Biosynthesis of peptide neurotransmitters: studies on the formation of peptide amides

A high proportion of peptide transmitters and peptide hormones terminate their peptide chain in a C-terminal amide group which is essential for their biological activity. The specificity of an enzyme that catalyses the formation of the amide was investigated with the aid of synthetic peptide substrates. With peptides containing l-amino acids the enzyme exhibited an essential requirement for glycine in the C-terminal position; amidation did not take place with peptides that had leucine, alanine, glutamic acid, lysine or N-methylglycine at the C-terminus and a peptide extended by the attachment of lysine to the C-terminal glycine did not act as a substrate. Amidation did occur with a peptide containing C-terminal D-alanine but no reaction was detected with peptides having C-terminal, D-serine or D-leucine. In tripeptides with a neutral amino acid in the penultimate position, amidation, took place readily but the reaction was slower when this position was occupied by an acidic or a basic residue. A series of overlapping peptides with C-terminal glycine, based on partial sequences of calcitonin, underwent amidation at similar rates, indicating that the amidating enzyme recognizes only a limited sequence at the C-terminus of its substrates. The results provide evidence that the amidating enzyme has a highly compact substrate binding site.     

Imprinting Effects of Proline Containing Dipeptides (Proline-Glycine, Proline-Leucine, Proline-Valine and Their Retro Variants) in Tetrahymena. Evolutionary Conclusions

Proline-glycine, proline-leucine and proline-valine dipeptides and their retro variants were used in the experiments to study the effects of pretreatment (imprinting) in Tetrahymena, by investigating fluorescein isothiocyanate (FITC)-conjugated peptide binding. The protozoan organism could differentiate between the proline-dipeptides containing different partner amino-acids and between the dipeptides having the amino acids in reversed positions. The effect of imprinting was positive or negative and this was dependent on the type of the partner amino acid and on its position. Pro-Gly and Pro-Leu induced positive imprinting (elevated FITC-dipeptide binding) and Pro-Val induced negative imprinting (decrease of FITC-peptide binding). There was positive imprinting induction in two cases for the retro FITC-peptide and in one case for the FITC-conjugate of the imprinter peptide itself. The highest positive imprinting (almost 60% increase) was induced by Pro-Gly for FITC-Gly-Pro. Considering earlier—chemotaxis—experiments, the results of the present—binding—studies run parallel with the physiological effects. The experiments call attention to the sharp differentiating ability of small peptides at a unicellular level, that could have some role in the selection of molecules for hormone formation, during evolution.     

Studies of hormone evolution. Can non-hormone oligopeptides induce a functional imprinting?

Certain non-hormone oligopeptides have a greater imprinting effect on Tetrahymena than others. The imprinting potential is unrelated to the length of the peptide chain, but seems greatly dependent on the amino acid sequence. The direct growth-stimulant action developed by the peptides at the first interaction is unrelated to their imprinting effect.     

Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: Complex interplay of electrostatic and hydrophobic interactions

Basic amino acids play a key role in the binding of membrane associated proteins to negatively charged membranes. However, side chains of basic amino acids like lysine do not only provide a positive charge, but also a flexible hydrocarbon spacer that enables hydrophobic interactions. We studied the influence of hydrophobic contributions to the binding by varying the side chain length of pentapeptides with ammonium groups starting with lysine to lysine analogs with shorter side chains, namely ornithine (Orn), α,γ-diaminobutyric acid (Dab) and α, β-diaminopropionic acid (Dap). The binding to negatively charged phosphatidylglycerol (PG) membranes was investigated by calorimetry, FT-infrared spectroscopy (FT-IR) and monolayer techniques. The binding was influenced by counteracting and sometimes compensating contributions. The influence of the bound peptides on the lipid phase behavior depends on the length of the peptide side chains. Isothermal titration calorimetry (ITC) experiments showed exothermic and endothermic effects compensating to a different extent as a function of side chain length. The increase in lipid phase transition temperature was more significant for peptides with shorter side chains. FTIR-spectroscopy revealed changes in hydration of the lipid bilayer interface after peptide binding. Using monolayer techniques, the contributions of electrostatic and hydrophobic effects could clearly be observed. Peptides with short side chains induced a pronounced decrease in surface pressure of PG monolayers whereas peptides with additional hydrophobic interactions decreased the surface pressure much less or even lead to an increase, indicating insertion of the hydrophobic part of the side chain into the lipid monolayer.     

Rat liver polysome N alpha-acetyltransferase: substrate specificity.

The substrate specificity of polysome rat liver N alpha-acetyltransferase (NAT) has been examined by utilizing a series of synthetic and natural substrates that has been systematically altered with respect to N-terminal sequence and length. Families of peptides of the structure S-Y-S-G-G-L-L-L were generated by successively replacing the N-terminal serine, the penultimate tyrosine, and the antepenultimate serine with all 19 commonly occurring amino acids, which were then assessed for their reactivity with the rat liver enzyme. Only peptides with N-terminal serine, alanine, methionine, leucine, and phenylalanine were modified. Glycine, lysine, arginine, valine, isoleucine, and tryptophan in the second position are (with N-terminal serine) strongly inhibitory, and proline completely blocks modification. Third-position substitutions have less of an effect on NAT activity with glycine, aspartic acid, glutamic acid, and tryptophan being most inhibiting (with N-terminal Ser-Tyr). These observations are generally in agreement with in situ modifications although there are some significant differences particularly with respect to the amino-terminal residues. Optimal chain length was determined to be 10-11 residues with either synthetic peptides of the structure S-Y-S-(G)n-L-L-L or adrenocorticotropin (ACTH) sequences ranging from 8 to 39 residues. The ACTH peptides were generally found to be severalfold better substrates than the corresponding synthetic ones. Activity was not affected by increased chain length beyond approximately 17 residues. These data support the view that polysome-catalyzed N alpha-acetylation occurs as a cotranslational event on nascent chains of about 20-40 amino acids in length.     

Taxon-dependence of receptor level cell-to-cell communication in Tetrahymena: possible explanation for the transmission of hormonal imprinting

Insulin treatment induced in Tetrahymena pyriformis a positive hormonal imprinting, and in Tetrahymena thermophila a negative imprinting, resulting in increased and decreased binding capacity, respectively, at re-exposure to the hormone. The imprinting, or the information associated with it, is transferred by the nutrient medium of the insulin-treated cells to those not treated. The issue of transfer depends on the nature of the receiver taxon, leading always to a positive imprinting in Tetrahymena pyriformis, and to a negative imprinting in Tetrahymena thermophila, regardless of the nature of the 'imprinted' transmitter taxon. The findings substantiate the transferability of hormonal imprinting by the nutrient medium at the unicellular level, the key role of the postreceptorial mechanism in determining the trend of imprinting and may explain the persistence of imprinting in the progeny generations.     

Effect of vanadate and ouabain on insulin binding and insulin imprinting in Tetrahymena.

Na-metavanadate and ouabain that act on Na+K(+)-ATPase had no influence on insulin binding to Tetrahymena immediately after treatment, but after 24 h considerably enhanced the binding capacity of generations of progeny. The increase in binding was of a similar magnitude to that elicited by insulin imprinting. Vanadate failed to increase the imprinting potential of insulin while ouabain even prevented insulin imprinting when administered together with insulin, but, did not affect imprinting when administered after insulin. By analogy with higher organisms it appears that inhibition of Na+K(+)-ATPase plays no role in the insulin-like effect of vanadate on the unicellular Tetrahymena, as judged also from the capacity to bind insulin of the generations of offspring.     

Critical functional requirement for the guanidinium group of the arginine 41 side chain of human epidermal growth factor as revealed by mutagenic inactivation and chemical reactivation.

In a preliminary study we demonstrated that the formation of the epidermal growth factor (EGF) receptor-ligand complex requires the participation of the highly conserved arginine 41 side chain of the growth factor peptide (Engler, D.A., Montelione, G.T., and Niyogi, S.K. (1990) FEBS Lett. 271, 47-50). In an attempt to gain further insight into the nature of this interaction(s), we used both site-directed mutagenesis and chemical modification reagents to produce human EGF (hEGF) analogues with altered chemical properties of the residue 41 side chain. Eight mutant analogues of hEGF were generated, substituting arginine 41 with lysine, glutamine, isoleucine, tyrosine, glycine, alanine, aspartate, or glutamate. Although each of the mutant analogues was able to displace wild-type hEGF fully in receptor competition binding assays, affinity of the receptor for the mutants was substantially reduced, varying from 0.4 to less than 0.01% of that observed for wild-type growth factor. At sufficiently high concentrations these mutants were able to stimulate DNA synthesis in mouse keratinocytes. Substitution of lysine for arginine 41 reduced the receptor affinity 250-fold from that observed for wild type, despite retention of the positive electrostatic charge. The lysine substitution leaves a reactive amine at position 41 and made it possible, using amine-specific chemical modification reagents, to produce selected arginine homologues that were tested for their effects on receptor binding, receptor tyrosine kinase activation, and stimulation of DNA synthesis in mouse keratinocytes. The reaction of lysine 41 with methyl acetimidate resulted in a lysineacetamidine product which only partially restored activity of the lysine hEGF mutant. However, reaction with O-methylisourea resulted in generation of an arginine 41 homologue (homoarginine) which restored full activity. The results indicate that the chemical properties inherent in the guanidinium group of the arginine 41 side chain of hEGF are responsible for optimal receptor-ligand association.     

Influence of hormone treatment applied or begun in different phases of the cell cycle on hormonal imprinting in Tetrahymena

Primary exposure to a hormone (hormonal imprinting) alters--in the case of the Tetrahymena increases--cellular response to re-exposure(s) to the same hormone. The intensity of hormonal imprinting depends on the phase of the cell cycle in which the primary exposure has taken place. The effect of imprinting was greater on the cells exposed to the hormone in phase G1 than on those exposed in phase S or G2. The response pattern of the progeny generations corresponded to that of the primarily exposed (imprinted) ancestor cell, irrespective of their own pre-exposure in phase G1, G2 or S of their cycle.     

Structural and biological studies on synthetic peptide analogues of a low-affinity calcium-binding site of skeletal troponin C

We have synthesized four oligopeptides that are structural analogues of a low-affinity Ca2+-specific binding site (site II) of rabbit skeletal troponin C. One analogue (peptide 3) was a dodecapeptide with a sequence corresponding to the 12-residue Ca2+-binding loop (residues 63-74 in troponin C), two (peptides 4 and 5) were 23-residue in length, corresponding to residues 52-74 of the protein, and the fourth (peptide 6) was a 25-residue peptide corresponding to residues 50-74. All four peptides had one amino acid substitution within the 12-residue binding loop in which phenylalanine at position 10 was replaced by tyrosine to provide a marker for spectroscopic studies. In addition, peptides 3 and 4 each had a second substitution within the binding loop where glycine at position 6 was replaced by alanine. The second substitution was motivated by the conservation of glycine at the position in the Ca2+-binding loops of all four Ca2+-binding sites in troponin C. The peptides were characterized by their intrinsic fluorescence, ability to enhance the emission of bound Tb3+, affinity for Ca2+ and Tb3+, and circular dichroism. The affinity for Ca2+ was in the range 10-10(2) M-1, and the affinity for Tb3+ was in the range 10(4)-10(5) M-1. The binding constants of the longer peptides were several-fold larger than that of the dodecapeptide. With peptides 4 and 5, substitution of glycine by alanine at position 6 within the 12-residue loop decreased the affinity for Ca2+ by a factor of four, but had little effect on the affinity for Tb3+. However, the mean residue ellipticity of peptide 4 was substantially higher than that of peptide 5. Since peptide 4 differs from peptide 5 only in the substitution of glycine at position 6 in the loop segment, the conservation of glycine at that position may serve a role in providing a suitable secondary structure of the binding sites for interaction with troponin I. Peptides 4 and 6, when present in a large excess, mimic troponin C in regulating fully reconstituted actomyosin ATPase by showing partial calcium sensitivity and activation of the ATPase. Since these peptides are the smallest peptides containing the Ca2+-binding loop of site II, their biological activity suggests that a Ca2+-dependent binding site of troponin C for troponin I could be as short as the segment comprising residues 52-62.     

Hormonal imprinting: phylogeny, ontogeny, diseases and possible role in present-day human evolution

Hormonal (chemical) imprinting which was first observed (and named) by us in the seventies of the last century, is a general biological phenomenon which takes place when the developing receptor meets its target hormone for the first time. Under the effect of imprinting, receptors mature and reach their maximal binding capacity. It also influences the cells' hormone production and different functions depending on receptors and hormones. Hormonal imprinting is present already at the unicellular level causing the development of specific receptors and helping the easier recognition of useful or harmful surrounding molecules. The phenomenon is an important factor in the survival of the species, as the effect of imprinting is transmitted to the progeny cell generations. At the same time it possibly helps the selection of molecules which are suitable for acting as hormones in higher ranked animals. In mammals, hormonal imprinting takes place perinatally and determines the function of receptor-signal-transduction systems as well as hormone production for life. However, there are other critical imprinting periods for continuously developing cells. Excess of the target hormones or presence of foreign molecules which are able to bind to the receptors, provoke faulty imprinting in the critical periods with life-long morphological, biochemical, functional or behavioural consequences. As many receptor-bound foreign molecules are used as medical treatments and many such molecules are present around us and inside us as environmental pollutants, they--causing faulty imprinting--are able to predispose the (human) organism to cardiovascular, endocrine, metabolic and cancerous diseases. It seems likely that this effect is connected with disturbance of DNA methylation process in the critical periods of life. There are some signs of the transgenerational effect of faulty imprinting and this could be manifested in the evolution of humans by an epigenetic route.     

Verification of epigenetic inheritance in a unicellular model system: multigenerational effects of hormonal imprinting

The unicellular Tetrahymena has receptors for hormones of higher vertebrates, produces these hormones, and their signal pathways are similar. The first encounter with a hormone in higher dose provokes the phenomenon of hormonal imprinting, by which the reaction of the cell is quantitatively modified. This modification is transmitted to the progeny generations. The duration of the single imprinter effect of two representative signal molecules, insulin and 5-HT (5-hydroxytryptamine), in two concentrations (10-6 and 10-15 M) were studied. The effects of imprinting were followed in 5 physiological indices: (i) insulin binding, (ii) 5-HT synthesis, (iii) swimming behaviour, (iv) cell growth and (v) chemotaxis in progeny generations 500 and 1000. The result of each index was different from the non-imprinted control functions, growth rate, swimming behaviour and chemotactic activity to insulin being enhanced, while others, e.g. synthesis and chemotactic responsiveness of 5-HT and the binding of insulin were reduced. This means that a function-specific heritable epigenetic change during imprinting occurs, and generally a single encounter with a femtomolar hormone concentration is enough for provoking durable and heritable imprinting in Tetrahymena. The experiments demonstrate the possibility of epigenetic effects at a unicellular level and call attention to the possibility that the character of unicellular organisms has changed through to the present day due to an enormous amount of non-physiological imprinter substances in their environment. The results - together with results obtained earlier in mammals - point to the validity of epigenetic imprinting effects throughout the animal world.     

Effects of L-alanine and L-alanine peptides on the chemotaxis of tetrahymena: Evolutionary conclusions

L-alanine and its peptides (L-Ala-2–6) do not attract or repulse Tetrahymena in a 10^–8M concentration. In 10^–10M concentration there is a consistent repellent effect. Twenty four hours after L-alanine or L-alanine-peptides' pretreatment (imprinting) the progeny generation of the cells react differently to the same materials. L-Alanine, L-alanine penta- and hexapeptide in both concentrations are chemoattractant, while L-alanine tetrapeptide is repellent. L-Alanine dipeptide is inert in 10^–10M and repellent at 10^–8M concentrations, while L-alanine tripeptide is strongly repellent at 10^–10M and attractant at 10^–8M concentrations. This means, that the first encounter (imprinting) with an exogeneous amino acid or peptide is decisive to the later reaction of the protozoan cell. The chain length is important in the imprinting, however the reaction is not consistent. The experiments call the attention to the significance of imprinting in the receptor and hormone evolution.     

Trypanosoma gambiense: membrane transport of amino acids.

Trypanosoma gambiense absorbed ¹⁴C-labeled lysine, arginine, glutamate, phenylalanine, methionine, threonine, glycine, and alanine by mediated transport systems. The interactions of these compounds as inhibitors or stimulators formed complex patterns of uptake which suggested the presence of five binding and/or transport loci: Locus A bound glutamate, arginine, and lysine, and the binding of glutamate or arginine stimulated the transport of lysine. Locus B transported threonine, glycine, and alanine and appeared to be partially sensitive to ouabain and Na⁺. Locus C transported glutamate, locus D transported phenylalanine and methionine, and locus E transported lysine and arginine.     

Contribution of conserved glycine residues to ATP action at human P2X1 receptors: mutagenesis indicates that the glycine at position 250 is important for channel function

Glycine residues can introduce flexibility in proteins, give rise to turns and breaks in secondary structure and are key components of some nucleotide binding motifs. In the P2X receptor extracellular ATP binding domain, 11 glycine residues are completely conserved and an additional five are conserved in at least five of the seven family members. We have mutated individual conserved glycine residues and determined their effect on the ATP sensitivity and time-course of P2X1 receptors expressed in Xenopus oocytes. In the majority of cases, replacement by alanine had no or a less than 3-fold effect on ATP sensitivity and time-course of responses. G71A resulted in a 6-fold decrease in ATP potency and ATP (10 mM) failed to evoke functional responses from G96A, G250A and G301A mutant receptors. However, proline or cysteine could substitute for glycine at positions 96 and 301, giving receptors that were essentially normal. At glycine 250 substitution by serine gave functional responses to ATP with no effect on ATP sensitivity but a reduction in peak amplitude; in contrast, functional responses were not recorded when glycine 250 was replaced by the amino acids alanine, cysteine, aspartate, phenylalanine, isoleucine, lysine, proline or asparagine. These results suggest that glycine 250 plays an important role in determining the function of P2X receptors.     

Probing the beta-chain hole of fibrinogen with synthetic peptides that differ at their amino termini

In a recent report, we showed that alanine can replace glycine at the amino terminus of synthetic B-knobs that bind to human fibrin(ogen). We now report a survey of 13 synthetic peptides with the general sequence XHRPYam, all tested with regard to their ability to delay fibrinolysis in an in vitro system activated by t-PA, the results being used as measures of binding affinity to the betaC hole. Unexpectedly, some large and bulky amino acids, including methionine and arginine, are effective binders. Amino acids that branch at the beta carbon (valine, isoleucine, and threonine) do not bind effectively. Crystal structures were determined for two of the peptides (GHRPYam and MHRPYam) complexed with fibrin fragment D-dimer; the modeling of various other side chains showed clashing in the cases of beta-carbon substituents. The two crystal structures also showed that the enhanced binding observed with pentapeptides with carboxyl-terminal tyrosine, compared with that of their tetrapeptide equivalents, is attributable to an interaction between the tyrosine side chain and a guanidino group of a nearby arginine (beta406). The equivalent position in gamma-chains of human fibrin(ogen) is occupied by a lysine (gamma338), but in chicken and lamprey fibrin(ogen), it is an arginine, just as occurs in beta chains. Accordingly, the peptides GPRPam and GPRPYam, which are surrogate A-knobs, were tested for their influence on fibrin polymerization with fibrinogen from lamprey and humans. In lampreys, GPRPYam is a significantly better inhibitor, but in humans, it is less effective than GPRPam, indicating that in the lamprey system the same tyrosine-arginine interaction can also occur in the gamma-chain setting.     

Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain

A unique, late-eluting “basic peak” (relative to the “main peak”) was observed by weak cation exchange-HPLC (WCX) for a recombinant monoclonal antibody (mAb) sample. Peak fractions were collected, desalted, and analyzed by high-resolution MS using a top-down characterization approach that provided accurate masses of intact mAb charge isoforms and a comprehensive profile of the structural heterogeneity. The individual light (L) and heavy (H) chain subunits from the main and basic peaks were analyzed by reversed-phase (RP) HPLC/MS after disulfide bond reduction and cysteine alkylation. Three mAb isoforms were detected, and their modifications were localized to H chain. Bottom-up characterization using RP-HPLC/MS peptide mapping and accurate mass measurements identified three distinct H chain C-terminal peptides ending in glycine, lysine, or α-amidated proline. The combined analyses showed that the main WCX peak mAb isoform contained two unmodified L chains and two H chains terminating in glycine. Each mAb isoform that coeluted in the basic peak consisted of two unmodified L chain subunits and a single H chain ending in glycine, but the second H chain terminated in lysine for one isoform and α-amidated proline for another isoform. The WCX elution positions of the isoforms were consistent with their respective net charge. To the best of our knowledge, the occurrence of C-terminal α-amidation in mAbs has not been reported previously.     

Channeling of vanadium in two species of fungi.

Aspergillus flavus and Penicillium italicum were cultivated on Dox medium amended with different concentrations of a vanadium salt; vanadium pentoxide (V2O5). The fungal isolates were found to absorb considerable quantities of vanadium. Fungal contents of carbohydrates and proteins were decreased by the presence of vanadium in the growth environment. Vanadium was incorporated into several types of low and high molecular weight protein(s) and peptides. In case of P. italicum, lysine, tyrosine, glycine, methionine, valine, alanine, proline, glutamic acid and histidine were detected in the fungal cell free extract. While in case of A. flavus, the following amino acids were detected: cystine, methionine, leucine, phenylalanine, glycine, alanine, tyrosine, lysine, histidine, arginine and valine.     

Antibacterial activity of phosphono dipeptides based on 1-amino-1-methylethanephosphonic acid

Phosphono dipeptides containing 1-amino-1-methylethanephosphonic acid (phosphonic acid analogue of alpha-methylalanine, MeAlaP) and glycine, alanine, valine, leucine phenylalanine, proline, methionine or lysine as N- terminal component were synthesized in order to determine their antibacterial properties. Peptides containing alanine, leucine, valine phenylalanine and methionine showed marked in vitro activity, especially against Escherichia coli and Serratia marcescens strains. There were, however, generally less potent than the respective phosphono dipeptides based on 1-aminoethanephosphonic acid (phosphonic acid analogue of alanine, AlaP). The possible mechanism of action of the peptides of MeAlaP involves their active transport into the bacterial cell, followed by intracellular release of MeAlaP, which most likely inhibits alanine racemase, a key enzyme in peptidoglycan biosynthesis. Studies on the uptake of AlaMeAlaP and LeuMeAlaP by Escherichia coli mutants defective in the oligopeptide permease suggest that these peptides are not transported by the oligopeptide transport system.     

Is the saccharide component of the insulin receptor involved in hormonal imprinting?

Hormonal imprinting takes place at the first interaction of a given hormone with the cell (the Tetrahymena in the present case) and accounts for a greater responsiveness to the hormone on re-exposure(s). The Tetrahymena is able to bind insulin and Concanavalin-A (Con-A) as well. Exposure to both ligands—simultaneously or in sequence—enhances the binding of both in the progeny generations. It follows that the lectin, which inhibits insulin binding by direct action, enhances rather than depresses the effect of insulin-induced imprinting.