Epigallocatechin Gallate Remodels Fibrils of Lattice Corneal Dystrophy Protein, Facilitating Proteolytic Degradation and Preventing Formation of Membrane-Permeabilizing Species

Abstract

Lattice corneal dystrophy is associated with painful recurrent corneal erosions and amyloid corneal opacities induced by transforming growth factor β-induced protein (TGFBIp) that impairs vision. The exact mechanism of amyloid fibril formation in corneal dystrophy is unknown but has been associated with destabilizing mutations in the fourth fasciclin 1 (Fas1-4) domain of TGFBIp. The green tea compound epigallocatechin gallate (EGCG) has been found to inhibit fibril formation of various amyloidogenic proteins in vitro. In this study, we investigated the effect of EGCG as a potential treatment in lattice corneal dystrophy (LCD) using Fas1-4 with the naturally occurring LCD-inducing A546T mutation. A fewfold molar excess of EGCG was found to inhibit fibril formation in vitro by directing Fas1-4 A546T into stable EGCG-bound protein oligomers. Incubation with 2 molar equiv of EGCG led to a 4-fold reduction in the aggregates' membrane disruptive potential, potentially indicative of significantly lower cytotoxicity with regard to corneal erosions. EGCG did not induce oligomer formation by wild-type Fas1-4, indicating that treatment with EGCG would not interfere with the native function of the wild-type protein. Addition of EGCG to 10-day-old fibrils reduced fibril content in a dose-dependent manner. Proteinase K was found to reduce the light scattering of nontreated fibrils by 31% but reduced that of fibrils treated with 8 molar equiv of EGCG by 85%. This suggests that EGCG remodeling of fibril structure can facilitate aggregate removal by endogenous proteases and thus alleviate the protein deposits' light scattering symptoms.

Epigallocatechin-3-gallate preferentially induces aggregation of amyloidogenic immunoglobulin light chains

“…EGCG, despite being infamous as a promiscuous ligand, fulfills these requirements: It precipitates preferentially the amyloidogenic V L domains. A similar selectivity of EGCG for amyloidogenic mutants was recently reported for TGFBIp 64 . At the same time, the EGCG binding site in functional antibodies is hidden, as the binding site is protected by the heavy chain.…”

Epigallocatechin-3-gallate remodels apolipoprotein A-I amyloid fibrils into soluble oligomers in the presence of heparin

“…In the above experiments, we set out to investigate whether EGCG exerts a potentially protective effect on apoA-I amyloid that is associated with atherosclerosis, as reported for several other amyloidogenic proteins associated with disease ( 49 , 50 ). Studies on Aβ40/42, α-synuclein, amylin, huntingtin, and transthyretin have all shown a common ability of EGCG to bind to the oligomeric and multimeric forms of amyloid, to inhibit fibril assembly, and to prevent formation of toxic structures ( 28 , 29 , 51 – 55 ). In the case of both Aβ40/42 and α-synuclein, EGCG prevents fibrillogenesis by binding preferentially to unfolded protein, directing assembly toward off-pathway nontoxic oligomers, and thereby preventing formation of toxic oligomers and protofibrils ( 29 ).…”

Proteomic Analysis of Amyloid Corneal Aggregates from TGFBI-H626R Lattice Corneal Dystrophy Patient Implicates Serine-Protease HTRA1 in Mutation-Specific Pathogenesis of TGFBIp

“…Often these mutations result in amino acid substitutions that alter protein charge, hydrophobicity or tendency to form β-sheet secondary structures . Indeed, in TGFBI -linked corneal dystrophy, the disease-associated mutations encode proteins that are either thermodynamically unstable (A546T) or overly stable (R555W [PDB 2LTC] and G623D) relative to the native protein. − Where unstable mutant proteins are produced, excessive proteolysis may generate highly amyloidogenic peptide fragments. Conversely, enhanced protein stability may increase the turn over time of the protein in the cornea and possibly prevent effective protein clearance which is highly essential to maintain transparency of the cornea .…”