Arginine Residues In Peptides Research Articles

Arginine selective reagents for ligation to peptides and proteins.

A new class of arginine-specific bioconjugation reagents for protein labeling has been developed. This method utilizes a triazolyl-phenylglyoxal group on the probe molecule that reacts selectively with the guandinyl group of Arg residues in a protein or peptide. The reaction proceeds in neutral to basic bicarbonate buffers and is selective for arginine residues in peptides and folded proteins. Importantly, the triazolyl-phenylglyoxal group can be introduced into complex molecules containing alkyne groups using CuAAC chemistry, providing a robust approach for the generation of phenylglyoxal reactive groups into molecules to be covalently attached onto the surface of proteins. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Porphyromonas gingivalis peptidylarginine deiminase substrate specificity

While a group of oral commensals have been implicated in the aetiology of chronic periodontitis; the asaccharolytic Gram negative anaerobe Porphyromonas gingivalis is most commonly reported to be associated with severe forms of the disease. Although a variety of human tissues can produce a number of peptidylarginine deiminase (PAD), enzymes that convert peptide bound arginine residues to citrulline, P. gingivalis is one of the few prokaryotes known to express PAD. Protein and peptide citrullination are important in the development of rheumatoid arthritis and in recent years a number of authors have suggested a possible link between periodontitis and rheumatoid arthritis (RA). Indeed, some have linked P. gingivalis directly to RA via the action of PAD. Accordingly, the prime purpose of this study was to further characterise PAD in P. gingivalis cells particular emphasis on substrate specificity, using arginine containing peptides and RA relevant proteins. MethodsP. ginigvalis W50 was anaerobically cultured in BHI broth, cells harvested and resuspended in assay buffer. A colourimetric assay was developed to measure citrulline and employed to determine enzyme activity using the substrate BAEE. The assay was employed to investigate the effects of environmental pH and temperature on activity. Citrullination of BAEE by sonicated cells allowed the proportion of intracellular enzyme to be estimated. Enzyme specificity and substrate preference were investigated by using various arginine containing peptides, proteins and arginine analogues, as substrates and measuring the rate of citrullination. The influence of gingipains on citrullination was assessed by measuring the rates of citrullination of bovine serum albumin in the presence of protease inhibitors. ResultsEnzyme activity decreased by 13% following exposure of cells to 60 °C for 10 min. A comparison of intact and disrupted cells indicated that 90% of PAD activity is cell surface associated and the remainder cytoplasmic. Optimal pH for enzyme activity was between pH 7.5 and 8. All small arginine-containing peptides were citrullinated with reaction rates faster than that for free arginine with rates that varied with arginine residue position and number. Arginine analogues exhibited minimal effect and influence when tested as either substrates or competitive inhibitors. Cells were able to citrullinate yeast enolase, human vimentin and fibrin at varying rates. All proteins were modified at slower rates than those for peptide substrates. Inhibition of gingipains had no influence on the rate of protein citrullination. ConclusionsP. gingivalis PAD is a primarily cell surface associated, heat stable, enzyme that exhibits optimal activity under alkaline conditions similar to those present in the inflammatory environment. The enzyme displays high specificity for arginine residues in peptides and modified arginine in all positions and the gingipains did not influence the rate of protein citrullination. The ability of the enzyme to convert arginine residues in all proteins tested would indicate that its presence in inflamed tissue may promote autoimmune reactions by creation of altered host epitopes.

Methylation of ethanolamine groups in phosphoethanolamines is relevant for L-arginine insertion in lipid membranes

The interaction of L-arginine with membranes composed by phospholipids with different degrees of methylation of the ethanolamine group was studied by means of surface and dipole potentials and surface pressure variations. The subsequent methylation of the amine head group appears to hinder the synergic response of the adsorption observed in phosphatidylethanolamine membranes. The kinetics of the binding process denotes that the methyl groups are relevant in regulating the specific interaction of the amino acid with the interface by hydrogen bonds. This response can be put in correlation with the function of signal transduction assigned previously to methyl lipids [F. Hirata and J. Axelrod, 1980] and appears to be relevant to understand the mechanism of insertion of arginine residues in peptides of biological interest.

Identification of arginine residues in peptides by 2D-IR echo spectroscopy.

The CN stretching vibrations of the guanidyl group in the arginine dipeptide side chain are examined by two-dimensional infrared spectroscopy. In D(2)O, the spectra display two distinct diagonal peaks. These nearly degenerate modes undergo ultrafast energy transfer. The energy-transfer rate was determined directly from the 2D-IR spectra to be 1/2.1 ps(-1). The cross peaks in 2D-IR arising from the energy transfer provide a definitive identification of arginine in larger proteins. An example of arginine in the transmembrane protein M2, found in influenza viruses, is given.

Variation in proton affinity of the guanidino group between free and blocked arginine

In this paper, the analog of arginine residues in peptides was synthesized and characterized by ESI-MS/MS (electrospray ionization with tandem mass spectrometry), (31)P NMR, (1)H NMR, IR and high-resolution mass spectrometry. When the Todd reaction activity of the guanidino group in free arginine and the arginine peptide analog were compared, it was found that the proton affinity of the guanidino group was decreased when both the N- and the C-terminal were blocked. As a result, the guanidino group of arginine residues in peptides could be phosphorylated under the Todd reaction condition, but not the free arginine. This result was further proved by the theoretical calculation of their proton affinity.

Derivatisation of arginine residues with malondialdehyde for the analysis of peptides and protein digests by LC‐ESI‐MS/MS

In this study a selective tagging strategy for the derivatisation of arginine residues in peptides is presented. It is based on the reaction of the guanidine group of the arginine side-chain with malondialdehyde (MDA) under strongly acidic conditions, in which a stable pyrimidine ring is formed. The reaction conditions have been optimised so that quantitative modification can be achieved for a variety of peptides. The label has a strong influence on the polarity and basicity of the arginine side-chain and thus on the chromatographic and mass spectrometric properties of arginine-containing peptides. For example, retention, particularly of small and polar peptides as well as arginine-rich peptides, is significantly increased by derivatisation, and therefore sensitivity is also enhanced in liquid chromatography-mass spectrometry (LC-MS). The arginine side-chain also has a strong impact on the fragmentation behaviour of peptides in tandem mass spectrometry. This has been investigated for standard peptides for which, in some cases, significantly more fragment ions were formed after derivatisation. Finally, the method was tested for tryptic digests of standard proteins to demonstrate how the tagging strategy can give improved or complementary information for protein identification.

Selective molecular recognition of arginine by anionic salt bridge formation with bis-phosphate crown ethers: implications for gas phase peptide acidity from adduct dissociation

Arginine forms a stable noncovalent anionic salt bridge complex with DP (a crown ether which contains two endocyclic dialkylhydrogenphosphate esters). Abundant adduct formation with DP is observed for complexes with arginine, YAKR, HPPGFSPFR, AAKRKAA, RR, RPPGFSPFR, RYLGYL, RGDS, and YGGFMRGL in electrospray ionization mass spectrometry (ESI-MS) experiments. DFT calculations predict a hydrogen bonded salt bridge structure with a protonated guanidinium flanked by two deprotonated phosphates to be the lowest energy structure. Dissociation of DP/peptide adducts reveals that, in general, the relative gas phase acidity of a peptide is dependent on peptide length, with longer peptides being more acidic. In particular, peptides that are six residues or more in length can stabilize the deprotonated C-terminus by extensive hydrogen bonding with the peptide backbone. Dissociation of DP/peptide complexes often yields the deprotonated peptide, allowing for the facile formation of anionic peptides that otherwise would be difficult to generate in high abundance. Although DP has a preference for binding to arginine residues in peptides, DP is also observed to form less abundant complexes with peptides containing multiple lysines. Lys-Xxx-Lys and Lys-Lys sequences form low abundance anionic adducts with DP. For example, KKKK exclusively forms a double adduct with one net negative charge on the complex.

Fragmentation pathways of N G-methylated and unmodified arginine residues in peptides studied by ESI-MS/MS and MALDI-MS

Protein methylation at arginine residues is a prevalent posttranslational modification in eukaryotic cells that has been implicated in processes from RNA-binding and transporting to protein sorting and transcription activation. Three main forms of methylarginine have been identified: N G-monomethylarginine (MMA), asymmetric N G,N G-dimethylarginine (aDMA), and symmetric N G,N′ G-dimethylarginine (sDMA). To investigate gas-phase fragmentations and characteristic ions arising from methylated and unmodified arginine residues in detail, we subjected peptides containing these residues to electrospray triple-quadrupole tandem mass spectrometry. A variety of low mass ions including (methylated) ammonium, carbodiimidium, and guanidinium ions were observed. Fragment ions resulting from the loss of the corresponding neutral fragments (amines, carbodiimide, and guanidine) from intact molecular ions as well as from N- and C-terminal fragment ions were also identified. Furthermore, the peptides containing either methylated or unmodified arginines gave rise to abundant fragment ions at m/ z 70, 112, and 115, for which cyclic ion structures are proposed. Electrospray ionization tandem mass spectra revealed that dimethylammonium ( m/ z 46) is a specific marker ion for aDMA. A precursor ion scanning method utilizing this fragment ion was developed, which allowed sensitive and specific detection of aDMA-containing peptides even in the presence of a five-fold excess of phosphorylase B digest. Interestingly, regular matrix-assisted laser desorption/ionization mass spectra recorded from aDMA- or sDMA-containing peptides showed metastable fragment ions resulting from cleavages of the arginine side chains. The neutral losses of mono- and dimethylamines permit the differentiation between aDMA and sDMA.

Fragmentation pathways of NG-methylated and unmodified arginine residues in peptides studied by ESI-MS/MS and MALDI-MS

Fragmentation pathways of NG-methylated and unmodified arginine residues in peptides studied by ESI-MS/MS and MALDI-MS

Fyn tyrosine kinase participates in the compact myelin sheath formation in the central nervous system.

The cellular mechanisms for spiral wrapping and compaction of myelin sheaths by oligodendrocytes are not known yet. In this study, we examined the role of fyn tyrosine kinase, which could be responsible for molecular events during the stage of myelination in the CNS. Western blot and immunohistochemical analyses revealed that fyn-deficient mice have significantly lower levels of myelin basic protein (MBP), which is required for intracellular membrane adhesion parts so-called major dense line (MDL) and thought to be essential for the stability of myelin sheath. Electron microscopy verified that the myelin ultrastructure could be used to distinguish fyn-deficient mice from wild-type mice, showing a thin and redundant myelin sheath in the corpus callosum. Further, the electron-dense 'major' line in myelin from the purified myelin fractions remained condensed, and myelin compaction was split opened in fyn-deficient mice. To determine whether there was a change in the microheterogeneity of MBP due to a post-translational event we first investigated peptidylarginine deiminase (PAD), which is an enzyme that converts arginine residues in peptides to citrulline residues. PAD immunoreactivity was observed both in the myelin from fyn-deficient and wild-type mice. By Western blot analysis we found an increase of the citrullined form of MBP. In addition, MBP from fyn-deficient mice did weakly induce vesicle aggregation properties of MBP-mediated adhesion. We concluded that although oligodendrocytes from fyn-deficient mice are able to wrap around the axon, they are unable to form compact myelin due to decreased MBP level and the presence of increased citrullinated MBP.

Biological and antigenic characterization of three BApNA‐hydrolyzing proteases from the culture supernatant of Porphyromonas gingivalis

Biological and antigenic distinction of 3-N-alpha-benzoyl-DL-arginine p-nitroanilide (BApNA)-hydrolyzing proteases (Pase-B, Pase-C and Pase-S) isolated from the culture supernatant of Porphyromonas gingivalis were determined. Immunoblotting analysis of these enzymes using a polyclonal antibody against Pase-S, which is a soluble, clostripain-like protease, revealed immunological distinction from Pase-C, a vesicle-associated thiol-protease. Pase-B, a vesicle-associated clostripain-like protease, reacted with the antibody and was also found to contain a considerable amount of carbohydrates in its structure, as compared with the others. Analysis of N-terminal amino acids of Pase-B provided a sequence not found in the SwissProt data bank or previously reported as N-terminal sequences of proteases from P. gingivalis. Pase-S, resembling Pase-B in its hydrolytic specificity, cleaved only arginine residues of peptides and degraded type IV and denatured type I collagen. Pase-C hydrolyzed N-alpha-benzoyl-DL-lysine p-nitroanilide and showed the strongest capacity of degrading native type I collagen. This enzyme was also the only one to possess hemagglutinating activity. Our findings suggest that Pase-S from P. gingivalis is less active than Pase-C and that the enzyme may be an isozyme of Pase-B.

Isolation and properties of an angiotensin II-cleaving peptidase from mesquite pollen.

Biochemical studies of pollen proteins have been focused, primarily, in investigating their roles as allergens. These molecules, some of which have enzymatic activity, act as antigens and initiate the production of IgE antibodies, leading to allergic and/or asthmatic responses. Included in this mixture of proteins are proteinases which, although they may or may not be allergenic, could still be involved in airway dysfunction. We have isolated an arginine-specific endopeptidase to homogeneity from mesquite (Prosopis velutina) pollen, a known wind-borne allergen, which has a molecular mass near 84 kDa by NaDodSO4-gel electrophoresis, a pH optimum in the neutral to alkaline range, and a requirement for Ca2+ for stabilization. The enzyme is inhibited by diisopropyl fluorophosphate (DFP) and N-p-tosyl-L-lysine chloromethylketone but not by N-p-tosyl-L-phenylalanine chloromethylketone, EDTA, or iodoacetamide. It was also not inhibited by human plasma proteinase inhibitors nor several other naturally occurring plant and animal inhibitors. Cleavage by the endopeptidase was primarily on the carboxy-terminal side of arginine residues in peptides, whereas proteins such as kallikrein and prothrombin were only activated and/or degraded extremely slowly. Several bioactive peptides that may be involved in maintaining normal lung function were readily fragmented, including angiotensin II, a vasoconstrictor, and atrial natriuretic peptide, a modulator of vascular permeability, both of which were rapidly cleaved at low enzyme:substrate molar ratios. Thus, the pollen endopeptidase could be involved in exacerbating the development of asthma by inactivating bioactive peptides that have ameliorating effects in maintaining lung airway homeostasis.

New nitroxide reagents for the selective spin-labelling at the guanidino moiety of arginine residues in peptides and proteins

The synthesis of three glyoxal derivatives carrying a nitroxide moiety was achieved by the α-oxidation of the corresponding methyl ketones using selenium dioxide. These reagents appear to be useful for the specific spin-labelling at arginine side-chains. Sixteen peptides and proteins were successfully labelled and the rotational correlation times, as deduced from ESR spectra, were correlated with the molecular weights of the target macromolecules.

Student Forum

While a group of oral commensals have been implicated in the aetiology of chronic periodontitis; the asaccharolytic Gram negative anaerobe Porphyromonas gingivalis is most commonly reported to be associated with severe forms of the disease. Although a variety of human tissues can produce a number of peptidylarginine deiminase (PAD), enzymes that convert peptide bound arginine residues to citrulline, P. gingivalis is one of the few prokaryotes known to express PAD. Protein and peptide citrullination are important in the development of rheumatoid arthritis and in recent years a number of authors have suggested a possible link between periodontitis and rheumatoid arthritis (RA). Indeed, some have linked P. gingivalis directly to RA via the action of PAD. Accordingly, the prime purpose of this study was to further characterise PAD in P. gingivalis cells particular emphasis on substrate specificity, using arginine containing peptides and RA relevant proteins.P. ginigvalis W50 was anaerobically cultured in BHI broth, cells harvested and resuspended in assay buffer. A colourimetric assay was developed to measure citrulline and employed to determine enzyme activity using the substrate BAEE. The assay was employed to investigate the effects of environmental pH and temperature on activity. Citrullination of BAEE by sonicated cells allowed the proportion of intracellular enzyme to be estimated. Enzyme specificity and substrate preference were investigated by using various arginine containing peptides, proteins and arginine analogues, as substrates and measuring the rate of citrullination. The influence of gingipains on citrullination was assessed by measuring the rates of citrullination of bovine serum albumin in the presence of protease inhibitors.Enzyme activity decreased by 13% following exposure of cells to 60 °C for 10 min. A comparison of intact and disrupted cells indicated that 90% of PAD activity is cell surface associated and the remainder cytoplasmic. Optimal pH for enzyme activity was between pH 7.5 and 8. All small arginine-containing peptides were citrullinated with reaction rates faster than that for free arginine with rates that varied with arginine residue position and number. Arginine analogues exhibited minimal effect and influence when tested as either substrates or competitive inhibitors. Cells were able to citrullinate yeast enolase, human vimentin and fibrin at varying rates. All proteins were modified at slower rates than those for peptide substrates. Inhibition of gingipains had no influence on the rate of protein citrullination.P. gingivalis PAD is a primarily cell surface associated, heat stable, enzyme that exhibits optimal activity under alkaline conditions similar to those present in the inflammatory environment. The enzyme displays high specificity for arginine residues in peptides and modified arginine in all positions and the gingipains did not influence the rate of protein citrullination. The ability of the enzyme to convert arginine residues in all proteins tested would indicate that its presence in inflamed tissue may promote autoimmune reactions by creation of altered host epitopes.