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Therapeutical human proteins
Nicole Moguilevsky, PhD
Myeloperoxidase: from structure to function
Mammalian peroxidases belong to a superfamily as both the primary and tertiary structure and the prosthetic group are unique. The mammalian peroxidases are involved in defense mechanisms against pathogenic bacteria, viruses, fungi and metazoan parasites (e.g. myeloperoxidase, MPO, eosinophil peroxidase, EPO, and lactoperoxidase, LPO) and biosynthesis (e.g. thyroid peroxidase, TPO). However, MPO differs from the other mammalian peroxidases by its distinct catalytic and spectroscopic features. MPO is the only human peroxidase, which is able to catalyze the oxidation of chloride to the bactericidal agent hypochlorous acid at a significant rate.
The question arose about the particularity of the MPO structure, leading to such powerful oxidative properties. The nature of the heme group and its binding mode with the protein chain has been studied. By site-directed mutagenesis, mutations in the close environment of the heme have been created, the expression of recombinant human MPO and bovine LPO established in Chinese hamster ovary (CHO) cell allowed us to conduct detailed investigations on the structure-function relationship and to establish the difference between MPO and the other mammalian peroxidases.
Recently, spectroscopic investigations indicated that two ester bonds link the heme group in MPO, LPO and EPO to protein carboxyl groups, maintaining the heme in a planar conformation. However, the MPO possesses a third covalent bond between the heme group and the protein moiety, which we were able to identify, as well as the residue involved in this bond, the methionine 409. This is consistent with the fact that a methionine at this position is the only residue, which is not conserved throughout the homologous mammalian peroxidase family. The three covalent linkages lead to a distortion of the heme plan, causing a bowed shape of the heme, probably responsible for the high oxidation potential of MPO. (in collaboration with Pr R.Wever, University of Amsterdam and Pr. R. Ferrari, University of Turino).
Myeloperoxidase and inflammation
The remarkably high oxidation potential of MPO can also bring some negative consequences, when the enzyme is present in high concentrations. These last years, an increasing number of pathologies have been associated directly or indirectly to the reactive oxygen species, in particular in chronic inflammatory diseases, such as rhumatoïd arthritis, which destroys joints, atherosclerosis, characterized by the obstruction of the arteries and necrotizing vasculitides, diseases in which autoantibodies against myeloperoxidase cause kidneys and lung destruction.
We propose that the interaction between neutrophils and macrophages induces a state of chronic inflammation, which contributes to the disease state. One of the central players is myeloperoxidase. Our studies showed that myeloperoxidase released from neutrophils, induces macrophages to secrete IL-I, IL-6, IL-10 and TNF and enhance the production of reactive oxygen intermediates.
A possible consequence of neutrophilic myeloperoxidase-macrophage interactions in relation to the chronicity of inflammation is arthritis.In order to investigate the above interaction, an experimental arthritis was induced in the rat joint. Myeloperoxidase could not initiate arthritis, however myeloperoxidase injected after swelling and erythema has returned to the baseline, could induce a dose-dependant exacerbation of periarticular swelling and production of TNFa (in collaboration with Dr D. Lefkowitz, USA).
Inflammation is also strongly associated with an increased risk of atherosclerosis. Atherosclerosis and its consequence, the cardiovascular diseases, constitute the first cause of mortality in the Western world. Recently, the presence of oxidized lipoproteins of low density (oxLDL)and of active myeloperoxidase has been proved in the atherosclerotic plaques, which obstruct the lumen of the arteries. Again, we have made the hypothesis that myeloperoxidase released from the neutrophils could interact with monocytes/macrophages and with the cells constituting the arteries wall, leading to their activation and to the production of reactive oxygen species and cytokines.
The origin of oxidized LDL remains unclear, although these phenomena can lead to the oxidation of the (LDL) and this oxidation is a prerequisite for the accumulation of cholesterol.
We have examined the oxidation of LDL by the myeloperoxidase system and produced monoclonal antibodies against different oxidized forms of LDL. Using these antibodies, we were able to analyze the susceptibility to oxydation of LDL ex vivo as well as to determine the level of circulating oxidized LDL. In a panel of 27 patients, we established a clear relation between the susceptibility of LDL to MPO oxidation and the percentage of blood monocytes. A subset of blood monocytes (CD14+/CD16+) has been identified with a decrease of MPO content, indicating a release of MPO into the circulation. In conclusion, since complexes of LDL-MPO have been previously described in plasma, our test could reflect the preformed complexes in blood stream, leading to accelerated LDL oxidation.
Moreover, the level of autoantibodies specific for LDL oxidized by the myeloperoxidase system could be measured, as well as the level of circulating oxidized LDL.
Between the different autoimmune diseases, the necrotizing vasculitis is of particular interest. Indeed, the treatments of autoimmune diseases, performed with the use of immunosuppressive drugs are difficult to control and present a high degree of risk.
We are now able to produce under the Good Manufactoring Practice (GMP) conditions the first pure autoantigen, that is the human recombinant myeloperoxidase. The extracorporeal specific immuno-adsorption of autoantibodies on recombinant myeloperoxidase could lead to a remission of the disease by suppressing the factor, which maintains the disease and validate the general concept of the treatment. The clinical trial will be performed in collaboration with University Hospitals of Cambridge (UK), Maastricht (NL) and Regensburg (G).
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