Adhesive Properties and Inflammatory Potential of Citrullinated Myelin Basic Protein Peptide 45-89

Deimination of arginyl residue of myelin basic protein (MBP) reduces cationicity of MBP and impedes the normal myelin membrane assembly. Less ordered structure of MBP is more susceptible to proteolytic attack that may lead to the release of highly immunogenic deiminated peptides into extracellular milieu. We have studied the association of peptides 45-89 derived from citrullinated MBP (C8 isomer) and phosphorylated MBP (C3 isomer) with the myelin lipids in a model membrane system using optical waveguide lightmode spectrometry. The analysis of association/dissociation kinetics to planar lipids under controlled hydrodynamic conditions has shown that MBP 45-89 peptide from citrullinated C8 isomer is less effectively adsorbed on the lipid membrane, than peptide from phosphorylated C3 isomer and packing densities for phosphorylated 45-89 MBP peptide is higher than for citrullinated forms. On the other hand, our results shown that continuous (24h) exposure of mixed oligodendrocyte/microglial cells to peptides 45-89 from MBP-C8 induces apoptosis via mitochondrial pathway. In addition, peptides 45-89 stimulated the secretion of nitric oxide from microglial cells via induction of iNOS and decreased the level of the inhibitory protein IkB, indicating involvement of the transcription factor NF-kB in these processes. Our results suggest that some citrullinated peptides, initially released from oligodendrocytes, might activate microglia, which produces reactive nitrogen species and generates in turn fatal feedbacks that kill oligodendrocyte

Phosphorilation of the main protein myelin and its peptides

The experiments have been performed on the not-pedigree albino laboratory rats. The purpose of the work: revealing the role of the phosphorilation reaction in the main protein myelin proteolysis and study of the action of the obtained protein fragments on the definite receptor apparatus of the neurons. It has been show that the cAMP-dependent proteinkinase, the modified form of the myelin main protein, possesses the more sensibility to the tripsinolysis. Fragments 74-92 and 61-112 cause the modulation of bonding (33H1) haloperydol, and fragments 74-140 and 93-135 bonding (33H1) dilthiazema with the synaptic membranes. Revealation of the receptor-modulating properties of the definite fragments of the main protein of myelin can have an essential significance in understanding of mechanisms of the autoantigen and post-infection sensibilization of thenervous and T-cells called forth by the proteins similar to the main protein of myelin, and in revealation of the etiology of the autoimmune pathologies of the central nervous systemAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Metabotropic glutamate receptor 5 may be involved in macrophage plasticity

Abstract Background Macrophages are a functionally heterogeneous cell population and depending on microenvironments they polarize in two main groups: M1 and M2. Glutamic acid and glutamate receptors may participate in the regulation of macrophage plasticity. To investigate the role of glutamatergic systems in macrophages physiology, we performed the transfection of mGluR5 cDNAs into RAW-264.7 cells. Results Comparative analysis of modified (RAW-mGluR5 macrophages) and non-modified macrophages (RAW-macrophages) has shown that the RAW-mGluR5 macrophages absorbed more glutamate than control cells and the amount of intracellular glutamate correlated with the expression of excitatory amino acid transporters -2 (EAAT-2). Besides, our results have shown that RAW-mGluR5 macrophages expressed a higher level of peroxisome proliferator-activated receptor γ (PPAR-γ) and secreted more IL-10, high mobility group box 1 proteins (HMGB1) and Galectin-3 than control RAW-macrophages. Conclusions We propose that elevation of intracellular glutamate and expression of mGluR5 may initiate the metabolic rearrangement in macrophages that could contribute to the formation of an immunosuppressive phenotype

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Additional file 1: Figure S1. Expression of mGluR5, EAAT-2, PPAR-γ and HMGB1 proteins in control and mGluR5-transfected macrophages. RAW 264.7 cells (RAW-NT) and mGluR5-transfected macrophages (RAW-mGluR5) (5–105 cells per well) were incubated with LPS (100 ng ⁄ ml) or IL-10 (20 nM) for 24 h, followed by the determination of EAAT-2 (b), PPAR-γ (d) and HMGB1 (f) expression by western blot analysis, as described in the “Methods” section. (h) β-Actin was also visualized by Western blotting to confirm equal loading of the fractions. Data shown are representative of three independent experiments. Quantification of EAAT2 blots shown in c, *P < 0.05, vs corresponding RAW-NT cells. Quantification of PPAR-γ shown in e, *P < 0.05, vs corresponding RAW-NT cells, **P < 0.05, vs mGluR5 control. Quantification of HMGB1 blots shown in g, *P < 0.05, vs corresponding RAW-NT cells

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Additional file 2: Figure S2. Effect of LPS and glutamate on NO and IL-10 secretion. RAW 264.7 cells (RAW-NT) and mGluR5-transfected macrophages (RAW-mGluR5) (5–105 cells per well) were incubated with LPS (100 ng ⁄ ml) or glutamate (40 μM) for 24 h, followed by determination of NO (a) and IL-10 (b) secretion, as described in the “Methods” section. Data represented are mean ± SEM of results from four. separate experiments performed in duplicate. *P < 0.05, vs corresponding RAW-NT cells. **P < 0.05, vs RAW-mGluR5 control cells

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Additional file 4: Figure S4. Effect of mGluR5 transfection on the HMGB1 and Gal-3 secretion. RAW 264.7 cells (RAW-NT) and mGluR5-transfected macrophages (RAW-mGluR5) (5–105 cells per well) were incubated with LPS (100 ng⁄ml) or IL-10 (20 nM) for 24 h, followed by the determination of HMGB1 proteins (a) and Gal-3 (b), as described in the “Methods” section. Data represented are mean ± SEM of results from four separate experiments performed in duplicate. *P < 0.05, vs RAW-NT control cells. **P < 0.05, vs RAW-mGlur5 control cells