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Abstract :
[fr] Six variants of human lysozyme (single-point mutations I56T, F57I, W64R, D67H and double
mutations F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic
amyloidosis. These proteins form extracellular amyloid fibrils that deposit in a wide range of tissues
and organs such as liver, spleen and kidneys where they cause damages [1]. It was shown that the
D67H and I56T mutations cause a loss in stability and more particularly a loss of global cooperativity
of protein [1]. Consequently, under physiologically relevant conditions, these variants can transiently
populate a partially unfolded state in which the beta-domain and the C-helix are cooperatively
unfolded while the rest of the protein remains native like [1]. The formation of intermolecular
interactions between the regions that are unfolded in this intermediate state is likely to be a
fundamental trigger of the aggregation process that ultimately leads to the formation and deposition of
fibrils in tissues. The binding of three variable domain of camelid antibodies – also named nanobodies
- (cAb-HuL 6 [2], cAb-HuL 5 and cAb-HuL 22 [3]) raised against the wild type human lysozyme
inhibit in vitro the formation of amyloid fibrils by the lysozyme variants. These three nanobodies bind
on different regions of lysozyme and act as amyloid fibrils inhibitor through different mechanisms. On
one hand, cAb-HuL 6 and cAb-HuL 22 stabilize the native state of the lysozyme variants thus
restoring the global cooperativity characteristic of the wild-type protein. On the other, cAb-HuL 5
probably acts by binding soluble prefibrillar aggregates. In the present work, sixteen other nanobodies
specific of human lysozyme have been generated. Competition experiments have shown that they bind
to five non overlapping epitopes. The effects of the binding of these nanobodies on the stability of the
D67H variant of human lysozyme and on its aggregation into amyloid fibrils will be discussed.
References
[1] Dumoulin et al, (2006) Acc. Chem. Res, 39, 603-610.
[2] Dumoulin et al, (2003) Nature, 424, 783-788.
[3] Chan et al. (2008) Biochemistry, 47,11041-11054.