Wouter Van Overbeke1, Jantana Wongsantichon2,§, Inge Everaert3,§, Adriaan Verhelle1, Olivier Zwaenepoel1, Ariane De Ganck1,#, Tino Hochepied4,5, Jody Haigh4,5, Claude Cuvelier6, Wim Derave3, Robert C. Robinson7,*, Jan Gettemans1,*
1 Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium.
2 Institute of Molecular and Cellular Biology, A*STAR, Biopolis, Singapore 138673.
3 Department of Movement and Sport Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium.
4 Department for Molecular Biomedical Research, VIB, Ghent, Belgium;
5 Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
6 Department of Pathology, Faculty of Medicine and Health Sciences, Ghent University.
7 Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117597.
§ J.W. and I.E. contributed equally to this work.
# Present address: Biogazelle, Ghent, Belgium.
* To whom correspondence should be addressed.
Email: firstname.lastname@example.org (J.G.) or email@example.com (R.C.R)
Published online ahead of print in Human Molecular Genetics on 18 January 2015.
Hereditary gelsolin amyloidosis is an autosomal dominantly inherited amyloid disorder. A point mutation in the GSN gene (G654A being the most common one) results in disturbed calcium binding by the second gelsolin domain (G2). As a result, the folding of G2 is hampered, rendering the mutant plasma gelsolin susceptible to a proteolytic cascade. Consecutive cleavage by furin and MT1-MMP- like proteases generates 8 and 5 kDa amyloidogenic peptides that cause neurological, ophthalmological and dermatological findings. To this day, no specific treatment is available to counter the pathogenesis. Using GSN nanobody 11 as a molecular chaperone, we aimed to protect mutant plasma gelsolin from furin proteolysis in the trans-Golgi network. We report a transgenic, GSN nanobody 11 secreting mouse that was used for crossbreeding with gelsolin amyloidosis mice. Insertion of the therapeutic nanobody gene into the gelsolin amyloidosis mouse genome resulted in improved muscle contractility. X-ray crystal structure determination of the gelsolin G2:Nb11 complex revealed that Nb11 does not directly block the furin cleavage site. We conclude that nanobodies can be used to shield substrates from aberrant proteolysis and this approach might establish a novel therapeutic strategy in amyloid diseases.
Figure Legend: Gelsolin G2:Nb11 crystal structure.
(A) GSN Nb11 binds G2 at a distant site relative to the furin cleavage site (A173). Calcium is shown as a dark grey sphere; D187 is the gelsolin amyloidosis mutation site. Furin cleavage site (A173) and MT1-MMP cleavage sites (R225 and M243) are shown in ball-and-stick representations. (B) Superposition of G2 (V159-D259) as determined in the G2-Nb11 structure and G2 derived from the N-terminal active gelsolin/actin complex (PDB: 3FFK). Ribbon representation shows G2 from G2-Nb11 in green and from 3FFK in blue. Cysteine residues forming a disulfide bond and the calcium binding site are presented as ball-and-stick with parental colors and calcium is in grey. (C) GSN G2:Nb11 was overlaid onto the active N-terminal half of gelsolin (PDB: 3FFK). The β-sheet harboring the furin cleavage site is shown in pink; the short α-helix and G2-G3 linker are shown in blue.
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