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PubMed2004

Uptake of cell-penetrating peptides is dependent on peptide-to-cell ratio rather than on peptide concentration.

The influence of the peptide-to-cell ratio and energy depletion on uptake and degradation of the cell-penetrating peptides (CPPs) MAP (model amphipathic peptide) was investigated. The intracellular concentration of the CPPs, MAP and penetratin was monitored while varying the number of cells at fixed peptide concentration and incubation volume, or changing the concentration and incubation volume at fixed cell number. The uptake of CPPs was shown to be dependent on the peptide/cell ratio. At given peptide concentration and incubation volume, the intracellular concentration of peptide increased with lower cell number. At given cell number, doubling of the incubation volume increased intracellular peptide concentration to a similar extent as the doubling in incubation concentration. From a practical view, this means that the peptide/cell ratio has at least the same importance for the uptake of CPPs as the used peptide concentration. No influence of the peptide/cell ratio was found for the cellular uptake of peptide nucleic acid (PNA), or a non-amphipathic MAP analogue, investigated in parallel for comparison purposes. Energy depletion resulted in significantly reduced quantities of intracellular fluorescence label. Moreover, we show that this difference is mainly due to a membrane-impermeable fluorescent-labelled degradation product, which is lacking in energy-depleted cells. The mechanism of its generation is not likely to be endosomal degradation of endocytosed material, as it is not chloroquine- or brefeldin-sensitive.

Amino Acid Sequence↗
PubMed2003

Effect of physicochemical conditions on peptide-peptide interactions in a tryptic hydrolysate of beta-lactoglobulin and identification of aggregating peptides.

The objective of this study was to characterize the changes in peptide solubility resulting from changing some physicochemical conditions in a tryptic hydrolysate of beta-lactoglobulin (beta-LG). The turbidity (500 nm) of a 1% solution of tryptic peptides was measured at pH 3-10, at 5, 25, and 50 degrees C, in the presence of different salt concentrations (0, 0.5, and 1 M NaCl), in the presence of denaturing and reducing agents (6 M urea, 5% SDS, or 5% beta-mercaptoethanol), and under an electric field (isoelectric focusing). The results reveal an increase in turbidity of the peptide solution at pH 4, but a slight increase in turbidity was also observed at pH 8, which is attributable to peptides linked by disulfide bridges. The effect of temperature and ionic strength on the turbidity occurring at pH 4 indicates that mainly hydrophobic interactions are involved in the aggregation process. The material in the precipitate at pH 4 was identified as the peptides beta-LG 1-8, 15-20, and 41-60 and non-hydrolyzed alpha-lactalbumin. These results suggest that a limited number of peptides are involved in the aggregation process observed at pH 4, some of which having bioactive (beta-LG 15-20, ACE inhibitor, and opioid) or emulsifying properties (beta-LG 41-60). Aggregation of these peptides at acidic pH indicates that a simple acidification step could represent an easy process for isolating peptidic fractions enriched in bioactive or functional peptides.

Animals↗
PubMed1988

Structural characterization of a high-molecular-mass form of calcitonin [procalcitonin-(60-116)-peptide] and its corresponding N-terminal flanking peptide [procalcitonin-(1-57)-peptide] in a human medullary thyroid carcinoma.

Four peptides derived from procalcitonin were isolated in high yield from an extract of a human medullary thyroid carcinoma. The peptides were identified as procalcitonin-(1-57)-peptide, procalcitonin-(60-91)-peptide (calcitonin), procalcitonin-(60-116)-peptide and procalcitonin-(96-116)-peptide (katacalcin). Determination of the amino acid sequence of procalcitonin-(1-57)-peptide has demonstrated that the Ala25-Ala26 bond in preprocalcitonin is the site of cleavage of the signal peptide. Procalcitonin-(60-116)-peptide represents calcitonin extended from its C-terminus by the sequence Gly-Lys-Lys-Arg-katacalcin, and its formation is indicative of an aberrant pathway of procalcitonin processing in the tumour cells.

Amino Acids↗
PubMed1995

Unprotected peptides as building blocks for branched peptides and peptide dendrimers.

We describe two new site-specific ligation methods for preparing branched peptide dendrimers such as multiple antigen peptide (MAP). Both methods are based on the general approach of exploiting the specific reaction between a weak base and an aldehyde under acidic conditions so that unprotected peptides can be used as building blocks. A weak base such as benzoyl hydrazine or 1,2-amino thiol of cysteine was attached to the N-terminal of an unprotected peptide as nucleophile to react with the alkyl aldehyde on the core matrix of MAP to form a stable hydrazone linkage or a five-membered thiazolidine ring, respectively. Two synthetic peptides rich in basic amino acids such as lysine and arginine were used as models in the ligation reactions in solution to give peptide dendrimers containing four or eight copies of peptide immunogens. The resulting macromolecules with the MW ranging from 5 to 16 kDa were unambiguously characterized by laser-desorption mass spectrometry. Furthermore, we also optimized the conditions of these ligation reactions using elevated temperature and a water-miscible organic co-solvent to give a combination of rate enhancement about 10 fold. These optimizations allowed the ligation reactions to be completed in < 4 h instead of 2-3 days. Our ligation approach also has the advantages of flexibility so that peptides can be attached through the amino or carboxyl terminus to the core matrix. The phenyl hydrazone linkage and the five-membered ring were found to be stable at physiological pH suitable for immunization. Thus our results provide two practical and useful methods for the synthesis of macromolecular peptide dendrimers for vaccines, artificial proteins, and enzymes.

Aldehydes↗
PubMed1996

The relative importance of individual DR binding motif positions as defined by peptide anchor analysis of influenza hemagglutinin peptide 306-318 and human myelin basic protein peptide 152-165 binding to several DR molecules: definition of a common extended DR binding motif.

Definition of peptide binding motifs for DR molecules has proven difficult as the peptides that bind to a DR molecule have shown extensive variability at putative motif positions. Recent studies suggest that specific peptide anchor residues (motif positions) and specific DR residues can differ in importance for peptide binding to a DR molecule. To assess further the relevance of individual peptide anchor residues, the binding of serial alanine-substituted analogs of influenza virus hemagglutinin (HA) 306-318 and human myelin basic protein (MBP) 152-165 to a panel of transfected wild-type DR molecules was examined. This analysis included DR molecules from a wide range of allelic families and, unlike most earlier studies, multiple members of single DR allelic families. The data show that different peptide residues serve as critical anchors for binding to different DR molecules. For example, MBP binding to DR(alpha, beta 1*0303) required peptide residues F154 (i), R159 (i + 5) and R162 (i + 8). In contrast, MBP binding to DR(alpha, beta 1*0102) required peptide residues I153 (i) and L156 (i + 3). More importantly, the combination of critical anchor residues in HA and MBP differed for binding to a single DR molecule [e.g. V309 (i) for HA and I153 (i) and L156 (i + 3) for MBP binding to DR(alpha, beta 1*0102)]. Although the location of the binding pocket in each DR molecule compared to the DR (alpha, beta 1 *0101) crystal is expected to be similar and suggests a common extended DR binding motif, the present results suggest that the relative importance of individual peptide anchor residues and of the corresponding DR binding pockets will differ for each DR/peptide complex.

Amino Acid Sequence↗
PubMed1991

Chemically unambiguous peptide immunogen: preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system.

We described a novel and simple approach to prepare chemically unambiguous peptide immunogen using the multiple antigen peptide (MAP) approach. This approach requires the conjugation of two purified components: a chloroacetylated oligomeric lysine core matrix and a synthetic peptide containing cysteine at either the carboxyl or amino terminus. The resulting MAP is structurally unambiguous and contains a quantifiable amount of peptide antigens. Furthermore, this method also provides a flexible strategy to link a peptide antigen to the core matrix at the desirable orientation to mimic the native molecule. The carboxyl fragment 43-50 of human transforming growth factor alpha (TGF alpha) was used as a test model for this approach. Antipeptide antibodies did not recognize the "reverse immunogen" in which the peptide was attached to the MAP core matrix at a reverse orientation. To determine the specificity of the antibodies, we used two series of point-substituted TGF alpha analogs containing either alanine or the corresponding D-amino acid replacement to map the antigenic site. The alanine analogs were used to determine the contribution of the side chain while the D-amino acid analogs were used to determine the importance of backbone conformation. The antigen site was found to consist of four residues (Asp47-Leu48-Leu49-Ala50) at the distal end of the peptide-MAP conjugate. The results provide a clear explanation for the specificity of the antipeptide antibodies and their failure to recognize the "reverse immunogen" since the distal and the flexible end of the peptide-MAP construct constitutes the antigenic site. Furthermore, our results also suggests a strategy of placing the antigenic portion of a short-peptide at the distal end in the MAP approach to prepare immunogen.

Amino Acid Sequence↗
PubMed1991

The neurotransmitter role of calcitonin gene-related peptide in the rat and guinea-pig ureter: effect of a calcitonin gene-related peptide antagonist and species-related differences in the action of omega conotoxin on calcitonin gene-related peptide release from primary afferents.

In the rat and guinea-pig isolated ureter electrical field stimulation of intrinsic nerves (10 Hz for 10 s) produces transient inhibition of evoked (20 mM KCl or 0.1-1 microM neurokinin A) rhythmic contractions by releasing transmitter(s) from peripheral endings of capsaicin-sensitive primary afferents. The C-terminal fragment of human calcitonin gene-related peptide (8-37) blocked the inhibitory effect of electrical field stimulation as well as that produced by exogenous calcitonin gene-related peptide, while leaving unaffected the inhibitory response to isoprenaline. Human calcitonin gene-related peptide (8-37) was devoid of any inhibitory activity of its own but enhanced the amplitude and frequency of KCl-evoked rhythmic contractions in the rat ureter, probably by antagonizing the inhibitory effect of endogenous calcitonin gene-related peptide released by KCl. Omega conotoxin fraction GVIA, a peptide which possesses a potent blocking activity of N-type voltage-sensitive calcium channels, prevented the inhibitory response to electrical stimulation in the guinea-pig ureter, while leaving the response unaffected in the rat ureter. Conotoxin had no effect toward the inhibition produced by exogenous calcitonin gene-related peptide indicating its prejunctional site of action, demonstrated previously in the guinea-pig ureter [Maggi et al. (1990) Neurosci, Lett. 114, 203-206]. Dermorphin, an amphibian peptide with potent agonist activity on mu-type opioid receptors, inhibited the response to electrical stimulation in the guinea-pig ureter but had no effect in the rat ureter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Isolation, characterization and biological properties of two kinin-like peptides (peptide-S and peptide-r) from Scaptocosa raptoria venom.

Two peptides with kinin-like biological properties were isolated by chromatography on a Sephadex G-10 column followed by high-performance liquid chromatography, from the venom of the spider Scaptocosa raptoria. The isolated peptides (peptide-S and peptide-R) were shown to cause contraction on the isolated guinea-pig ileum at amounts equivalent to those shown by bradykinin. Both peptides relaxed the isolated rat duodenum, increased the capillary permeability, caused decreasing and biphasic effect of the arterial blood pressure in conscious rats and induced oedema in the rat paw. The peptides had activity and structural similarities to other peptides (kinin-like) isolated from venoms. The complete amino acid analysis gave peptide-S a structure with 36 amino acid residues and peptide-R 22 amino acid residues. The mol. wts were estimated to be in the range of 4000 and 2870, respectively.

Amino Acid Sequence↗
PubMed1987

Application of peptide-mediated ring current shifts to the study of neurophysin-peptide interactions: a partial model of the neurophysin-peptide complex.

Perdeuteriated peptides were synthesized that are capable of binding to the hormone binding site of neurophysin but that differ in the position of aromatic residues. The binding of these peptides to bovine neurophysin I and its des-1-8 derivative was studied by proton nuclear magnetic resonance spectroscopy in order to identify protein residues near the binding site through the observation of differential ring current effects on assignable protein resonances. Phenylalanine in position 3 of bound peptides was shown to induce significant ring current shifts in several resonances assignable to the 1-8 sequence, including those of Leu-3 and/or Leu-5, but was without effect on Tyr-49 ring protons. The magnitude of these shifts was dependent on the identity of peptide residue 1. By contrast, the sole demonstrable direct effect of an aromatic residue in position 1 was a downfield shift in Tyr-49 ring protons. Study of peptide binding to des-1-8-neurophysin demonstrated similar conformations of native and des-1-8 complexes except for the environment of Tyr-49, confirmed the peptide-induced ring current shift assignments in native neurophysin, and indicated an effect of binding on Thr-9. These observations are integrated with other results to provide a partial model of neurophysin-peptide complexes that places the ring of Tyr-49 at a distance 5-10 A from residue 1 of bound peptide and that places both the 1-8 sequence and the protein backbone region containing Tyr-49 proximal to each other and to peptide residue 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Indicators and Reagents↗
PubMedDate not supplied

Peptide self-aggregation and peptide complementarity as bases for the evolution of peptide receptors: a review.

This paper reviews the three major theories of peptide receptor evolution: (1) Dwyer's theory that peptide receptors evolved from self-aggregating peptides; (2) Root-Bernstein's theory that peptide receptors evolved from functionally and structurally complementary peptides; and (3) Blalock's theory that receptors evolved from hydropathically complementary sequences encoded in the antisense strand of the DNA encoding each peptide. The evidence to date suggests that the co-yevolution of peptides and their receptors is strongly constrained by one or more of these physicochemically based mechanisms, which argues against a random or frozen accident' model. The data also suggest that structure and function are integrally related from the earliest steps of receptor-ligand evolution so that peptide functionality is non-random and highly conserved in its origin. The result is a molecular paleontology' that reveals the evolutionary constraints that shaped the interaction of structure and function.

Amino Acid Sequence↗
PubMed2006

Novel snake venom ligand dendroaspis natriuretic peptide is selective for natriuretic peptide receptor-A in human heart: downregulation of natriuretic peptide receptor-A in heart failure.

The natriuretic peptides are considered to be cardioprotective; however, their receptors have not been identified in human myocardium using radiolabeled analogs. Dendroaspis natriuretic peptide (DNP) has been recently identified as a new member of this peptide family and is thought to be less susceptible to enzymatic degradation. Therefore, we have developed the novel radiolabeled analog [125I]-DNP and used this to localize high-affinity (K(D)=0.2 nmol/L), saturable, specific binding sites in adult human heart (n=6) and coronary artery (n=8). In competition binding experiments, atrial natriuretic peptide and brain type natriuretic peptide had greater affinity for [125I]-DNP binding sites than C-type natriuretic peptide and the natriuretic peptide receptor (NPR)-C ligand, cANF. This rank order of potency suggested binding of [125I]-DNP was specific to NPR-A. Messenger RNA encoding NPR-A was identified in left ventricle and coronary artery smooth muscle, and expression was confirmed by immunocytochemical studies at the protein level. In addition, fluorescence dual labeling immunocytochemistry localized NPR-A protein to cardiomyocytes, endocardial endothelial cells, and smooth muscle of intramyocardial vessels. Importantly, we demonstrated a significant downregulation in the density of NPR-A in heart and coronary artery of patients with ischemic heart disease that may explain, in part, the attenuated natriuretic peptide response reported in this patient group.

Adult↗
PubMed2003

A peptide from heat shock protein 60 is the dominant peptide bound to Qa-1 in the absence of the MHC class Ia leader sequence peptide Qdm.

The MHC class Ib molecule Qa-1 binds specifically and predominantly to a single 9-aa peptide (AMAPRTLLL) derived from the leader sequence of many MHC class Ia proteins. This peptide is referred to as Qdm. In this study, we report the isolation and sequencing of a heat shock protein 60-derived peptide (GMKFDRGYI) from Qa-1. This peptide is the dominant peptide bound to Qa-1 in the absence of Qdm. A Qa-1-restricted CTL clone recognizes this heat shock protein 60 peptide, further verifying that it binds to Qa-1 and a peptide from the homologous Salmonella typhimurium protein GroEL (GMQFDRGYL). These observations have implications for how Qa-1 can influence NK cell and T cell effector function via the TCR and CD94/NKG2 family members, and how this effect can change under conditions that cause the peptides bound to Qa-1 to change.

Animals↗
PubMed1987

Isolation, characterization, and pharmacological actions of peptide histidine valine 42, a novel prepro-vasoactive intestinal peptide-derived peptide.

The primary structure and biological activity of a novel prepro-vasoactive intestinal peptide (prepro-VIP)-derived peptide has been determined from an adrenal pheochromocytoma. The peptide was purified sufficiently for characterization by fast atom bombardment mapping after cation-exchange and reverse-phase fast protein liquid chromatography. The sequence of this novel peptide corresponds exactly to prepro-VIP-81-122 and has been designated peptide histidine valine 42 (PHV-42). Synthetic PHV-42 reduced both the force and frequency of spontaneous contractions of isolated rat uterus and was at least 12 times more potent than peptide histidine methionine (prepro-VIP-81-107), and over a hundred times more potent than noradrenaline. PHV-42 was also more potent than peptide histidine methionine in relaxing smooth muscle preparations of rat stomach and guinea pig trachea, but was approximately 4-fold less potent in reducing blood pressure than VIP. PHV-42 thus forms a separate subsystem in the VIP family of peptides and may be the most biologically active product of prepro-VIP in certain tissues such as the uterus and trachea.

Adrenal Gland Neoplasms↗
PubMed1994

Crystal structure of a peptide complex of anti-influenza peptide antibody Fab 26/9. Comparison of two different antibodies bound to the same peptide antigen.

The three-dimensional structure of the complex of a second anti-peptide antibody (Fab 26/9) that recognizes the same six-residue epitope of an immunogenic peptide from influenza virus hemagglutinin (HA1; 75-110) as Fab 17/9 with the peptide has been determined at 2.8 A resolution. The amino acid sequence of the variable region of the 26/9 antibody differs in 24 positions from that of 17/9, the first antibody in this series for which several ligand-bound and free structures have been determined and refined. Comparison of the 26/9-peptide with the 17/9-peptide complex structures shows that the two Fabs are very similar (r.m.s.d. 0.5 to 0.8 A) and that the peptide antigen (101-107) has virtually the same conformation (r.m.s.d. 0.3 to 0.8 A) when bound to both antibodies. A sequence difference in the 26/9 binding pocket (L94; His in 26/9, Asn in 17/9) results in an interaction with a bound water molecule that is not seen in the 17/9 structures. Epitope mapping shows that the relative specificity of 26/9 and 17/9 antibodies for individual positions of the peptide antigen are slightly different. Amino acid substitutions in the peptide, particularly at position SerP107, are tolerated to different extents by 17/9 and 26/9. Structural and sequence analysis suggests that amino acid differences near the peptide-binding site are responsible for altering slightly the specificity of 26/9 for three peptide residues and illustrates how amino acid substitutions can modify antibody-antigen interactions and thereby modulate antibody specificity.

Amino Acid Sequence↗
PubMed2006

Interaction of a G protein-coupled receptor with a G protein-derived peptide induces structural changes in both peptide and receptor: a Fourier-transform infrared study using isotopically labeled peptides.

G protein-coupled receptor signaling involves productive interaction between agonist-activated receptor and G protein. We have used Fourier-transform infrared difference spectroscopy to examine the interaction between the active Meta II state of the visual pigment rhodopsin with a peptide analogue corresponding to the C terminus of the alpha-subunit of the G protein transducin. Formation of the receptor-peptide complex evokes a spectral signature consisting of conformationally sensitive amide I and amide II difference bands. In order to distinguish between amide backbone contributions of the peptide and of the receptor moiety to the vibrational spectra, we employed complete (13)C,(15)N-labeling of the peptide. This isotopic labeling downshifts selectively the bands of the peptide, which can thus be extracted. Our results show that formation of the complex between the activated Meta II receptor state and the peptide is accompanied by structural changes of the peptide, and of the receptor, indicating that the conformation of the Meta II.peptide complex is different from that of Meta II. This result implies that the activated receptor state has conformational flexibility. Binding of the peptide to the activated receptor state stabilizes a substate that deviates from that stabilized only by the agonist.

Amino Acid Sequence↗
PubMed1974

The selective isolation of an active-site histidine peptide from chymotrypsin-alpha by diagonal peptide 'mapping'. An N-tau-carboxymethylhistidine diagonal peptide "mapping.".

1. The selective isolation of an ;active' histidine peptide from reduced and cyanoethylated chymotrypsin-alpha inhibited with Tos-Phe-CH(2)Cl (l-1-tosylamido-2-phenylethyl chloromethyl ketone) was obtained with a His(tauCm) (N(tau)-carboxymethylhistidine) diagonal peptide-;mapping' technique. Performic acid vapours, used between the first and second dimensions of the diagonal peptide ;map', resulted in a peracid rearrangement of the alkylated (Tos-Phe-CH(2))-histidine-57 residue into an N(tau)-carboxymethylhistidine residue. The consequent change in electrophoretic mobility allowed isolation of peptides that contained the ;active' histidine. 2. Peptides containing methionine or S-cyanoethylcysteine were oxidized to their sulphones during the treatment. Peptides in which these residues were N-terminal were selectively isolated on the basis of the change in electrophoretic mobility at pH6.5 which was due to the depression of the pK of the terminal amino group by the inductive effect of the sulphonyl group. 3. An attempt to apply the method to subtilisin BPN' inhibited with l-1-benzyloxycarbonylamido-2-phenylethyl bromomethyl ketone failed to yield a peptide containing N(tau)-carboxymethylhistidine, although peptides containing N-terminal methionine were isolated by the procedure.

Amino Acid Sequence↗
PubMed1995

Requirements for peptide binding to the human transporter associated with antigen processing revealed by peptide scans and complex peptide libraries.

Antigenic peptides are translocated into the lumen of the endoplasmic reticulum by the action of the transporter associated with antigen processing (TAP), where they are subsequently needed for the correct assembly of major histocompatibility complex molecules. The transport function was reconstituted in insect cells by expression of both TAP genes. On the basis of this over-expression system, substrate selection was analyzed in detail by a direct biomolecular peptide binding assay. Competition assays with peptide variants, including substitutions of residues with alanine or structurally related amino acids, underline the broad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides and scans with bulky, hydrophobic amino acids. Complex nonapeptide libraries were used to determine the contribution of each residue to stabilize peptide-TAP complexes. For the first time, this approach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transporter.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

A peptide analogue of thrombin receptor-activating peptide inhibits thrombin and thrombin-receptor-activating peptide-induced vascular smooth muscle cell proliferation.

The serine protease thrombin, in addition to its pivotal role in the coagulation cascade, plays an important role in the development of atherosclerosis and restenosis by inducing smooth cell proliferation. Thrombin exerts its cellular effects mainly by cleaving its own receptor, leaving a new NH2-terminus that can act as a tethered ligand to activate the thrombin receptor. Peptides derived from the new NH2-terminus are able to fully activate thrombin receptor and mimic cellular effects of thrombin. Peptides with structural similarities to the tethered ligand have been tested for their ability to prevent thrombin- and tethered ligand-induced platelet aggregation and thrombus formation. We synthesized a peptide with multiple alanine substitutions in both critical and noncritical residues of tethered ligand that specifically inhibited platelet aggregation induced by thrombin and thrombin receptor-activating peptide and prevented thrombus formation in a rabbit thrombosis model. In the present study we demonstrate that this peptide inhibited only thrombin- and tethered ligand-induced human vascular smooth muscle cell proliferation as determined by (3H)-thymidine incorporation and has no effect on platelet-derived growth factor and serum-induced smooth muscle cell proliferation. The inhibitory effect of this peptide is dependent on the concentration of the antagonist used and length of preincubation time. The possible mechanism by which this peptide exerts its inhibitory effect may by desensitizing the thrombin receptor. The results of the present study suggest that apart from being antithrombotic, tethered ligand antagonist peptides can also act as antiatherosclerotic or antirestenotic agents.

Cell Count↗