Methionine Residue In Position Research Articles

Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile Deinococcus radiodurans.

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.

Methionine 35 sulphoxide reduces toxicity of Aβ in red blood cell.

The oxidation of methionine residue in position 35 of Ab to sulphoxide (Ab-sulphoxide) has the ability to deeply modify wild-type Ab 1-42 (Ab) neurotoxic action. Our previous studies suggest that in nucleated cells, lower toxicity of Ab-sulphoxide might result not from structural alteration, but from elevation of methionine sulphoxide reductase A (MsrA) activity and mRNA levels. On this basis, we hypothesised that red blood cell (RBC), a cell devoid almost completely of MsrA activity, shares with nucleated cells an antioxidant system induced by methionine 35 sulphoxide, responsible for the lower toxicity of Ab-sulphoxide in RBC. (Results) Supporting this hypothesis, we found that the low toxicity of Ab-sulphoxide in RBC correlated with pentose phosphate pathway (PPP) flux increase, and this event was associated with a low level of methionine oxidation in total proteins. None of these effects were observed when cells were exposed to Ab native. These results outline the importance of the redox state of methionine 35 in the modulation of Ab-mediated events and suggest an important protective role for PPP in RBC of patients affected by Alzheimer's disease.

Oxidation of methionine 35 reduces toxicity of the amyloid beta-peptide(1–42) in neuroblastoma cells (IMR-32) via enzyme methionine sulfoxide reductase A expression and function

The beta amyloid peptide (Aβ), the major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of free radicals that may lead to neurodegeneration. Our recent evidence suggest that the redox state of methionine residue in position 35 (Met-35) of Aβ has the ability to deeply modify peptide's neurotoxic actions. Reversible oxidation of methionine in proteins involving the enzyme methionine sulfoxide reductase type A (MsrA) is postulated to serve a general antioxidant role and a decrease in MsrA has been implicated in Alzheimer's disease. In rat neuroblastoma cells (IMR-32), we used Aβ(1–42), in which the Met-35 is present in the reduced state, with a modified peptide with oxidized Met-35 (Aβ(1–42)Met35 OX), as well as an Aβ-derivative in which Met-35 is substituted with norleucine (Aβ(1–42)Nle35) to investigate the relationship between Met-35 redox state, expression and function of MsrA and reactive oxygen species (ROS) generation. The obtained results shown that MsrA activity, as well as mRNA levels, increase in IMR-32 cells treated with Aβ(1–42)Met35 OX, differently to that shown by the reduced derivative. The increase in MsrA function and expression was associated with a decline of ROS levels. None of these effects were observed when cells were exposed to Aβ containing oxidized Met35 (Aβ1–42)Met35 OX. Taken together, the results of the present study indicate that the differential toxicity of Aβ peptides containing reduced or oxidised Met-35 depends on the ability of the latter form to reduce ROS generation by enhancing MsrA gene expression and function and suggests the therapeutic potential of MsrA in Alzheimer's disease.

New weak toxins from the cobra venom

A protein with M 7485 Da containing five disulfide bonds was isolated from the venom of cobra Naja oxiana using various types of liquid chromatography. The complete amino acid sequence of the protein was determined by protein chemistry methods, which permitted us to assign it to the group of weak toxins. This is the first weak toxin isolated from the venom of N. oxiana. In a similar way, two new toxins with M 7628 and 7559 Da, which fall into the range of weak toxin masses, were isolated from the venom of the cobra N. kaouthia. The characterization of these proteins using Edman degradation and MALDI mass spectrometry has shown that one of these proteins is a novel weak toxin and the other is the known weak toxin WTX with an oxidized methionine residue in position 9. Such a modification was detected in weak toxins for the first time. A study of the biological activity of the toxin from N. oxiana showed that, like other weak toxins, it can be bound by muscle nicotinic cholinoreceptors and alpha7 nicotinic cholinoreceptors.

Role of methionine 35 in the intracellular Ca2+ homeostasis dysregulation and Ca2+‐dependent apoptosis induced by amyloid β‐peptide in human neuroblastoma IMR32 cells

Amyloid beta-peptide (Abeta) plays a fundamental role in the pathogenesis of Alzheimer's disease. We recently reported that the redox state of the methionine residue in position 35 of amyloid beta-peptide (Abeta) 1-42 (Met35) strongly affects the peptide's ability to trigger apoptosis and is thus a major determinant of its neurotoxicity. Dysregulation of intracellular Ca(2+) homeostasis resulting in the activation of pro-apoptotic pathways has been proposed as a mechanism underlying Abeta toxicity. Therefore, we investigated correlations between the Met35 redox state, Abeta toxicity, and altered intracellular Ca(2+) signaling in human neuroblastoma IMR32 cells. Cells incubated for 6-24 h with 10 microM Abeta1-42 exhibited significantly increased KCl-induced Ca(2+) transient amplitudes and resting free Ca(2+) concentrations. Nifedipine-sensitive Ca(2+) current densities and Ca(v)1 channel expression were markedly enhanced by Abeta1-42. None of these effects were observed when cells were exposed to Abeta containing oxidized Met35 (Abeta1-42(Met35-Ox)). Cell pre-treatment with the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (1 microM) or the Ca(v)1 channel blocker nifedipine (5 microM) significantly attenuated Abeta1-42-induced apoptosis but had no effect on Abeta1-42(Met35-Ox) toxicity. Collectively, these data suggest that reduced Met35 plays a critical role in Abeta1-42 toxicity by rendering the peptide capable of disrupting intracellular Ca(2+) homeostasis and thereby provoking apoptotic cell death.

New analogue of cyclolinopeptide B modified by amphiphilic residue of alpha-hydroxymethylmethionine.

In order to evaluate the role and influence of the methionine residue on the biological activity of cyclolinopeptide B, an analogue with methionine residue in position 7 replaced by the amphiphilic (S)-alpha-hydroxymethylmethionine residue was synthesized. This peptide exhibits high immunosuppressive activity in the cellular, and to a lesser degree in the humoral immune response, comparable to that of CsA. In addition, the peptide was devoid of toxicity, even at high doses.

New analogue of cyclolinopeptide B modified by amphiphilic residue of alpha-hydroxymethylmethionine.

In order to evaluate the role and influence of the methionine residue on the biological activity of cyclolinopeptide B, an analogue with methionine residue in position 7 replaced by the amphiphilic (S)-alpha-hydroxymethylmethionine residue was synthesized. This peptide exhibits high immunosuppressive activity in the cellular, and to a lesser degree in the humoral immune response, comparable to that of CsA. In addition, the peptide was devoid of toxicity, even at high doses.

A beta-peptides enhance vasoconstriction in cerebral circulation.

Amyloid-beta (A beta)-peptides are involved in the pathophysiology of Alzheimer's dementia. We studied the effects of A beta on selected constrictor responses of cerebral circulation. Mice were anesthetized (by using urethane-chloralose) and equipped with a cranial window. Arterial pressure and blood gases were monitored and controlled. Cerebral blood flow (CBF) was monitored by a laser Doppler probe. Topical superfusion with A beta 1-40 (0.1-10 microM), but not with the reverse peptide A beta 40-1, reduced resting CBF (-29 +/- 4% at 5 microM; P < 0.05) and augmented the reduction in CBF produced by the thromboxane analog U-46619 (+45 +/- 3% at 5 microM; P < 0.05). A beta 1-40 or A beta 1-42 did not affect the reduction in CBF produced by hypocapnia. The reduction in resting CBF and the enhancement of vasoconstriction were reversed by treatment with the free radical scavengers superoxide dismutase or manganic(I-II)meso-tetrakis(4-benzoic acid)porphyrin. Substitution of the methionine residue in position 35 with norleucine, a mutation that abolishes the ability of A beta to produce free radicals, abolished its vascular effects. Nanomolar concentrations of A beta 1-40 constricted isolated pressurized middle cerebral artery segments with intrinsic tone (-16 +/- 3% at 100 nM; P < 0.05). We conclude that A beta acts directly on cerebral arteries to produce vasoconstriction and to enhance selected constrictor responses. The evidence supports the idea that A beta-induced production of reactive oxygen species plays a role in this effect. The vascular actions of A beta may contribute to the deleterious effects resulting from accumulation of this peptide in Alzheimer's dementia.

The sulfoxide of thymosin beta 4 almost lacks the polymerization-inhibiting capacity for actin.

Thymosin beta 4 (T beta 4), a peptide of 43 amino acids, binds to actin monomers and inhibits filament formation. In preparations of T beta 4 from bovine lung tissue, the peptide is accompanied by a derivative in which the methionine residue in position 6 is replaced by its sulfoxide. T beta 4 sulfoxide inhibits actin polymerization to an extent approximately 20-times less than T beta 4. While an equimolar amount of T beta 4 prevented actin polymerization almost completely, polymerization with the corresponding amount of the sulfoxide proceeded in a manner similar to that of pure actin, except for a slight retardation. We showed that the decrease in the inhibitory activity is reflected by a 20-times lower affinity to actin. Interestingly, under non-polymerizing conditions, the affinity of T beta 4 sulfoxide for actin is as high as that of T beta 4 (approximately 1 microM). In accordance with this, no differences were found between T beta 4 and the sulfoxide in cross-linking experiments with the monomer, where both forms of the peptide yielded similar amounts of a 47-kDa band representing conjugates of actin and beta-thymosin, as proved by Western-blotting analysis. Likewise, both, T beta 4 and the sulfoxide retarded the exchange of G-actin-bound nucleotide to similar extents. Although the sulfoxide is presumably a product of autoxidation, it is attractive to speculate that oxidation of the methionine residue in T beta 4 may represent a regulatory switch for starting filament formation in non-muscle cells.

Characterization of the Macrophage Migration Inhibitory Factor-Binding Site of Sarcolectin and Its Relationship to Human Serum Albumin

The sialic acid-binding protein sarcolectin from human placenta specifically interacts with the lymphokine macrophage migration inhibitory factor, enabling its convenient purification and histochemical localization. After cyanogen bromide-mediated cleavage of sarcolectin one polypeptide with an apparent molecular weight of approximately 15,000 exhibited binding capacity to the labelled lymphokine, as revealed by ligand blotting. The N-terminal sequence stretch of this peptide is identical to the respective sequence of human serum albumin, following the internal methionine residue in position 298. Cleavage at a methionine moiety in position 446 can explain the size of the 15 KDa product of chemical degradation. Close similarity of circular dichroism of sarcolectin and human serum albumin added further evidence to their structural similarity, calling for further studies to rigorously define their relationship.

Neuropeptide Y in guinea pig, rabbit, rat and man. Identical amino acid sequence and oxidation of methionine-17

Neuropeptide Y (NPY) was isolated and characterised from acid-ethanol extracts of rabbit and guinea pig brain. In both instances the chromatographic purification was a two-step procedure of gel filtration followed by reverse-phase high-performance liquid chromatography. The amino acid sequence of rabbit and guinea pig NPY was found to be identical to human and rat NPY as deduced from the cDNA structures. With the exception of the porcine peptide, all mammalian NPYs characterised to date have a methionine residue in position 17. This methionine residue is readily oxidized as indicated by the high degree of spontaneous oxidation of peptides found in the rabbit and guinea pig brain extracts and in NPY extracted from a rat phaeochromocytoma cell line. It is concluded that NPY is among the most highly conserved peptides and that NPYs containing methionine in position 17 are prone to oxidation.

Characterization of a polypeptide from human seminal plasma with inhibin (inhibition of FSH secretion)-like activity

A polypeptide in preparations of ‘large’ form ( M r ∼14 000) material with inhibin-like activity (inhibiting FSH secretion) has been isolated from human seminal plasma. Its amino acid composition, cleavage pattern with CNBr, N-terminal sequence, and properties on reverse-phase high-performance liquid chromatography establish this inhibin-like preparation to be homogeneous. The polypeptide contains close to 130 residues, has a free N-terminal serine residue, a methionine residue in position 19, and a dibasic structure (Arg—Lys) in positions 16–17.

Immunochemistry of cytochrome c. Identification of antigenic determinants through the study of hybrid molecules.

Antibodies elicited in rabbits against horse cytochrome c cross-react in varying degree with the cytochromes c of other species. Radioimmunoassay experiments are described in which 23% of the immunoglobulin shown to bind to horse cytochrome c fails to react with beef cytochrome c, and 14% of that bound by beef cytochrome c does not react with rabbit cytochrome c. Studies of hybrid cytochromes, prepared by a reconstitution procedure involving the cleavage and re-formation of peptide bond 65-66, show that these differences result from single amino acid replacements, Beef cytochrome c differs in binding capacity from horse cytochrome c as a result of the substitution of a glycine for a lysine residue in sequence position 60, and the difference between rabbit cytochrome c and beef cytochrome c reflects the substitution of a valine for a proline residue in position 44. Reconstituted horse cytochrome c and reconstituted beef cytochrome c have binding capacities indistinguishable from those of the parent proteins. The presence of a homoserine rather than a methionine residue in position 65 results, in the case of the reconstituted horse molecule, in a slightly lower affinity for the antibody population directed against the lysine-60 region. A corresponding difference is not observed in the case of beef cytochrome c and its reconstitution product, which do not bind the population in question.

The vasoactive intestinal peptide (VIP): VI. The 17-norleucine analog of the sequence 14–28

A pentadecapeptide amide with the C-terminal sequence (14–28) of the vasoactive intestinal peptide (VIP), but with the methionine residue in position 17 replaced by l-norleucine, was synthesized. The synthesis was carried out through stepwise chain lengthening, by the in situ technique. The norleucine-containing pentadecapeptide, l-arginyl- l-lysyl- l-glutaminyl- l-norleucyl- l-alanyl- l-valyl- l-lysyl- l-lysyl- l-tyrosyl- l-leucyl- l-asparaginyl- l-seryl- l-isoleucyl- l-leucyl- l-asparaginamide, was as active in relaxing different smooth-muscle preparations as the methionine-containing parent sequence.

Uniformity and Species‐Specific Features of the N‐Terminal Amino‐Acid Sequence of Porcine Immunoglobulin λ‐Chains

Cleavage with cyanogen bromide was employed to investigate the N‐terminal amino acid sequence of the λ‐chains of normal porcine immunoglobulin. The N‐terminal homoserine‐containing nonapeptide was isolated from all fractions of the λ‐chains in a yield of 60% of theory. Its amino acid sequence was determined by mass spectrometry and was found to be uniform. Other homoserine‐containing fragments were obtained from the cyanogen bromide hydrolyzate of the λ‐chains with split disulfide bonds. Partial amino acid sequence of these fragments provided evidence that methionine in position 9 is present in more than 60% of variants and obviously has an invariant character. Variants of the λ‐chains exist (by estimate not more than 15% of the total λ‐chains) which, in addition to the invariant methionine residue in position 9, possess a variable methionine residue in the section between positions 46 and 51.The data obtained indicate that the sequence of 23 amino acid residues from the N‐terminus of porcine λ‐chains is uniform with the exception of a single replacement (in position 13). Moreover, in comparison with the sequence of λ‐chains of man, mouse and birds, the sequence displays several species‐specific replacements and a deletion of threonine which occupies position 5 in the chains of the other species. In the pig, the λ‐chains represent the predominant type of light chains (some 70%). Hence it follows that some 50% of the light chains in this species possess a uniform amino acid sequence in the N‐terminal section. These facts are difficult to reconcile with the germline theories of genetic control so that mechanisms of somatic diversification of immunoglobulins must be envisaged.

Cleavage of cytochrome c with cyanogen bromide

Heme peptides and nonheme peptides suitable for use in binding studies have been prepared by cleavage of horse cytochrome c with cyanogen bromide. These derivatives include a heme peptide of 65 residues and a peptide of 39 residues obtained by division of the protein at the bond following methionine residue 65. Other useful products include: (1) a peptide corresponding to residues 66–104 of the parent molecule, but with a homoserine residue substituted for the methionine residue in position 80; (2) smaller peptides consisting of residues 66–80 and 81–104; and (3) a heme peptide of 80 residues containing a homoserine instead of a methionine residue in position 65. Unlike other instances in which conversion of a methionine to a homoserine residue has been observed to occur without peptide-bond cleavage, neither of the methionine residues of horse cytochrome c is followed by a threonine or serine residue. A mechanism similar to that for the hydrolysis of N-phenyliminolactone is proposed.

The structure of sheep hemoglobins: II. The amino acid composition of the tryptic peptides of the non-α chains of hemoglobins A, B, C, and F

This paper contains detailed data of the amino acid compositions of the peptides from tryptic digests of the non-α chains of the three adult sheep hemoglobins A, B, and C and of sheep fetal hemoglobin. Sequential arrangements of amino acids of some of these peptides are also presented. The peptides were isolated from 2- and 24-hour tryptic digests by Dowex-50 chromatography and purified on Dowex-1 resin. The β chains of the adult hemoglobins A and B and the γ chains of fetal hemoglobin contain likely 145 residues with methionine in amino terminal position. The minimal number of differences between the two adult chains is 6; these differences include a methionine residue in position 76 and a proline residue in position 58. The β chain of hemoglobin C, which replaces the β chain of hemoglobin A in sheep homozygous for hemoglobin A during severe anemia, may contain 141 residues with proline in amino terminal position. Excluding the first 8 residues from the comparison, at least 12 differences are likely to exist between the β chains of the hemoglobins A and C. The γ chain differs from the β chain of hemoglobin B minimally in 27 residues. A comparison of the putative structures of these chains with the corresponding bovine polypeptide chains revealed a greater resemblance than noted with any other non-α chain from species studied. Since the structural differences between the non-α chains of the hemoglobins A, B, and C are located in various positions in the total chain structures, it seems indicated (a) that the allelic genes governing the structural synthesis of the β chains of the hemoglobins A and B are greatly different in structure, and (b) that the synthesis of the β chain of hemoglobin C is controlled by a structurally different and “silent” gene, which is activated during severe anemia.