N-terminal Peptide Fragment Research Articles

Proline Conformation-Dependent Antimicrobial Activity of a Proline-Rich Histone H1 N-Terminal Peptide Fragment Isolated from the Skin Mucus of Atlantic Salmon

A 30-residue N-terminally acetylated peptide derived from the N-terminal part of histone H1 was identified as the dominant antimicrobial peptide in skin mucus from Atlantic salmon (Salmo salar). The peptide (termed salmon antimicrobial peptide [SAMP H1]) was purified to homogeneity by a combination of reversed-phase and cation-exchange chromatographies. By Edman degradation of the deacetylated peptide and by sequencing of the PCR-amplified DNA that encodes the peptide, the complete amino acid sequence was determined to be AEVAPAPAAAAPAKAPKKKAAAKPKKAGPS. The theoretical molecular weight of N-terminally acetylated SAMP H1 was calculated to be 2,836, which is the same as that determined by matrix-assisted laser desorption ionization mass spectrometry. The peptide was active against both gram-negative and -positive bacteria. The N-terminal acetyl group was not necessary for activity since deacetylation did not reduce the activity. A synthetic peptide whose sequence was identical to that of the isolated fragment was initially inactive but could be activated by binding it to a cation-exchange column. Treatment of the synthetic peptide when it was bound to the exchange column with peptidylproline cis-trans-isomerase increased the amount of active peptide, indicating that isomerization of the proline peptide bond(s) was necessary for activation of the synthetic peptide. Comparison of the active and inactive forms by circular dichroism and chromatographic analyses suggests that the active form, both the natural and the synthetic forms, is more structured, condensed, and rigid than the inactive form, which has a more nonstructured conformation. This work shows for the first time the importance of proline isomers in the activity of an antimicrobial peptide.

Proteolysis of Xenopus laevis egg envelope ZPA triggers envelope hardening

The egg envelope of most animal eggs is modified following fertilization, resulting in the prevention of polyspermy and hardening of the egg envelope. In frogs and mammals a prominent feature of envelope modification is N-terminal proteolysis of the envelope glycoprotein ZPA. We have purified the ZPA protease from Xenopus laevis eggs and characterized it as a zinc metalloprotease. Proteolysis of isolated egg envelopes by the isolated protease resulted in envelope hardening. The N-terminal peptide fragment of ZPA remained disulfide bond linked to the ZPA glycoprotein moiety following proteolysis. We propose a mechanism for egg envelope hardening involving ZPA proteolysis by an egg metalloprotease as a triggering event followed by induction of global conformational changes in egg envelope glycoproteins.

Aβ1-28 Fragment of the Amyloid Peptide Predominantly Adopts a Polyproline II Conformation in an Acidic Solution

To structurally characterize the nonaggregated state of the amyloid beta peptide, which assembles into the hallmark fibrils of Alzheimer disease, we investigated the conformation of the N-terminal extracellular peptide fragment Abeta(1-28) in D(2)O at acidic pD by utilizing combined FTIR and isotropic and anisotropic Raman spectra measured between 1550 and 1750 cm(-1). Peptide aggregation is avoided under the conditions chosen. The amide I' band was found to exhibit a significant noncoincidence effect in that the first moment of the anisotropic Raman and of the IR band profile appears red-shifted from that of the isotropic Raman scattering. A simulation based on a coupled oscillator model involving all 27 amide I' modes of the peptide reveals that the peptide adopts a predominantly polyproline II conformation. Our results are inconsistent with the notion that the monomeric form of Abeta(1-28) is a totally disordered, random-coil structure. Generally, they underscore the notion that polyproline II is a characteristic motif of the unfolded state of proteins and peptides.

Structural Characterization of the HIV-1 Vpr N Terminus: EVIDENCE OF cis/trans-PROLINE ISOMERISM

The 96-residue human immunodeficiency virus (HIV) accessory protein Vpr serves manifold functions in the retroviral life cycle including augmentation of viral replication in non-dividing host cells, induction of G2 cell cycle arrest, and modulation of HIV-induced apoptosis. Using a combination of dynamic light scattering, circular dichroism, and NMR spectroscopy the N terminus of Vpr is shown to be a unique domain of the molecule that behaves differently from the C-terminal domain in terms of self-association and secondary structure folding. Interestingly, the four highly conserved proline residues in the N terminus are predicted to have a high propensity for cis/trans isomerism. Thus the high resolution structure and folding of a synthetic N-terminal peptide (Vpr1-40) and smaller fragments thereof have been investigated. 1H NMR data indicate Vpr1-40 possesses helical structure between residues 17-32, and for the first time, this helix, which is bound by proline residues, was observed even in aqueous solution devoid of any detergent supplements. In addition, NMR data revealed that all of the proline residues undergo a cis/ trans isomerism to such an extent that approximately 40% of all Vpr molecules possess at least one proline in a cis conformation. This phenomenon of cis/trans isomerism, which is unprecedented for HIV-1 Vpr, not only provides an explanation for the molecular heterogeneity observed in the full-length molecule but also indicates that in vivo the folding and function of Vpr should depend on a cis/trans-proline isomerase activity, particularly as two of the proline residues in positions 14 and 35 show considerable amounts of cis isomers. This prediction correlates well with our recent observation (Zander, K., Sherman, M. P., Tessmer, U., Bruns, K., Wray, V., Prechtel, A. T., Schubert, E., Henklein, P., Luban, J., Neidleman, J., Greene, W. C., and Schubert, U. (2003) J. Biol. Chem. 278, 43170-43181) of a functional interaction between the major cellular isomerase cyclophilin A and Vpr, both of which are incorporated into HIV-1 virions.

Tracing glycoprotein structures: electrospray ionization tandem mass spectrometric analysis of sugar-peptide adducts.

Owing to the diversity of carbohydrate structures and their significance for the function of many biopolymers, structural analysis of various carbohydrate-related compounds is of great importance. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to establish the fragmentation behaviour of a range of sugar-peptide adducts as model compounds of widespread glycoprotein structures. The compounds used in this study were chosen to provide correlation of distinct fragment ions with specific structural differences, namely position and type of carbohydrate-peptide bond and structure of the sugar moiety. All compounds show N- and C-terminal sequence ions along with losses of up to three water molecules. Fructose-related Amadori compounds exhibit M + 78 modified N-terminal peptide fragment ions. Fragmentation of glucose-peptide esters is characterized by the sugar ring fragmentation. Additionally, under the ESI-MS conditions applied, the esters studied undergo intramolecular reaction giving cyclic sugar-peptide structures that can be traced by the presence of N-terminal peptide M + 42 adducts. Detailed analysis of cyclic fructose-related compound comprising structural features of both studied groups revealed a rich fragmentation pattern derived from amino acid residues and water molecules losses from [M - 2H(2)O + H](+) ion. Also, some interesting differences were found with respect to the nature of carbohydrate moieties.

Radioimmunoassay for N-terminal probrain natriuretic peptide in human plasma

Measurement of plasma levels of natriuretic peptides has been used to assess left ventricular dysfunction and prognosis. Recently levels of the N-terminal peptide fragment of the precursor of brain natriuretic peptide have been reported to be present in peripheral plasma and to be increased in chronic heart failure patients. Our aim in this study was to develop a radioimmunoassay for N-terminal proBNP, to compare its plasma concentrations in control subjects and in patients with end-stage heart failure and to define its relation to brain natriuretic peptide (BNP). A polyclonal antibody was raised in rabbits against human N-terminal proBNP fragment (amino acid 1-21). The plasma N-terminal proBNP concentrations were assayed directly without extraction. No detectable cross-reactivity existed with other natriuretic peptides: BNP, ANP or N-terminal proANP. The assay had a detection limit (2 SD from zero) of 9.7 pmol/L. Plasma N-terminal proBNP was 29 (13-75) (median (range)) pmol/L in the control group. There were no gender difference, male: 28 (13-61) vs. female 33 (13-75) pmol/L, p= NS, but there was a positive correlation to age (r=0.52, p<0.0001). In patients with end-stage heart failure the median N-terminal proBNP levels were increased significantly 616 (114-2781) pmol/L (p<0.001) and in pooled data N-terminal proBNP showed a close correlation to BNP (r=0.96, p<0.0001). Size exclusion of plasma extracts indicated that proBNP (1-108) may circulate both as intact prohormone and as split products, N-terminal proBNP (1-76) and BNP (77-108). Our results support the concept that N-terminal proBNP measurement could be a valuable tool in the biochemical indication of increased cardiac wall stress.

Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum.

Glutamate dehydrogenase (GDH) was purified from rough endoplasmic reticulum (RER) in rat liver using anion-exchange and affinity chromatography. As GDH has been known as an enzyme that exists mainly in the matrix of mitochondria, the properties of purified GDH were compared with those of mitochondrial GDH. The GDH activity in 0. 1% Triton X-100-treated RER subcellular fraction was nearly the same as intact RER, whereas that of the mitochondrial fraction increased by 50% after the detergent treatment. In kinetic values, in addition, mitochondrial GDH had a higher K(m) value for NADP(+) than NAD(+), whereas the K(m) value for NAD(+) was higher than that for NADP(+) in the case of GDH of RER, which showed a difference in specificity to cofactors. Moreover, when two GDH isoproteins were incubated at 42 degrees C or treated with trypsin, GDH from RER was more stable against heat inactivation and less susceptible to proteolysis than mitochondrial GDH in both cases. In addition, GDH of RER had at least five amino acids different from mitochondrial GDH when sequences of N-terminal and several internal peptide fragments were analyzed. These results showed that GDH of RER is another isoprotein of GDH, of whose properties are different from those of mitochondrial GDH.

Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum

Glutamate dehydrogenase (GDH) was purified from rough endoplasmic reticulum (RER) in rat liver using anion-exchange and affinity chromatography. As GDH has been known as an enzyme that exists mainly in the matrix of mitochondria, the properties of purified GDH were compared with those of mitochondrial GDH. The GDH activity in 0. 1% Triton X-100-treated RER subcellular fraction was nearly the same as intact RER, whereas that of the mitochondrial fraction increased by 50% after the detergent treatment. In kinetic values, in addition, mitochondrial GDH had a higher K(m) value for NADP(+) than NAD(+), whereas the K(m) value for NAD(+) was higher than that for NADP(+) in the case of GDH of RER, which showed a difference in specificity to cofactors. Moreover, when two GDH isoproteins were incubated at 42 degrees C or treated with trypsin, GDH from RER was more stable against heat inactivation and less susceptible to proteolysis than mitochondrial GDH in both cases. In addition, GDH of RER had at least five amino acids different from mitochondrial GDH when sequences of N-terminal and several internal peptide fragments were analyzed. These results showed that GDH of RER is another isoprotein of GDH, of whose properties are different from those of mitochondrial GDH.

On the effect of xenin and xenin fragments on exocrine pancreas secretion in vivo

A stimulatory effect on exocrine pancreas secretion could be demonstrated with high concentrations of the 25-amino-acid peptide xenin in non-anesthetized dogs. This peptide has been isolated from gastric mucosa and it is part of a structural coat protein. It has close structural similarities to neurotensin. The longer C-terminal fragments xenin-(13–25) and xenin-(18–25) are essential for the stimulation of exocrine pancreas secretion in vivo. The smaller peptide fragments xenin-(21–25) and xenin-(22–25) failed to stimulate the pancreas as well as the N-terminal peptide fragment xenin-(1–23). The stimulatory effects of xenin may be mediated via neural neurotensin pathways, because neurotensin receptor blockade abolished the stimulatory effect on pancreatic secretion. Cholinergic pathways are not involved, because atropine had no inhibiting effect.

Subcellular Localization and Protein-Protein Interaction Regions of Ku Proteins

The Ku protein is a complex of Ku70 and Ku80 subunits and is capable of binding promoters in a sequence-specific manner, although it remains unclear whether Ku is involved in transcriptional regulation. We examined the subcellular localization and determined the interaction regions of Ku. Our results indicate that heterodimers of Ku70 and Ku80 are localized in the nucleus, and that the stretches from amino acid (aa) 378 to 482 of Ku70 and from aa 374 to 502 of Ku80 are necessary for heterodimerization. These interaction regions do not contain any previously recognized protein-protein interaction motifs. To determine whether Ku contains a potential transcriptional activation domain, we examined N- and C-terminal deletion mutants of Ku70 and Ku80 for their ability to activate transcription in the GAL4-based one-hybrid system. We found that the whole Ku protein had no transcriptional activity, although the N-terminal peptide fragment of Ku70 was capable of activating transcription of theHIS3andlacZreporter genes in yeast cells.

Monoclonal antibody, a novel probe for protein folding

Two monoclonal antibodies (McAb2C9, McAb1E5) against Staphylococcal nuclease R (SNase R) and its N-terminal peptide fragments were prepared, purified and characterized. Further studies show that the intact enzyme SNase R and its seven N-terminal peptide fragments differ in their interaction with McAb2C9. SNase R, SNR121, SNR102, SNR79 and SNR52 can bind to McAb2C9 readily, while fragments of SNR141, SNR135, SNR110 react with the antibody poorly. If this difference is due to diverse extent of exposure of the specific epitope in the fragments, it is suggested that the conformation of the peptide is subjected to continuous adjustments through chain elongation until the biologically active protein is formed. This result supports Tsou's hypothesis of nascent peptide folding experimentally.

Peptidic and Non-peptidic Neurotensin Analogs

The structure-activity relationship studies of neurotensin and its analogs have revealed that i) the C-terminal hexapeptide NT (8-13) (H-ArgB­ Arg9-Pro1o_Tyr1LIIe12-Leu13.QH) is equipotent to or more potent than the native NT(1-13) and retains all the intrinsic biological properties of neurotensin, ii) the N-terminal peptide fragment up to NT(1-7) does not significantly contribute to the binding potency, iii) Pro1o that can form a reverse-turn conformation is required, iv) an aromatic amino acid is important at Tyr11 position, v) the lipophilic side chain of Leu 13 is also crucial to the biological activities, and, vi) L-form amino acids are essential at critical positions for the binding potency. In addition to many peptidic neurotensin analogs, highly potent, orally active, and selective non-peptidic analogs as antagonists of NT receptors, which have long lasting effects and the ability to cross the blood­ brain barrier (e. g. SR 142948 A and SR 48692), have been discovered.

Synthesis and FTIR Conformational Studies of Peptides From the Basic Region of c-Jun: a Critical Analysis on the Basis of CD and NMR Data

The peptide (35 residues) corresponding to the basic subdomain (bSD) of c-Jun (residues 252–281) and its fragments NP (N-terminal peptide, 1–19) and CP (C-terminal peptide, 1635) were synthesized in stepwise solid-phase using the tert-butyloxycarbonyl/benzyl strategy. In a previous paper, we have shown that during its binding to the DNA site CRE (cAMP- responsive element) the bSD structure was converted into α-helix from an initial random coil conformation [Krebs, D., Dahmani, B., El Antri, S., Monnot, M., Convert, O., Mauffret, O., Troalen, F. & Fermandjian, S. Eur. J. Biochem. 231, 370–380 (1995)]. Our results suggested both a high flexibility and a helical potential in bSD, these two properties seeming crucial for the accommodation of the basic subdomain of c-Jun to its specific DNA targets. In this work, we assessed the conformational variability of bSD through the study of the secondary structures of its NP and CP fragments in trifluoroethanol (TFE)/2H2O mixtures, using Fourier transform infrared (FTIR) spectroscopy. The IR results were critically analyzed in light of our previously reported circular dichroism (CD) and NMR data [Krebs, D., Dahmani, B., Monnot, M., Mauffret, O., Troalen, F. & Fermandjian, S. Eur. J. Biochem. 235, 699–712 (1996)]. Upon addition of TFE, the relative areas of the seven components of the amide I band (1700–1620 cm−1) reflected the conversion of a large amount of random coil conformation into α-helix for the two fragments and bSD. This effect was accompanied by more subtle variations of the less populated structures, in agreement with the results of CD and NMR experiments. The IR results stipulated the conservation of the parent bSD secondary structures in both fragments; however, NP and CP peptides did not display similar random-to-α-helix stabilization pattern upon additions of TFE to aqueous solutions. The profile from CD signal at 222 nm was found sigmoidal for NP and almost linear for CP, while that corresponding to the parent peptide bSD was just in between those of its fragments. Thus, the present study confirms the high flexibility and helix propensity of the c-Jun basic subdomain and suggests that the N- and C-terminal parts of the peptide do not follow the same random-to-helix conversion profile during their complexation with DNA.

The interaction between Alzheimer amyloid β(1–40) peptide and ganglioside G M1-containing membranes

The interaction between Alzheimer amyloid peptide Aβ(1–40) and membrane lipids was studied by circular dichroism spectroscopy under the conditions of physiologically relevant ionic strength and neutral pH. The peptide binds to the membranes containing ganglioside G M1 and upon binding undergoes a conformational transition from random coil to an ordered structure rich in β-sheet. This interaction appears to be ganglioside-specific as no changes in Aβ(1–40) conformation were found in the presence of various phospholipids or sphingomyelin. The isolated oligosaccharide moiety of the ganglioside was ineffective in inducing alterations in the secondary structure of Aβ(1–40). No interaction was observed between ganglioside G M1 and the N-terminal peptide fragment Aβ(1–28). Binding to the ganglioside is likely to modulate the neurotoxic and/or amyloidogenic properties of Aβ(1–40).

1124 A neurotrophic action of midkine, a developmentally regulated protein, was examined on cerebelar neurons using a defined culture system

Ovarian factors that promote growth of vascular smooth muscle cells were investigated. Two distinct heparin-binding polypeptides were isolated from bovine follicular fluid by successive chromatographies. N-Terminal and tryptic peptide fragment analysis of these polypeptides revealed that they are identical to midkine (MK) and pleiotrophin (PTN), respectively, which form a new family of heparinbinding growth/differentiation factors. Two N-terminally distinct forms of midkine were also identified in the bovine follicular fluid. The concentrations of MK and PTN in the bovine follicular fluid were estimated to be 125 μg/l and 400 μg/l, respectively. The present findings that MK and PTN are quite rich in the follicular fluid will provide a new insight into so far unclarified functions of MK and PTN, especially their roles in the maturation of ovarian follicles.

Succinimide-mediated pathway for peptide bond cleavage: Kinetic study on an Asn-Sar containing peptide

The cleavage reaction of the peptide bond next to the Asn residue has been studied in the pH range 7.4–13.8 at 37°C and μ = 1M. This reaction yields an N-terminal peptide fragment having at its C-terminus a succinimide ring, which rapidly hydrolyses to both asparagine and iso-asparagine residues. For both the two consecutive reactions, peptide bond cleavage and the succinimide hydrolysis, the general trend is an increase of the reaction rate with the pH. However, for the hydrolysis reaction there is a small decrease in the pH range 10–11 caused by the deprotonation of the succinimide nitrogen atom. Kinetic evidence indicates that the cleavage reaction is a multistep process with a change in the rate-determining step at pH 8.5–9.0. The mechanism involves preequilibrium deprotonation of the NH2 amide group of the Asn side chain, followed by nucleophilic attack of the nitrogen atom on the carbonyl carbon atom of the same asparagine residue, giving a cyclic intermediate. Then, general acid-catalyzed departure of the leaving group gives the final reaction product. At pH < 8.5, the formation of the cyclic intermediate is rate determining, whereas, at higher pH, it is the departure of the leaving group. © 1997 John Wiley & Sons, Inc. Biopoly 40: 543–551, 1996

Influence of bone marrow hexapeptides (myelopeptides) on antibody production and algesthesia in mice and the dependence of these effects on naloxone

It is shown that synthetic analogs of two bone marrow hexapeptides (myelopeptides 1 and 2), which are identical in structure to the N-terminal peptide fragments of hemoglobin α- and β-chains, are characterized by naloxone-independent antibody-stimulating activity. The antibody-stimulating effect of the myelopeptides becomes naloxone-dependent and stronger against the background of immunosuppression provoked by the hot plate test carried out before immunization. Intraperitoneal administration of both myelopeptides in doses of 10−13–10−8 g/mouse causes a naloxone-dependent modulatory effect on mouse algesthesia threshold with a predominant analgetic effect.

Unilateral deficits induced in rats by MPP + are markedly reduced by an N-terminal peptide fragment of dopamine-releasing protein

Dopamine-releasing protein (DARP) is a novel factor involved in the function and development of catecholaminergic systems. To test whether a peptide synthesized from the N-terminus of DARP (DARP-36aa) ameliorates deficits in nigrostriatal dopamine, rats were unilaterally lesioned with MPP +, 1 day before administration of DARP-36aa began. Striatal delivery of 1 μg DARP-36aa daily for 10 days elevated striatal dopamine ( P < 0.01) and and reduced amphetamine-induced rotations ( P < 0.05) relative to controls, both indicating the protection or restoration of dopaminergic function.

Isolation and identification of midkine and pleiotrophin in bovine follicular fluid

Ovarian factors that promote growth of vascular smooth muscle cells were investigated. Two distinct heparin-binding polypeptides were isolated from bovine follicular fluid by successive chromatographies. N-Terminal and tryptic peptide fragment analysis of these polypeptides revealed that they are identical to midkine (MK) and pleiotrophin (PTN), respectively, which form a new family of heparinbinding growth/differentiation factors. Two N-terminally distinct forms of midkine were also identified in the bovine follicular fluid. The concentrations of MK and PTN in the bovine follicular fluid were estimated to be 125 μg/l and 400 μg/l, respectively. The present findings that MK and PTN are quite rich in the follicular fluid will provide a new insight into so far unclarified functions of MK and PTN, especially their roles in the maturation of ovarian follicles.

Cloning and characterization of ppiB, a Bacillus subtilis gene which encodes a cyclosporine A‐sensitive peptidyl‐prolyl cis‐trans isomerase

Sequencing of N-terminal and internal peptide fragments of the purified 17 kDa Bacillus subtilis peptidyl-prolyl cis-trans isomerase (PPIase) revealed sequence identity to conserved regions of a number of eukaryotic and prokaryotic cyclophilins. Using two oligonucleotide primers corresponding to the N-terminus and a highly conserved internal amino acid sequence, polymerase chain reactions (PCR) with B. subtilis genomic DNA were carried out. The resultant PCR fragment of 335 bp was cloned, sequenced and subsequently used as a probe for screening a lambda Zap II gene library of B. subtilis. Two overlapping positive clones of 5 and 7 kb containing the B. subtilis PPIase gene (ppiB), which is 432 bp in length and encodes a protein of 144 amino acid residues, were identified and two distinct transcriptional initiation sites at the 5' end of ppiB were mapped. The entire region (35 kb) between spoVA and serA was recently sequenced in B. subtilis, and an open reading frame (ORF) that encodes a putative peptidyl-prolyl cis-trans isomerase at about 210 degrees on the B. subtilis genetic map was located. This putative PPIase is identical to PPiB. We have overexpressed the ppiB gene in Escherichia coli, purified the encoded protein to apparent homology and shown that it exhibits PPIase activity. In addition, the recombinant PPiB shows a significant inhibition of PPIase activity by cyclosporin A (CsA) at a level comparable to that observed for the B. subtilis enzyme. Interestingly the B. subtilis PPIase shows about 40% identity to eukaryotic PPIases and less similarity to those of Gram-negative bacteria (27-32% identity). Like other interruption mutants of yeast and Neurospora, which lack a functional cyclophilin gene, a B. subtilis mutant containing ppiB::cat, a cat-interrupted copy of ppiB in the chromosome, is viable.