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Nanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme.
Dumont, Janice; Menzer, Linda; Pardon, Els et al.
2010Single Domain Antibodies Come of Age
 

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Keywords :
Amyloid Fibril; Nanobodies; Lysozyme
Abstract :
[en] 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 amyloidose. 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Dumont, Janice ;  Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
Menzer, Linda ;  Université de Liège - ULiège > 2e an. master bioch. & biol. moléc. & cell., fin. appr.
Pardon, Els;  Vrije Universiteit Brussel - VUB > Departement ultrastructure
Wyns, Lode;  Vrije Universiteit Brussel - VUB
Steyaert, Jan;  Vrije Universiteit Brussel - VUB > Departement Ultrastructure
Dobson, Christopher;  University of Cambridge > Departement of chemistry
Dumoulin, Mireille  ;  Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
Language :
English
Title :
Nanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme.
Publication date :
2010
Event name :
Single Domain Antibodies Come of Age
Event organizer :
Ablynx
Event place :
Ghent, Belgium
Event date :
14-15 octobre 2010
Audience :
International
Available on ORBi :
since 25 November 2011

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