C-terminal Arginine Residue Research Articles

Identification of a new genetic variant (G231N, E232T, N235D) of peptidylarginine deiminase from P. gingivalis in advanced periodontitis.

Chronic periodontitis (CP), an inflammatory disease of periodontal tissues driven by a dysbiotic subgingival bacterial biofilm, is also associated with several systemic diseases, including rheumatoid arthritis (RA). Porphyromonas gingivalis, one of the bacterial species implicated in CP as a keystone pathogen produces peptidyl arginine deiminase (PPAD) that citrullinates C-terminal arginine residues in proteins and peptides. Autoimmunity to citrullinated epitopes is crucial in RA, hence PPAD activity is considered a possible mechanistic link between CP and RA. Here we determined the PPAD enzymatic activity produced by clinical isolates of P. gingivalis, sequenced the ppad gene, and correlated the results with clinical determinants of CP in patients from whom the bacteria were isolated. The analysis revealed variations in PPAD activity and genetic diversity of the ppad gene in clinical P. gingivalis isolates. Interestingly, the severity of CP was correlated with a higher level of PPAD activity that was associated with the presence of a triple mutation (G231N, E232T, N235D) in PPAD in comparison to W83 and ATCC 33277 type strains. The relation between mutations and enhanced activity was verified by directed mutagenesis which showed that all three amino acid residue substitutions must be introduced into PPAD expressed by the type strains to obtain the super-active enzyme. Cumulatively, these results may lead to the development of novel prognostic tools to assess the progress of CP in the context of associated RA by analyzing the ppad genotype in CP patients infected with P. gingivalis.

A protease activity-based machine-learning approach as a complementary tool for conventional diagnosis of diarrhea-predominant irritable bowel syndrome.

Irritable bowel syndrome (IBS) has no clinically accepted biomarkers even though it affects a large number of individuals worldwide. To address this lack of understanding, we evaluated peptidase activity in fecal samples from 35 patients with diarrheal IBS without symptom exacerbation (IBS-n) and 35 healthy subjects using a library of 384 fluorescent enzymatic substrate probes. IBS-n patients had high trypsin-like peptidase activity for cleavage of C-terminal lysine and arginine residues and low elastase-like activity for cleavage of C-terminal serine and glycine residues. These fluorescent probe library data, together with diagnostic machine-learning techniques, were able to accurately predict IBS-n. This approach can be used to diagnose diseases where no clinically accepted biomarkers exist, in which fecal enzyme activity is altered and also suggests that the development of new therapies targeting enzyme activities is possible.

Structure of AQEE-30 of VGF Neuropeptide in Membrane-Mimicking Environments.

AQEE-30 is one of the VGF peptides, which are derived from the VGF polypeptide precursor, and related to various physiological phenomena including neuroprotective effects in Huntington's disease and amyotrophic lateral sclerosis (ALS). Although various functions of AQEE-30 have been reported so far, the structure of this peptide has not been reported yet. In this study, the structure of human AQEE-30 was investigated in hexafluoroisopropanol (HFIP) and dodecyl phosphocholine (DPC) micelle solutions, using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. CD results showed that AQEE-30 had a partial helical structure in aqueous buffer, and the helical structure was stabilized in the HFIP and DPC micelle solutions. The 3D structures determined by NMR spectroscopy showed that AQEE-30 adopted mainly α-helical structure in both the HFIP and DPC micelle solutions. The surface of AQEE-30 showed that it was predominantly negatively charged. The residues from 601 to 611 in both the HFIP and DPC micelle solutions showed amphiphilicity with four negatively charged residues, glutamate. The C-terminal consecutive arginine residues formed a partial positively charged surface. These results suggest an α-helical active structure of AQEE-30 in the cell-membrane environment.

Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

Lack of simple and robust methods to determine complement activation in human serum induced by antigen–antibody complexes is a major hurdle for monitoring therapeutic antibody drug quality and stability. Dezamizumab is a humanized IgG1 monoclonal antibody that binds to serum amyloid P component (SAP) for potential treatment of systemic amyloidosis. The mechanism of action of Dezamizumab includes the binding of SAP, complement activation through classical pathway, and phagocytosis; however, the steps in this process cannot be easily monitored. We developed two novel methods to determine Dezamizumab-SAP complex-induced complement activation. Complement component 3 (C3) depletion was detected by homogeneous time-resolved fluorescence (HTRF), and C3a desArg fragment, formed after the cleavage of C3 to yield C3a followed by removal of its C-terminal arginine residue, was determined using Meso Scale Discovery (MSD) technology. We found that the presence of both Dezamizumab and SAP was required for complement activation via both methods. The optimal molar ratio of Dezamizumab:SAP was 6:1 in order to obtain maximal complement activation. The relative potency from both methods showed a good correlation to Dezamizumab-SAP-dependent complement component 1q (C1q) binding activity in Dezamizumab thermal-stressed samples. Both SAP and C1q binding, as determined by surface plasmon resonance and the two complement activation potency methods described here, reflect the mechanism of action of Dezamizumab. We conclude that these methods can be used to monitor Dezamizumab quality for drug release and stability testing, and the novel potency methods reported here can be potentially used to evaluate complement activity induced by other antigen–antibody complexes.

A Systematic Review of Molecular Imaging Agents Targeting Bradykinin B1 and B2 Receptors.

Kinins, bradykinin and kallidin are vasoactive peptides that signal through the bradykinin B1 and B2 receptors (B1R and B2R). B2R is constitutively expressed in healthy tissues and mediates responses such as vasodilation, fluid balance and retention, smooth muscle contraction, and algesia, while B1R is absent in normal tissues and is induced by tissue trauma or inflammation. B2R is activated by kinins, while B1R is activated by kinins that lack the C-terminal arginine residue. Perturbations of the kinin system have been implicated in inflammation, chronic pain, vasculopathy, neuropathy, obesity, diabetes, and cancer. In general, excess activation and signaling of the kinin system lead to a pro-inflammatory state. Depending on the disease context, agonism or antagonism of the bradykinin receptors have been considered as therapeutic options. In this review, we summarize molecular imaging agents targeting these G protein-coupled receptors, including optical and radioactive probes that have been used to interrogate B1R/B2R expression at the cellular and anatomical levels, respectively. Several of these preclinical agents, described herein, have the potential to guide therapeutic interventions for these receptors.

Features of Tryptic Peptides Providing Their Detection and Identification by MALDI Mass Spectrometry

Characteristics of tryptic peptides that provide the detection and identification of these compounds and corresponding proteins by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with α-cyano-4-hydroxycinnamic acid as a matrix are determined and discussed. Twenty-five proteins are identified; the features of reliably identified peptides are compared to those of the compounds that might be produced by trypsinolysis but were not detected. Two key factors enhancing a possibility of the detection of tryptic peptides are determined. One refers to the basicity of peptides in the gas phase, which is the highest for peptides with the C-terminal arginine residue; this reason for the emergence of significant analytical signals is well known. Another factor contributing to the ionization of peptides in study under MALDI conditions is their hydrophilicity. This result did not correlate with the most conclusions of earlier relevant studies discussed in the article.

Chemical synthesis and racemic crystallization of rat C5a-desArg

The deletion of the C-terminal arginine of the anaphylatoxin protein C5a reduces it receptor binding affinity. Understanding how C-terminal arginine affects the structure and bioactivity of C5a is important for the development of C5a C-terminal mimics as drug candidates. Herein, we report the total chemical synthesis of rat C5a and its d-enantiomer with its C-terminal arginine deleted, namely l-rC5a-desArg and d-rC5a-desArg. The structure of rC5a-desArg was then determined by racemic crystallography for the first time. The C-terminal residues of rC5a-Arg were found to expand from the fourth helix in a continuous helical conformation. This C-terminal conformation is significantly different from that of the previously reported full-length of C5a, indicating that the deletion of C-terminal arginine residue could result in the destruction of a positively charged surface formed by two adjacent Arg residues in C5a.

The secreted Candida albicans protein Pra1 disrupts host defense by broadly targeting and blocking complement C3 and C3 activation fragments

Candida albicans the most frequently isolated clinical fungal pathogen can cause local as well as systemic and life-threatening infections particularly in immune-compromised individuals. A better and more detailed understanding how C. albicans evades human immune attack is therefore needed for identifying fungal immune-evasive proteins and develop new therapies. Here, we identified Pra1, the pH-regulated C. albicans antigen as a hierarchical complement inhibitor that targets C3, the central human complement component. Pra1 cleaved C3 at a unique site and further inhibited effector function of the activation fragments. The newly formed C3a-like peptide lacked the C-terminal arginine residue needed for C3a-receptor binding and activation. Moreover, Pra1 also blocked C3a-like antifungal activity as shown in survival assays, and the C3b-like molecule formed by Pra1 was degraded by the host protease Factor I. Pra1 also bound to C3a and C3b generated by human convertases and blocked their effector functions, like C3a antifungal activity shown by fungal survival, blocked C3a binding to human C3a receptor-expressing HEK cells, activation of Fura2-AM loaded cells, intracellular Ca2+ signaling, IL-8 release, C3b deposition, as well as opsonophagocytosis and killing by human neutrophils. Thus, upon infection C. albicans uses Pra1 to destroy C3 and to disrupt host complement attack. In conclusion, candida Pra1 represents the first fungal C3-cleaving protease identified and functions as a fungal master regulator of innate immunity and as a central fungal immune-escape protein.

Structure‐function relationships in thrombin‐activatable fibrinolysis inhibitor

Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator in the balance of coagulation and fibrinolysis. TAFI is a metallocarboxypeptidase that circulates in plasma as zymogen. Activated TAFI (TAFIa) cleaves C-terminal lysine or arginine residues from peptide substrates. The removal of C-terminal lysine residues from partially degraded fibrin leads to reduced plasmin formation and thus attenuation of fibrinolysis. TAFI also plays a role in inflammatory processes via the removal of C-terminal arginine or lysine residues from bradykinin, thrombin-cleaved osteopontin, C3a, C5a and chemerin. TAFI has been studied extensively over the past three decades and recent publications provide a wealth of information, including crystal structures, mutants and structural data obtained with antibodies and peptides. In this review, we combined and compared available data on structure/function relationships of TAFI.

Crystal structure of Porphyromonas gingivalis peptidylarginine deiminase: implications for autoimmunity in rheumatoid arthritis

BackgroundPeriodontitis (PD) is a known risk factor for rheumatoid arthritis (RA) and there is increasing evidence that the link between the two diseases is due to citrullination by the unique...

PAS-cal: A repetitive peptide sequence calibration standard for MALDI mass spectrometry.

Mass spectrometers equipped with matrix-assisted laser desorption/ionization (MALDI-MS) require frequent multipoint calibration to obtain good mass accuracy over a wide mass range and across large numbers of samples. In this study, we introduce a new synthetic peptide mass calibration standard termed PAS-cal tailored for MALDI-MS based bottom-up proteomics. This standard consists of 30 peptides between 8 and 37 amino acids long and each constructed to contain repetitive sequences of Pro, Ala and Ser as well as one C-terminal arginine residue. MALDI spectra thus cover a mass range between 750 and 3200 m/z in MS mode and between 100 and 3200 m/z in MS/MS mode. Our results show that multipoint calibration of MS spectra using PAS-cal peptides compares well to current commercial reagents for protein identification by PMF. Calibration of tandem mass spectra from LC-MALDI experiments using the longest peptide, PAS-cal37, resulted in smaller fragment ion mass errors, more matching fragment ions and more protein and peptide identifications compared to commercial standards, making the PAS-cal standard generically useful for bottom-up proteomics.

Modified Cysteine-Deleted Tachyplesin (CDT) Analogs as Linear Antimicrobial Peptides: Influence of Chain Length, Positive Charge, and Hydrophobicity on Antimicrobial and Hemolytic Activity

Due to increasing resistance of bacteria to traditional antibiotics, antimicrobial peptides are being investigated as a promising alternative. Tachyplesin, an antimicrobial peptide isolated from horseshoe crab, inhibits the growth of many different types of bacteria with its ability to permeabilize the cell membrane. Starting with a previously reported linear tachyplesin analog lacking cysteine (cysteine-deleted tachyplesin, CDT, KWFRVYRGIYRRR-CONH2), this study examines the systematic deletion of the C-terminal arginines and the N-terminal lysine, addition of positively charged N-and C-terminal residues, replacement of arginine with similarly-charged lysine, and replacement of hydrophobic residues with aliphatic, aromatic, fluoro-substituted aromatic, and bicyclic amino acids to examine effects on activity. The 16 modified CDT analogs were tested for their ability to disrupt model liposomes, and minimum inhibitory concentrations were determined for gram-positive and gram-negative bacterial strains. Hemolytic activity also was assessed. Overall, results indicate that elimination of two C-terminal arginine residues results in a peptide ([des-Arg12,13]CDT) with preserved antimicrobial activity but a reduction in hemolysis, a selectivity desirable for a therapeutic agent. Additional deletion was not tolerated, nor was addition of residues at the termini. Analysis of the 16 analogs also reveals the importance of hydrophobicity, not necessarily aromaticity, as an analog with hydrophobic isoleucine residues placed throughout the sequence ([Ile2,3,6,10]CDT) displayed comparable antimicrobial activity to CDT with lower hemolysis, representing a promising antimicrobial peptide with lowered toxicity.

Influence of kinin peptides on monocyte-endothelial cell adhesion

Adhesion of leukocytes to vascular endothelium in response to proinflammatory mediators is an important component of the overall inflammatory reaction. In the current work, we used a retinoic acid-differentiated human promonocytic cell line, U937 and a human microvascular endothelial cell line, HMEC-1 to analyze the effect of the potent pro-inflammatory bradykinin-related peptides (kinins) on cell adhesion. Bradykinin (BK) and kinin metabolites without the C-terminal arginine residue enhanced adhesion of the monocyte-like cells to fibronectin and to the HMEC-1 cells. Expression of adhesion proteins on the surface of both cell types was altered by the kinin peptides. In the monocyte-like cells, expression of CD11b, a subunit of Mac-1 integrin, was significantly increased whilst in the endothelial cells, a strong increase in the production of intercellular adhesion molecule 1 (ICAM-1) was observed. The positive bradykinin-induced effect on the cell-cell interaction was reversed by a carboxypeptidase inhibitor (MGTA), hence we suspected a significant role of the des-Arg kinin metabolites, which acted through the kinin receptor type 1. Indeed, the expression of this receptor was up-regulated not only by agonists but also by interferon-γ and bradykinin. Kinin peptides also regulated signal transducer and activator of transcription proteins (STATs) activated by cytokines. Taken together, the above observations support our hypothesis that kinins stimulate monocyte adhesion to the vessel wall, especially during pathological states of the circulatory system accompanied by proinflammatory cytokine release.

Structural and functional characterization of human and murine C5a anaphylatoxins.

Complement is an ancient part of the innate immune system that plays a pivotal role in protection against invading pathogens and helps to clear apoptotic and necrotic cells. Upon complement activation, a cascade of proteolytic events generates the complement effectors, including the anaphylatoxins C3a and C5a. Signalling through their cognate G-protein coupled receptors, C3aR and C5aR, leads to a wide range of biological events promoting inflammation at the site of complement activation. The function of anaphylatoxins is regulated by circulating carboxypeptidases that remove their C-terminal arginine residue, yielding C3a-desArg and C5a-desArg. Whereas human C3a and C3a-desArg adopt a canonical four-helix bundle fold, the conformation of human C5a-desArg has recently been described as a three-helix bundle. Here, the crystal structures of an antagonist version of human C5a, A8(Δ71-73), and of murine C5a and C5a-desArg are reported. Whereas A8(Δ71-73) adopts a three-helix bundle conformation similar to human C5a-desArg, the two murine proteins form a four-helix bundle. A cell-based functional assay reveals that murine C5a-desArg, in contrast to its human counterpart, exerts the same level of activition as murine C5a on its cognate receptor. The role of the different C5a conformations is discussed in relation to the differential activation of C5a receptors across species.

A plain method of prediction of visibility of peptides in mass spectrometry with electrospray ionization

A new method for screening of essential peptides for protein detection and quantification analysis in the direct positive electrospray mass spectrometry has been proposed. Our method is based on the prediction of the normalized abundance of the mass spectrometric peaks using a linear regression model. This method has the following limitations: (i) selected peptides should be taken so that at pH 2.5 the tested peptides must be presented mainly as the 2+ and 3+ ions; (ii) only peptides having C-terminal lysine or arginine residues are considered. The amino acid composition of the peptide, the peptide concentration, the ratio of the polar surface of peptide to common surface and ratio of the polar volume to common volume are used as independent variables in equation. Several combinations of variables were considered and the best linear regression model had a determination coefficient in leave-one-out validation procedure equal 0.54. This model confidently discriminates peptides with high response ability and peptides with low response ability, and therefore it allows to select only the most promising peptides. This screening method, a plain and fast, can be successfully applied to reduce the list of observed peptides.

Unblocking the sink: improved CID-based analysis of phosphorylated peptides by enzymatic removal of the basic C-terminal residue.

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H3PO4), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H3PO4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H3PO4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available. Figureᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-013-0770-2) contains supplementary material, which is available to authorized users.

SUPPRESSOR OF LLP1 1‐mediated C–terminal processing is critical for CLE19 peptide activity

Cell-to-cell communication is essential for the coordinated development of multicellular organisms. Members of the CLAVATA3/EMBRYO-SURROUNDING REGION-RELATED (CLE) family, a group of small secretory peptides, are involved in these processes in plants. Although post-translational modifications are considered to be indispensable for their activity, the detailed mechanisms governing these modifications are not well understood. Here, we report that SUPPRESSOR OF LLP1 1 (SOL1), a putative Zn²⁺ carboxypeptidase previously isolated as a suppressor of the CLE19 over-expression phenotype, functions in C-terminal processing of the CLE19 proprotein to produce the functional CLE19 peptide. Newly isolated sol1 mutants are resistant to CLE19 over-expression, consistent with the previous report (Casamitjana-Martinez, E., Hofhuis, H.F., Xu, J., Liu, C.M., Heidstra, R. and Scheres, B. (2003) Curr. Biol. 13, 1435-1441). As expected, our experiment using synthetic CLE19 peptide revealed that the sol1 mutation does not compromise CLE signal transduction pathways per se. SOL1 possesses enzymatic activity to remove the C-terminal arginine residue of CLE19 proprotein in vitro, and SOL1-dependent cleavage of the C-terminal arginine residue is necessary for CLE19 activity in vivo. Additionally, the endosomal localization of SOL1 suggests that this processing occurs in endosomes in the secretory pathway. Thus, our data indicate the importance of C-terminal processing of CLE proproteins to ensure CLE activities.

Kinin-generating cellular model obtained from human glioblastoma cell line U-373.

Kinins, a group of important pro-inflammatory peptides, are abundantly found in tissues and biological fluids of cancer patients. Bradykinin, the major representative of kinins, induces vascular permeability and, in consequence, promotes tumor expansion. Additionally, the kinin-induced inflammatory responses, especially those mediated by kinin metabolites without the C-terminal arginine residue, lead to enhanced tumor growth. The present study aimed at analyzing the ability of the human glioblastoma cell line U-373, derived from a malignant tumor, to produce kinin peptides. The proteins involved in kinin generation, i.e., the kininogens and the kallikreins, were shown to be expressed in these cells. Moreover, tumor necrosis factor α, a proinflammatory cytokine that mediates tumorigenesis, was found to enhance the expression of enzymes associated with kinin production. The strong binding of kininogen to the cell surface and the enzymatic degradation of this protein by cells suggest the activation of kinin-generating systems. Indeed, glioblastoma cells, pre-treated with tumor necrosis factor α, released kinin peptides from exogenous kininogen. The expression of kinin receptors in these cells was also shown to increase under the influence of this cytokine. Our results suggest that the human glioblastoma cell line U-373 constitutes a good cellular model that can be helpful in cancer research focused on kinin-induced inflammation. Furthermore, our findings can contribute to new approaches in cancer treatment with the use of kinin receptor antagonists and inhibitors of kinin production.

Flavinylation and assembly of succinate dehydrogenase are dependent on the C-terminal tail of the flavoprotein subunit.

Succinate dehydrogenase (SDH) requires a covalent addition of FAD for catalytic function. Mutational analyses of Sdh1 implicate C-terminal region Arg residues involvement in covalent flavinylation and SDH assembly. SDH assembly is dependent on FAD binding to Sdh1 but not covalent binding. These results document the basis for the SDH deficiency and pathology seen with mutations in human Sdh1. The enzymatic function of succinate dehydrogenase (SDH) is dependent on covalent attachment of FAD on the ~70-kDa flavoprotein subunit Sdh1. We show presently that flavinylation of the Sdh1 subunit of succinate dehydrogenase is dependent on a set of two spatially close C-terminal arginine residues that are distant from the FAD binding site. Mutation of Arg(582) in yeast Sdh1 precludes flavinylation as well as assembly of the tetrameric enzyme complex. Mutation of Arg(638) compromises SDH function only when present in combination with a Cys(630) substitution. Mutations of either Arg(582) or Arg(638)/Cys(630) do not markedly destabilize the Sdh1 polypeptide; however, the steady-state level of Sdh5 is markedly attenuated in the Sdh1 mutant cells. With each mutant Sdh1, second-site Sdh1 suppressor mutations were recovered in Sdh1 permitting flavinylation, stabilization of Sdh5 and SDH tetramer assembly. SDH assembly appears to require FAD binding but not necessarily covalent FAD attachment. The Arg residues may be important not only for Sdh5 association but also in the recruitment and/or guidance of FAD and or succinate to the substrate site for the flavinylation reaction. The impaired assembly of SDH with the C-terminal Sdh1 mutants suggests that FAD binding is important to stabilize the Sdh1 conformation enabling association with Sdh2 and the membrane anchor subunits.

Binding of carboxypeptidase N to fibrinogen and fibrin

The ultimate step in the blood coagulation cascade is the formation of fibrin. Several proteins are known to bind to fibrin and may thereby change clot properties or clot function. Our previous studies identified carboxypeptidase N (CPN) as a novel plasma clot component. CPN cleaves C-terminal lysine and arginine residues from several proteins. The activity of CPN is increased upon its proteolysis by several proteases. The aim of this study is to investigate the presence of CPN in a plasma clot in more detail. Plasma clots were formed by adding thrombin, CaCl2 and aprotinin to citrated plasma. Unbound proteins were washed away and non-covalently bound proteins were extracted and analyzed with 2D gel electrophoresis and mass spectrometry. The identification of CPN as a fibrin clot-bound protein was verified using Western blotting. Clot-bound CPN consisted of the same molecular forms as CPN in plasma and its content was approximately 30ng/ml plasma clot. Using surface plasmon resonance we showed that CPN can bind to fibrinogen as well as to fibrin. In conclusion, CPN binds to fibrinogen and is present in a fibrin clot prepared from plasma. Because CPN binds to a fibrin clot, there could be a possible role for CPN as a fibrinolysis inhibitor.