Protein oligomerization induced by oleic acid at the solid–liquid interface – equine lysozyme cytotoxic complexes

Abstract

Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self‐assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid–liquid interface within an ion‐exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed similarity with both amyloid oligomers and the complexes with oleic acid and its structural homologous protein α‐lactalbumin, known as humanα‐lactalbumin made lethal for tumour cells (HAMLET). As determined by NMR diffusion measurements, ELOA may consist of 4–30 lysozyme molecules. Each lysozyme molecule is able to bind 11–48 oleic acids in various preparations. Equine lysozyme acquired a partially unfolded conformation in ELOA, as evident from its ability to bind hydrophobic dye 8‐anilinonaphthalene‐1‐sulfonate. CD and NMR spectra. Similar to amyloid oligomers, ELOA also interacts with thioflavin‐T dye, shows a spherical morphology, assembles into ring‐shaped structures, as monitored by atomic force microscopy, and exerts a toxic effect in cells. Studies of well‐populated ELOA shed light on the nature of the amyloid oligomers and HAMLET complexes, suggesting that they constitute one large family of cytotoxic proteinaceous species. The hydrophobic surfaces can be used profitably to produce complexes with very distinct properties compared to their precursor proteins.

The cytotoxicity of fatty acid/α‐lactalbumin complexes depends on the amount and type of fatty acid

“…However, apart from these quite specific effects of HAMLET, there is also evidence of a more crude effect connected to membrane interactions and alterations. -LA binds to the membrane, but HAMLET causes loss of membrane integrity and leakage of lipid membranes [35] and ELOA, the cytotoxic complex between equine lysozyme and OA, has also been shown to induce loss of membrane integrity with ELOA accumulating at the cell membrane until the cells rupture and ELOA fills the cells [11,36].…”

“…Furthermore, equine lysozyme, a structural homolog to -LA, can also be converted to a cytotoxic complex on an OA pretreated anion exchange column [10,11].…”

Structural Origin of ELOA Toxicity – Implication for HAMLET-Type Protein Complexes with Oleic Acid

“…ELOA complex remains stable in its lyophilized form suitable for long storage as well as it can be kept in solution for up to a week. It is also important to note, that co-incubation of a 50 fold excess of OA mixed with EL in solution at room temperature did not lead to ELOA formation as was evident from the lack of characteristic ELOA conformational transitions monitored by near-UV CD [4]. Thus, the application of a solid-liquid interface facilitating protein self-assembly and protein-OA interactions proved to be an efficient approach in production of both ELOA and HAMLET complexes.…”

“…In the absence of OA, free EL was eluted as a narrow peak at a low NaCl concentration [4]. EL was subjected to column chromatography without decalcification as it has been performed with human -lactalbumin during original HAMLET production, indicating a difference in the generic properties of EL and -lactalbumin.…”

“…Most notably, the newly obtained features include the ability to specifically kill tumor cells while sparing the healthy, normally functioning ones, as it is the case with human or bovine α-lactalbumin made lethal to tumor cells (HAMLET or BAMLET) [1,2] or to indiscriminately induce cell death in all tested cell lines, as it is the case with equine lysozyme (EL) complex with oleic acid (ELOA) [3,4]. While extensive information has been accumulated on the structural, functional and therapeutic properties of protein complexes with OA, many questions remain still unanswered, such as what is the structural origin of their toxicity, what are the specific targets at the cell surface and/or the cellular interior, what are the mechanisms of cellular uptake?…”