The effect of amyloid fibrils on the structural and functional state of human erythrocyte membranes in vitro was studied. The process of the formation of amyloid fibrils from hen egg lysozyme was monitored by the fluorescence method with a specific thioflavin T probe. Experiments were carried out on the blood erythrocytes of virtually healthy donors. Cells were incubated in a solution containing amyloid fibrils, then erythrocyte membranes were isolated from the cells. The structure of human erythrocyte membranes was estimated by the degree of cell vesiculation under metabolic depletion, protein modification of erythrocyte membranes was assessed by the fluorescence parameters of N- (1-pyrene) maleimide (PM) probes, reflecting the level of sulfhydryl groups, and 4-4, -diacetamide 2,2, -stilbendisulphonate (DIDS), specifically binding to protein of lane 3. Erythrocytes incubated under the same conditions in medium containing intact lysozyme, as well as in medium devoid of protein components were used as the controls. A three-hour incubation of cells in a solution containing amyloid fibrils from lysozyme was found to accelerate the erythrocyte vesiculation process upon their metabolic depletion, decrease the fluorescence intensity of the protein SH-groups-specific N-1- (pyrene) maleimide and lane 3 protein associated 4-4, -diacetamido-2,2, -stilbendisulphonate, compared with control cells. The results allow us to conclude that amyloid fibrils obtained from lysozyme, when acting upon human erythrocytes in vitro, cause a modification of their membrane structure, which manifests in an increase in the separation of the membrane material from the red blood cells - vesicles, a decrease in the level of protein SH-groups and modification of the lane 3 protein structure in human erythrocyte membranes.
erythrocytes, amyloid fibrils, lysozyme, membrane proteins
1. Shavlovskiy M.M. Molekulyarnye i geneticheskie osnovy etiopatogeneza amiloidozov. Medicinskiy akademicheskiy zhurnal, 2010, t. 10, № 4, s. 63-81. @@[Schavlovsky M.M. Ethiology and pathogenesis of amyloidoses: the molecular and genetic basis. Med. Acad. Journ., 2010, vol. 10, no. 4, pp. 63-81. (In Russ.)]
2. Chiti F., Dobson C. Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade. Annu. Rev. Biochem., 2017, vol. 86, pp. 27-68.
3. Swaminathan R., Ravi V.K., Kumar S., Kumar M.V., Chandra N. Lysozyme: a model protein for amyloid research. Adv Protein Chem Struct Biol., 2011, vol. 84, pp. 63-111.
4. Zou Y., Hao W., Li H., Gao Y., Sun Y., Ma G. New insight into amyloid fibril formation of hen egg white lysozyme using a two-step temperature-dependent FTIR approach. J. Phys. Chem. B, 2014, vol. 118, no. 33, pp. 9834-9843.
5. Fazili N.A., Bhat I.A., Bhat W.F., Naeem A. Anti-fibrillation propensity of a flavonoid baicalein against the fibrils of hen egg white lysozyme: potential therapeutics for lysozyme amyloidosis. J Biomol Struct Dyn., 2016, vol. 34, no. 10, pp. 2102-14.
6. Dodge G.T., Mitchell C., Hanahan D.J. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch. Biochem. Biophys., 1963, vol. 100, pp. 119-130.
7. Markwell M.A., Haas S.M., Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein sample. Analyt. Biochem., 1978, vol. 87, no. 2, pp. 206-210.
8. Zubrickaya G.P., Luk'yanenko L.M., Slobozhanina E.I., Kozarezova T.I., Klimkovich N.N. Processy mikrovezikulyacii eritrocitov u detey pri gematologicheskih zabolevaniyah. Novosti mediko-biol. nauk, 2004, № 1, s. 143-146. @@[Zubritskaya G.P., Lukyanenko L.M., Slobozhanina E.I., Kozarezova T.I., Klimkovich N.N. Microvesicular process in erythrocytes of children with aplastic anemia and myelodysplastic syndrome. News of biomedical sciences, 2004, no. 1, pp. 143-146. (In Russ.)]
9. LeVine H. Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci., 1993, vol. 2, no. 3, pp. 404-410.
