Studies on cytochrome C. XII. Synthesis of the protected undecapeptide (sequence 66-76) and pentadecapeptide (sequence 66-80) of horse heart cytochrome c.

This paper is part of a series on synthesis of suitably protected peptides covering the 66-104 sequence of horse heart cytochrome c. It describes the preparation, by conventional procedures, of a partially protected N alpha-benzyloxycarbonyl-undecapeptide hydrazide corresponding to the sequence from 66 to 76 (Fragment F), which represents a building block for the synthesis of the entire 66-104 sequence. Moreover, the preparation is described of a partially protected pentadecapeptide corresponding to the sequence region 66 to 80, which represents the key peptide for the semisynthesis of the same COOH-terminal sequence utilizing the natural 81-104 N epsilon-trifluoroacetylated CNBr fragment.     

脊髓灰质炎病毒合成肽的免疫诱导作用

 本文报道了模拟脊髓灰质类Ⅰ型病毒Mahoney株外壳蛋白的氨基酸顺序,用化学方法合成了位于VP1 70～75、95～102、93～103、70～75+93～103和VP 3132～143的五段肽。合成的肽段与载体蛋白偶联后免疫家兎能产生明显的免疫记忆作用。结果表明这些合成肽均能被脊髓灰质炎多克隆抗血清所识别,具有一定的免疫原性。     

Functionally distinct agretopic and epitopic sites. Analysis of the dominant T cell determinant of moth and pigeon cytochromes c with the use of synthetic peptide antigens

The dominant T cell determinant on moth and pigeon cytochromes c in B10.A (E beta k:E alpha k) mice is located in the C-terminal portion of the protein, contained within residues 93-103 or 93-104. Thirty-seven antigen analogs, containing single amino acid substitutions at positions 98, 99, 101, 102, 103, and 104, were synthesized. The effects of the substitutions on in vitro antigenicity and in vivo immunogenicity were determined. Functional assays with T cell clones identified residues 99, 101, 102, and 103 as critical, based on their effect on antigenic potency. Peptides containing substitutions at residues 99, 101, and 102 were capable of eliciting unique clones upon immunization of B10.A mice. This was consistent with the identification of these residues as part of the epitope, the site on the antigen that interacts with the T cell receptor. Immunization with peptides substituted at residue 103, however, failed to elicit clones with unique specificity for the immunogen. When these peptides were tested for their ability to stimulate the T cell clones with antigen-presenting cells from B10.A(5R) mice expressing the E beta b:E alpha k Ia molecule, a consistent change in the relative antigenic potency was observed with 50% of the peptides. The effect of the Ia molecule on the antigenic potency ruled out the possibility that residue 103 nonspecifically affected antigen uptake or processing and identified residue 103 as part of the agretope, the site that interacts with the Ia molecule. The locations of the agretope and the epitope on this antigenic determinant appear to be fixed, even in the presence of large numbers of amino acid substitutions. However, some substitutions were found to affect both the agretope and the epitope, placing limits on the functional independence of the two sites. The results are discussed in terms of the trimolecular complex model of T cell activation and the implications of these data for antigen-Ia molecule interactions.     

Substrate specificity of phospholipid/Ca2+-dependent protein kinase as probed with synthetic peptide fragments of the bovine myelin basic protein

The substrate specificity of phospholipid/Ca2+-dependent protein kinase (protein kinase C) was studied using synthetic peptides, in particular those corresponding to the amino acid sequence around serine 115 in bovine myelin basic protein (MBP). It was found that MBP (104-118) and MBP (104-123) were substrates for the enzyme, with apparent Km values of 14 and 10 microM, respectively. Neither MBP (111-118) nor MBP (111-123) were phosphorylated, indicating that an additional segment of sequence extending toward the N terminus, but not toward the C terminus, was essential for the substrate activity of the peptides. Of the alanine-substituted analogs examined, [Ala 105] MBP (104-118) was comparable to the parent peptide, whereas [Ala 107] MBP (104-118) and [Ala 113] MBP-(104-118) were much poorer substrates. These findings indicated that lysine 105 was not essential, but both arginine 107 and arginine 113 were important specificity determinants. Initial studies revealed that [Ala 113] MBP (104-118) inhibited phosphorylation by the enzyme of the parent peptide and, to a lesser extent, the intact MBP(1-170). Serine 115 was the only site phosphorylated in the analog peptides [Ala 105] MBP (104-118) and [Ala 107]MBP (104-118). In the parent peptide, serine 115 was the initial site of phosphorylation but after prolonged phosphorylation other sites became phosphorylated (serine 110 and/or serine 112), further supporting the concept that arginine residues act as essential substrate specificity determinants for phospholipid/Ca2+-dependent protein kinase.     

Characterization of periplasmic protein BP26 epitopes of Brucella melitensis reacting with murine monoclonal and sheep antibodies

More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues ⁹³DRDLQTGGI¹⁰¹ (position 93 to 101) or residues ¹⁰⁴QPIYVYPD¹¹¹, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65–70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90.     

Synthetic myelin basic protein peptide analogs are specific inhibitors of phospholipid/calcium-dependent protein kinase (protein kinase C)

Synthetic peptide analogs of the bovine myelin basic protein (MBP) corresponding to residues 104-118 were found to specifically inhibit phospholipid/ Ca2+-dependent protein kinase (protein kinase C). The peptides [Ala107]MBP (104-118) and [Ala113]MBP (104-118) inhibited protein phosphorylation of intact MBP, histone H1 and peptide phosphorylation with MBP(104-123), MBP(104-118) or [Ala105]MBP (104-118) as substrates. The inhibitor peptides [Ala107]MBP(104-118) and [Ala113]MBP (104-118), containing alanine in place of the arginine recognition sites, apparently inhibited the enzyme noncompetitively with respect to substrates, with IC50 values ranging from 46-145 and 28-62 microM, respectively. These peptide analogs did not inhibit cyclic AMP-dependent protein kinase or myosin light chain kinase but inhibited phospholipid/Ca2+-dependent phosphorylation of endogenous proteins in the total, solubilized fraction of rat brain.     

Specific cyanylation and cleavage at cysteine-104 human hemoglobin alpha-chain. A novel approach to the problem of the alpha-chain tryptic core in the study of haemoglobin variants by "fingerprinting" methods.

1. A new approach to the analysis, by "fingerprinting", of the tryptic core region of human haemoglobin alpha-chain is described. 2. The alpha-chain is cyanylated at its single cysteine residue (alpha104) and then split, by exposure to mild alkali, at the N-peptide bond of the resulting beta-thiocyanoalanine residue. 3. The two cleavage fragments, alpha1-103 and alpha104-141, are separated by gel filtration, and the fragment alpha104-141, which contains all the residues of the alpha-chain tryptic core, is digested with pepsin. 4. Preparative "fingerprints" of these peptic peptides yield eight major peptides, which provide complete sequence information for the whole region alpha104-141. 5. The utility of the method is demonstrated by repeating the determination of the substitution in haemoglobin Hopkins-2, a known alpha-chain core variant in which histidine-alpha112 (G19) is replaced by an aspartic acid residue.     

Rat liver HMG CoA reductase, a glycoprotein of the endoplasmic reticulum, is in equilibrium between monomeric and dimeric forms

Incubation of rat hepatocytes with [35S]methionine in pulse and pulse-chase experiments followed by immunoprecipitation of the HMG CoA reductase and SDS-PAGE results in two labelled polypeptides of 104 and 180 Kdaltons. These two polypeptides have half lives of 80 and 46 minutes respectively. When hepatocytes are incubated with mevalonolactone, and a pulse of [35S]methionine is given, the rate of synthesis of both the 180 and 104 Kd peptides is strongly diminished. After treatment of the [35S] labelled immunoprecipitates with endoglycosidase H, the 180 Kd reductase splits into two labelled peptides of 110 and 97 Kd. We suggest that in addition to the 104 Kd reductase, the endoplasmic reticulum contains the dimer of two reductases linked by a carbohydrate chain. The equilibrium monomer-dimer probably regulates the rate of degradation of reductase.     

Solid-phase synthesis of the native sequence of horse heart cytochrome c-(66-104)-nonatriacontapeptide and of a C-terminal carboxyamide analog selectively modified at Met-80

The peptide corresponding to the (66-104) sequence of horse heart cytochrome c and its carboxyamide analog, selectively modified at the critical Met80 residue, have been synthesized by stepwise solid-phase methods on PAM and BHA resins respectively. The correctness of the growing peptide chain as well as the homogeneity of the final products have been monitored by several analytical methods including quantitative Edman degradation. After HF cleavage both peptides were purified by semipreparative HPLC. The overall yields were 24% for the native (66-104) and 10% for the carboxyamide analog. The homogeneity of the purified synthetic peptides have been determined by different criteria including HPLC, amino acid composition, Edman degradation, electrophoresis, and tryptic peptide mapping. The synthetic fragments have been utilized for preliminary semisynthesis experiments with the native [Hse greater than 65] (1-65)H heme-sequence.     

Effects of ATP and CTP on the conformation of the regulatory subunit of Escherichia coli aspartate transcarbamylase in solution: a medium-resolution hydrogen exchange study

Medium-resolution hydrogen exchange methods have been used to examine the solvent accessibility of seven peptides in the regulatory subunit (r2) of Escherichia coli aspartate transcarbamylase in the presence and absence of ATP and CTP. Both nucleotides are allosteric effectors of the holoenzyme; binding of ATP increases the affinity of the holoenzyme for the substrate L-Asp, while CTP has the opposite effect. Following Rosa and Richards (1979, 1981, 1982) and Englander et al. (1983, 1985), exchange-out curves for individual peptides were generated by adjusting the pH to 2.7 to quench exchange-out, digesting the protein with pepsin, separating peptides by reverse-phase HPLC, determining their radioactivity, and correcting for radioactivity lost during the analysis. Sixteen peptides from segments 1-11 and 76-153 were identified by amino acid and N-terminal analysis. Nine fell in regions where background was too high or were present at too low concentrations for exchange to be monitored. The number of protons whose exchange could be followed in peptides 1-11, 76-91, 78-90, 84-101, 93-112, 108-114, and 115-125 ranged from approximately 1 (1-11, 108-114) to 10 (84-101) and 11 (93-112). The pattern of results obtained suggests that the structure of r2 in solution is similar to that of the regulatory subunits in crystalline ATCase. Both CTP and ATP reduce rates of exchange from all seven peptides except 115-125. Although CTP slows exchange more than ATP, the effect is small except for peptides 76-91 and 78-90 which are near the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation and ion binding properties of peptides related to calcium binding domain III of bovine brain calmodulin

The conformational and ion binding properties of the sequences 93-104, 96-104, and 93-98 of domain III of bovine brain calmodulin (CaM) have been studied by CD and Tb3+-mediated fluorescence. In aqueous solution the interaction of all fragments with Ca2+ and Mg2+ ions is very weak and without any effect on the peptide conformation, which remains always random. In trifluoroethanol the interaction is very strong and the different fragments exhibit very distinct binding properties. In particular, the dodecapeptide fragment 93-104, and its N-terminal hexapeptide 98-104, bind calcium and magnesium with a very high binding constant (Kb greater than 10(5) M-1), undergoing a substantial conformational change. The structural rearrangement is particularly evident in the hexapeptide fragment, which tend to form a beta-bend. The C-terminal nonapeptide fragment 96-104 interacts with calcium and magnesium more weakly, and the binding process causes a decrease of ordered structure. These results suggest that, even in the entire dodecapeptide sequence corresponding to the loop of domain III of CaM, the calcium binding site is shifted toward the N-terminal hexapeptide segment. This interpretation is consistent with the results of crystallographic studies of CaM, which show that the calcium ions are located toward the amino terminal portion of the loop.     

Selection of phage-displayed peptides that bind to a particular ligand-bound antibody

Phage-displayed peptides that selectively bind to aldolase catalytic antibody 93F3 when bound to a particular 1,3-diketone hapten derivative have been developed using designed selection strategies with libraries containing 7-12 randomized amino acid residues. These phage-displayed peptides discriminated the particular 93F3-diketone complex from ligand-free 93F3 and from 93F3 bound to other 1,3-diketone hapten derivatives. By altering the selection procedures, phage-displayed peptides that bind to antibody 93F3 in the absence of 1,3-diketone hapten derivatives have also been developed. With using these phage-displayed peptides, ligand-bound states of the antibody were distinguished from each other. A docking model of one of the peptides bound to the antibody 93F3-diketone complex was created using a sequential divide-and-conquer peptide docking strategy; the model suggests that the peptide interacts with both the antibody and the ligand through a delicate hydrogen bonding network.     

Orientation of the cleavage site of the herpes simplex virus glycoprotein G-2.

During the synthesis of glycoprotein G-2 (gG-2) of herpes simplex virus type 2, the 104,000-Da gG-2 precursor (104K precursor) is cleaved to generate the 72K and the 31K intermediates. The 72K product is processed to generate the mature gG-2 (molecular mass, 108,000 Da), while the 31K product is additionally processed and secreted into the extracellular medium as the 34K component (H. K. Su, R. Eberle, and R. J. Courtney, J. Virol. 61:1735-1737, 1987). In this study, the orientations of the 31K and 72K products on the 104K precursor were determined by using two antipeptide sera produced in rabbits and a monoclonal antibody, 13 alpha C6, directed against gG-2. The sera prepared against synthetic peptides corresponding to the terminal amino acid residues 67 to 78 and an internal peptide at amino acids 247 to 260 of gG-2 recognized the 104K precursor and the 31K cleavage product but not the 72K intermediate. In contrast, 13 alpha C6 detected the 72K cleavage product and the uncleaved precursor but not the 31K cleavage component. The epitope recognized by 13 alpha C6 was mapped within amino acids 486 to 566. These results suggest that the 31K cleavage product is derived from the amino-terminal portion of the 104K precursor molecule and that the 72K intermediate is derived from the carboxyl terminus. In support of our model described above for the synthesis of gG-2, antibodies recognizing either of the cleavage products reacted with the uncleaved precursor but not with the other cleavage product. By using partial endo-beta-N-acetylglucosaminidase H analysis, two N-linked glycosylation sites were found on each of the cleavage products. The distribution of the N-linked glycosylation sites and the reactivities of the antipeptide sera allowed the cleavage region on the precursor to be mapped to within amino acids 260 to 437.     

MAGE-A1-, MAGE-A10-, and gp100-derived peptides are immunogenic when combined with granulocyte-macrophage colony-stimulating factor and montanide ISA-51 adjuvant and administered as part of a multipeptide vaccine for melanoma

Twelve peptides derived from melanocyte differentiation proteins and cancer-testis Ags were combined and administered in a single mixture to patients with resected stage IIB, III, or IV melanoma. Five of the 12 peptides included in this mixture had not previously been evaluated for their immunogenicity in vivo following vaccination. We report in this study that at least three of these five peptides (MAGE-A1(96-104), MAGE-A10(254-262), and gp100(614-622)) are immunogenic when administered with GM-CSF in Montanide ISA-51 adjuvant. T cells secreting IFN-gamma in response to peptide-pulsed target cells were detected in peripheral blood and in the sentinel immunized node, the node draining a vaccine site, after three weekly injections. The magnitude of response typically reached a maximum after two vaccines, and though sometimes diminished thereafter, those responses typically were still detectable 6 wks after the last vaccines. Most importantly, tumor cell lines expressing the appropriate HLA-A restriction element and MAGE-A1, MAGE-A10, or gp100 proteins were lysed by corresponding CTL. This report supports the continued use of the MAGE-A1(96-104), MAGE-A10(254-262), and gp100(614-622) epitopes in peptide-based melanoma vaccines and thus expands the list of immunogenic peptide Ags available for human use. Cancer-testis Ags are expressed in multiple types of cancer; thus the MAGE-A1(96-104) and MAGE-A10(254-262) peptides may be considered for inclusion in vaccines against cancers of other histologic types, in addition to melanoma.     

Synthesis of palmitoyl-thioester T-cell epitopes of myelin proteolipid protein (PLP). Comparison of two thiol protecting groups (StBu and Mmt) for on-resin acylation.

In order to test the effect of thiopalmitoylation on the encephalitogenic properties of two proteolipid protein (PLP) T-cell epitopes, we have studied the on-resin S-palmitoylation of peptides, synthesized using the Fmoc/tBu strategy. The use of two Cys protecting groups was investigated: the tert-butylsulfenyl (StBu) and the methoxytrityl (Mmt). Our studies show that the ease of deprotection of the thiol protected with StBu was sequence dependent. The deprotection of Cys(StBu) was difficult in the case of the two peptides PLP(104-117) and PLP(139-151). Neither of the two Cys(StBu) (Cys108 and Cys140, respectively) could be deprotected with tributylphosphine. Beta-mercaptoethanol was only efficient for the deprotection of Cys(StBu)140 at 85 degrees C and at 135 degrees C for Cys108. The two palmitoylated peptides could be obtained in good yield starting from Cys protected with Mmt. Our conclusion is that the Mmt group is the more versatile protecting group of the thiol for use in the on-resin synthesis of thiopalmitoylated peptides.     

Identification of covalent binding sites of ethyl 2-cyanoacrylate,methyl methacrylate and 2-hydroxyethyl methacrylate in human hemoglobin using LC/MS/MS techniques

Acrylates are used in vast quantities, for instance in paints, adhesive glues, molding. They are potent contact allergens and known to cause respiratory hypersensitivity and asthma. Here we study ethyl 2-cyanoacrylate (ECA), methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate (HEMA). There are only limited possibilities to measure the exposure to acrylates, especially for biological monitoring. The aim of the present study was to investigate the chemical structures of adducts formed after reaction of hemoglobin (Hb) with ECA, MMA, and HEMA. This information may be used to identify adducted Hb peptides for biological monitoring of exposure to acrylates. Hb-conjugates with ECA, MMA, and HEMA were synthesized in vitro. The conjugates were digested by trypsin and pronase E. Adducted peptides were characterized and analyzed by liquid chromatography and nano electro spray/hybrid quadrupole time-of-flight mass spectrometry (MS) as well as tandem quadrupole MS. The search for the adducted peptides was facilitated by visualizing the MS data by different computer programs. The results showed that ECA binds covalently to cysteines at the 104 position in the α and the position 112 in the β-chains in Hb. MMA and HEMA bound to all the cysteines in both chains, Cys¹⁰⁴ in the α-chain and Cys⁹³ and 112 in the β-chain. The full-length spectra of in un-digested Hb confirmed this binding pattern. There was no reaction with N-acetyl-l-lysine at physiological pH. The adducted peptides were possible to measure using LC/MS/MS in selected reaction monitoring mode. These peptides may be used for biological monitoring of exposure to ECA, MMA and HEMA.     

Total synthesis of S-carbamoylmethyl bovine apocytochrome c by segment coupling

Three peptide segments corresponding to the complete sequence of the 104 amino acid protein bovine apocytochrome c were synthesized by the solid-phase method. The peptides Ac-[Cys(Cam)14,17, GlyS23]-apocytochrome c-(1-23) (I), CF3CO-[GlyS60]-apocytochrome c-(24-60) (II), and CF3CO-apocytochrome c-(61-104) (III) were purified by chromatography on CM-cellulose, partition chromatography and/or HPLC. Each of the peptides was reacted with citraconic anhydride to block all of the lysine side chains, and the 61-104 peptide was treated with 10% hydrazine to remove the trifluoroacetyl group, to give the corresponding peptides Ia, IIa, and IIIa. Peptides IIa and IIIa were coupled together by reaction with silver nitrate/N-hydroxysuccinimide to give the 24-104 sequence. After removal of the trifluoroacetyl group from the amino terminus, peptide Ia was also coupled. Treatment of the peptide mixture with aqueous acetic acid removed the citraconyl groups, and purification by chromatography on CM-cellulose and HPLC gave a 0.6% yield of [Cys(Cam)14,17]-apocytochrome c. The synthetic product was shown to be identical to a sample derived from native bovine cytochrome c by paper or gel electrophoresis, HPLC and by chymotryptic or tryptic map.     

Identification of a peptide of the membrane protein of tobacco mosaic virus, cross-linked with intraviral RNA under the effect of UV-radiation

As reported previously, UV-irradiation induces crosslinking between tobacco mosaic virus (TMV) coat protein molecules and intraviral RNA nucleotides. We have irradiated [3H]-uridine labeled TMV and isolated TMV coat protein subunits with the attached nucleotide label. These TMV protein subunits were hydrolyzed with trypsin. The tryptic peptides were separated by high-performance liquid chromatography and [3H]-labeled peptides were identified. The UV-irradiation of TMV was found to result in crosslinking to intraviral RNA of the T8 tryptic peptide (residues 93-112) of TMV coat protein.     

RNA associated with murine intracisternal type A particles codes for the main particle protein.

Intracisternal type A particles were isolated from MOPC-104E myeloma grown subcutaneously and from N 4 neuroblastoma cells in culture. Polyadenylated RNA was prepared from the particles and tested in a cell-free translation system derived from rabbit reticulocytes. RNA from the two sources directed the synthesis of multiple polypeptides with similar distributions of electrophoretic mobilities in sodium dodecyl sulfate-containing polyacrylamide gels, including one conponent of the same size as the major A-particle structural protein (73,000 daltons). Analysis of the RNAs by electrophoresis in methyl mercury-containing agarose gels revealed a 35S component common to A-particles from both cell types. This was a major component of the N4 preparations, whereas a 28S species predominated in the case of MOPC-104E. These two RNAs (35S from N4 cells and 28S from MOPC-104E), when isolated on isokinetic sucrose gradients, each directed the synthesis of a 73,000-molecular-weight polypeptide that comigrated on gels with authentic A-particle structural protein. Idnetity of the cell-free product was confirmed by two-dimensional analysis of the [35S]methionine-labeled tryptic peptides. The N4 RNA preparations also contained a major32S component which did not code effectively for the A-particle structural protein.     

Mutations in the E1 hydrophobic domain of rubella virus impair virus infectivity but not virus assembly

Rubella virus (RV) virions contain three structural proteins, a capsid protein that interacts with viral genomic RNA to form a nucleocapsid and two membrane glycoproteins, E2 and E1. We found that substitution of either an aspartic acid residue at Gly93 (G93D) or a glycine residue at Pro104 (P104G) in the internal hydrophobic domain of E1 affected virus infectivity but not virus assembly. Viruses carrying G93D and P104G mutations had impaired infectivity, reduced 1,000-fold and 10-fold, respectively. A revertant was isolated from the G93D mutant. Sequencing analysis showed that the substituted aspartic acid residue in G93D mutant had reverted to the original glycine residue, suggesting the involvement of Gly93 in membrane fusion during viral entry.