Protein misfolding studies on human fibrinogen alpha chain and hen egg white lysozyme

Sivalingam, Vishwanath and Patel, Basant Kumar (2017) Protein misfolding studies on human fibrinogen alpha chain and hen egg white lysozyme. PhD thesis, Indian institute of technology Hyderabad.

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Abstract

Aggregation of misfolded proteins into fibrillar, β-sheet-rich structures, termed amyloid, causes debilitating diseases like Alzheimer’s disease, prion disease, & renal amyloidosis. Hereditary renal amyloidoses are a set of autosomal-dominant diseases where the primary clinical manifestation is amyloid deposition mutated forms of proteins (such as fibrinogen α chain, lysozyme, apolipoproteins I and II) in kidneys. Strikingly, a notable feature of amyloid formation is generation of distinct but stable variant amyloid conformations by misfolding of the same protein with the identical amino acid sequence into alternate conformations, called “variants” or “strains”. Fibrinogen is a 340 kDa glycoprotein involved in the formation of blood clots in wounds. Mutant fibrinogen α chain (FGA) was identified to cause some familial forms of renal amyloidosis. Amyloid material extracted from affected individuals showed presence of mutant forms of FGA fragments of length 27-81 residues, mainly through FGA amino acid 500-580 residues. In Chapter II and III, after recombinant expression of wild-type and two disease-related point mutants E540V and R554L in FGA 496-581 fragment, we examined their amyloidogenic properties. AFM imaging revealed that E540V mutant formed amyloid-like fibrillar aggregates exhibiting height of ~ 4-5 nm and length of ~ 200 nm, while the R554L mutant protein aggregates displayed predominantly globular aggregates with sizes ranging 50-200 nm and displaying ~ 30-40 nm heights. Lysozyme is a bacteriolytic enzyme present in human saliva, tears glands and milk. Mutated human lysozyme was found to be associated with familial non-neuropathic systemic amyloid deposition in kidney, liver and gastro-intestinal tract. Hen egg white lysozyme (HEWL) which has around 60% sequence similarity with human lysozyme can also form amyloid aggregates under a few different incubation conditions, but whether these aggregates represent any prion- like conformational variants remains to be investigated. In Chapter IV, HEWL lysozyme amyloid-like aggregation was achieved at pH 2.0 and 37°C under agitated and static conditions, which were then compared for prion-like self seeding abilities. The HEWL amyloid aggregates prepared in agitated and static conditions displayed distinguishable properties in ThT binding as well as in stability assays protease, pH and detergent-resistance. While static HEWL aggregates showed about 10-fold increase in ThT fluorescence intensity, agitated HEWL showed over 40-fold increase compared to soluble control. CD spectroscopy revealed agitated HEWL contained relatively higher β-sheet content compared to that of the static HEWL aggregates. AFM studies revealed that agitated HEWL formed thinner fibrils (~ 2-2.5 nm) than static HEWL (~ 2.5-3 nm). Upon investigating strain formation by amyloid seeding assay, agitated HEWL showed rapid increase in ThT fluorescence upon self-seeding with agitated seeds, but not with static seeds. Similarly, static seeds showed increase in ThT fluorescence upon self-seeding with static seeds, but did not get cross-seeded by agitated seeds, thereby confirming that HEWL is capable of forming faithfully self-seeding conformational variants under different conditions, as observed in prion variants.

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