A llama-derived gelsolin single-domain antibody blocks gelsolin–G-actin interaction

Abstract

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca(2+)-activated gelsolin (K (d) approximately 10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca(2+) (K (d) approximately 5 nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/capping or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated.

Chaperone Nanobodies Protect Gelsolin Against MT1-MMP Degradation and Alleviate Amyloid Burden in the Gelsolin Amyloidosis Mouse Model

“…[20][21][22][23]30 In this study, detailed insight into the proteolytic gelsolin amyloidosis cascade 12,13 enabled us to target the C68 precursor that gives rise to amyloidogenic peptides. Preventing C68 proteolysis by MT1-MMP may embody a therapeutically preferred strategy since metalloproteases participate in many diverse physiological processes.…”

A Nanobody‐Based Approach to Amyloid Diseases, the Gelsolin Case Study

“…Both gelsolin and nanobody are abundantly present in the cytoplasm of unstimulated MCF-7 cells, preventing straightforward assessment of their co-localization. However, when the cells were stimulat-ed with epidermal growth factor (EGF), both endogenous gelsolin and the nanobodies extensively decorated membrane ruffles [47]. In view of the binding region of Nb11 in gelsolin, it was surmised that it could interfere with furin-mediated degradation.…”

Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein

“…Camelidae antibodies are a class of immunoglobulins that lack light chains; they are named heavy-chain antibodies and are also referred to as nanobodies (Nbs; Van den Abbeele et al, 2010). Nbs, which are composed of a single domain, are thus some of the smallest antigen-binding fragments and possess all of the features of conventional antibodies (Lam et al, 2009).…”