UNSPECIFIED (2018) UNSPECIFIED Doctoral thesis, The Tamilnadu Dr.M.G.R. Medical University, Chennai.
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Abstract
Differentially expressed proteins were screened in serum of Eales' disease patients by 2DGE and native electrophoresis. The 88 kDa protein in serum, that is differentially expressed in Eales' disease, is composed of haptoglobin, complement C3 and galectin-1. It also contains complement regulators and proteins involved in blood coagulation. Nano LC coupled ESI MS analysis of purified 88 kDa protein confirms it to be a complex of haptoglobin, complement C3 and galectin-1. These proteins were validated by ELISA. Haptoglobin levels were increased; galectin-1 levels were decreased whereas levels of complement C3 were unaltered in Eales' disease. The complement C3 and haptoglobin present in 88 kDa protein complex were identified as interacting partners by MS analysis and western blot. In addition, haptoglobin of Eales' disease patients had shown increased amount of β-(1-6) GlcNAc branching and reduced sialic acid content which were identified by lectin blot and mass spectrometry. The β-(1-6) GlcNAc branching with reduced sialic acid capping indicates the favourable sites of galectin-1 binding. The disease conditions were mimicked in ARPE-19 and HUVEC cells by treating them with haptoglobin and silencing galectin-1. Although complement pathway proteins were observed in serum of Eales' disease patients, due to their unaltered levels, no further investigations were carried out. In serum of Eales' disease patients, haptoglobin phenotype was screened and Hp 2-2 phenotype were widely seen among the groups. The presence of Hp 2-2 phenotype is reported to cause iron mediated oxidative stress. To understand alterations in iron and copper metabolism, levels of iron, ferritin, transferrin and soluble transferrin receptor were estimated in serum of Eales' disease patients. Ferritin levels were found to be elevated and transferrin levels were reduced in Eales' disease patients while levels of iron and soluble transferrin receptors were unaffected. Since copper is also associated to Eales' disease and angiogenesis, levels of CTR1 were estimated and found to be elevated. The Hp 2-2 phenotype of haptoglobin was tested for its anti inflammatory effect on LPS induced ARPE-19 cells. Since RPE forms blood retinal barrier and carries out macrophage like action in the ocular environment, human RPE derived cell line ARPE-19 was used for studying inflammation. Angiogenic potential of haptoglobin, is evaluvated in human endothelial cells. Angiogenic and antiinflammatory effects of haptoglobin are identified in tested concentrations. Haptoglobin activates p38 MAPK and suppresses NFκB. Haptoglobin might act through NFκB to carry out its anti inflammatory function. Galectin-1 was cloned to overexpress and to identify its interacting partners. Annexin, serum peptidase inhibitor, RNA binding protein were found to be interacting with galectin-1. In addition to the above findings, presence of PE35 protein of Mycobacterium tuberculosis were identified in PBMCs' Eales' Disease patients by mass spectrometry. The presence of PE35 gene of Mycobacterium tuberculosis is confirmed by the PCR. Inflammatory roles of PE35 were seen in ARPE -19 cells. It increased the levels of IL-8, MCP-1, TNF-α and also the 88 kDa proteins - complement C3 and galectin-1. To mimic Eales' disease, galectin-1 was silenced and PE35 was treated. In these cells, levels of proinflammatory cytokines, complement C3 and haptoglobin were increased. The levels of these cytokines were found to be elevated in vitreous and in serum of patients with Eales' disease (14, 15, 44, 185). Hence, the inflammation of RPE cells in Eales' disease may be due to the presence of Mycobacterium tuberculosis. This, inflammatory response of RPE cells may eventually leas to the occlusion of the vascular lumen resulting in retinal ischemia. Ischemic retinal vasculitis is commonly seen, in patients with Eales disease and triggers the neo angiogenesis response (23). This is the first work which connects, tubercular proteins, roles of haptoglobin, galectin-1, iron and copper in association with the Eales' disease pathogenesis. CONCLUSION: The 88 kDa protein, a potential biomarker of Eales' disease, is identified as complex of haptoglobin, complement C3 and galectin-1. The complement C3 and haptoglobin are interacting partners and galectin-1 binds to haptoglobin due to decreased sialic acid residues. The levels of haptoglobin were elevated whereas the galectin-1 levels were reduced in serum of Eales' disease patients which might be due to the binding of galectin-1 with haptoglobin. Haptoglobin 2-2 phenotype is widely seen in the population which 171 was found to be anti inflammatory and angiogenic in tested concentrations. It reduces inflammation by suppressing NFκB pathway. In PBMCs of Eales' disease patients, bacterial PE35 protein was identified and its treatment in APRE-19, increases pro inflammatory cytokines. Galectin-1 silencing further amplifies the production of proinflammatory cytokine induced by PE35. Increased ferritin, CTR1, and decreased transferrin were identified in Eales' disease patients. Inflammation caused by PE35 may be the reason for the elevated levels of these proteins. Haptoglobin can be developed as a therapeutic molecule to target inflammation in Eales' disease.
| Item Type: | Thesis (Doctoral) |
|---|---|
| Additional Information: | Ref.No.28209/2011 |
| Uncontrolled Keywords: | Identification, Molecular Cloning and Characterization of Differentially Expressed Proteins Found in the Serum of Patients with Eales' disease. |
| Depositing User: | Subramani R |
| Date Deposited: | 01 Jan 2019 13:51 |
| Last Modified: | 20 Oct 2022 09:10 |
| URI: | http://repository-tnmgrmu.ac.in/id/eprint/10293 |
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