. Therefore, tolerance deletes high-affinity T cells directed against the TAAs, leaving only a low-affinity repertoire. We have demonstrated previously that the T cell repertoire against the immunodominant p773-782 A2.1-Her-2/neu-restricted peptide has low affinity in A2xneu mice (Her-2/neu mice crossed with A2.1/Kb mice), compared with A2xFVB mice (A2.1/Kb crossed with FVB-wild-type mice). Immunizations with this peptide have a minor impact in preventing tumor growth in A2xneu mice. Therefore, attempts to expand these responses may be of little clinical value. We hypothesized that if not all possible cross-reactive peptides (CPs) are naturally processed and presented, the possibility exists that T cells against these CPs persist in the repertoire and can be used to induce antitumor responses with higher avidity against native epitopes present on the tumor cells. We have used the positional scanning synthetic peptide combinatorial library methodology to screen the p773-782 T cell clone. The screening data identified potential amino acids that can be substituted in the primary sequences of the p773-782 peptide. The designed CPs induce CTL responses of higher affinity in A2xneu mice compared with the native p773-783 peptide. These CTLs recognize A2+-Her-2/neu(+) tumors with high efficiency. Moreover, multiple immunizations with CPs significantly prolonged the survival of tumor-bearing A2xneu mice. These results have demonstrated that it was possible to circumvent tolerance with the identification of CPs and that these peptides could be of significant clinical value.     

Pro domain peptide of HGCP-Iv cysteine proteinase inhibits nematode cysteine proteinases

Cysteine proteinases (CPs) are synthesized as zymogens and converted to mature proteinase forms by proteolytic cleavage and release of their pro domain peptides. A cDNA encoding a papain-like CP, called hgcp-Iv, was isolated from a Heterodera glycines J2 cDNA library, expressed and utilized to assess the ability of its propeptide to inhibit proteinase in its active form. The hgcp-Iv cDNA sequence encodes a polypeptide of 374 amino acids with the same domain organization as other cathepsin L-like CPs, including a hydrophobic signal sequence and a pro domain region. HGCP-Iv, produced in Escherichia coli as a fusion protein with thioredoxin, degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin and is inhibited by E-64, a substrate and inhibitor commonly used for functional characterization of CPs. Recombinant propeptides of HGCP-Iv, expressed in E. coli, presented high inhibitory activity in vitro towards its cognate enzyme and proteinase activity of Meloidogyne incognita females, suggesting its usefulness in inhibiting nematode CPs in biological systems. Cysteine proteinases from other species produced no noticeable activity.     

Degradation of Dynorphin-Related Peptides by the Puromycin-Sensitive Aminopeptidase and Aminopeptidase M

Abstract: The degradation of dynorphin-related peptides by the puromycin-sensitive aminopeptidase and aminopeptidase M was examined using these peptides as alternate substrate inhibitors. K _i determinations showed that both aminopeptidases exhibit a higher affinity for longer dynorphin-related peptides, i.e., K _i for dynorphin A-17 = 23–30 n M with the K _i increasing to 25–50 µ M for the enkephalin pentapeptides. Binding appears dependent not only on peptide length, but also on its sequence. With aminopeptidase M, as the peptide size increases from five to 10 amino acids, k _cat remains relatively constant; however, as the peptide size increases beyond a decapeptide, k _cat decreases significantly. With the puromycin-sensitive aminopeptidase, similar results were obtained except that k _cat was greatest for the pentapeptide. Thus, if one considers k _cat/ K _m as the relevant kinetic constant for estimating in vivo peptide hydrolysis, these results are consistent with the involvement of aminopeptidase M and the puromycin-sensitive aminopeptidase in the degradation of extended dynorphin-related peptides.     

Peptide-dependent Conformational Fluctuation Determines the Stability of the Human Leukocyte Antigen Class I Complex

In immune-mediated control of pathogens, human leukocyte antigen (HLA) class I presents various antigenic peptides to CD8⁺ T-cells. Long-lived peptide presentation is important for efficient antigen-specific T-cell activation. Presentation time depends on the peptide sequence and the stability of the peptide-HLA complex (pHLA). However, the determinant of peptide-dependent pHLA stability remains elusive. Here, to reveal the pHLA stabilization mechanism, we examined the crystal structures of an HLA class I allomorph in complex with HIV-derived peptides and evaluated site-specific conformational fluctuations using NMR. Although the crystal structures of various pHLAs were almost identical independent of the peptides, fluctuation analyses identified a peptide-dependent minor state that would be more tightly packed toward the peptide. The minor population correlated well with the thermostability and cell surface presentation of pHLA, indicating that this newly identified minor state is important for stabilizing the pHLA and facilitating T-cell recognition.     

Human C5a-related synthetic peptides as neutrophil chemotactic factors.

The pentapeptide L-methionyl-L-glutaminyl-L-leucyl-glycyl-L-arginine, which mimics the C-terminal sequence of human C5a anaphylatoxin, and two additional N-formylmethionyl derivatives of this peptide have been assessed for their ability to simulate C5a-related biological activities. Only the N-formylated peptides display chemotactic activity or induce lysosomal enzyme release when assayed with human neutrophils. Additional studies indicate that the active peptides, although designed after the C-terminal structure of the human C5a molecule, were apparently active because of their interaction with the N-formylmethionyl peptide receptor rather than the C5a receptor on neutrophils.