Dataset Information

Selection and characterization of llama single domain antibodies against N-terminal huntingtin.

ABSTRACT: Huntington disease is caused by expansion of a CAG repeat in the huntingtin gene that is translated into an elongated polyglutamine stretch within the N-terminal domain of the huntingtin protein. The mutation is thought to introduce a gain-of-toxic function in the mutant huntingtin protein, and blocking this toxicity by antibody binding could alleviate Huntington disease pathology. Llama single domain antibodies (VHH) directed against mutant huntingtin are interesting candidates as therapeutic agents or research tools in Huntington disease because of their small size, high thermostability, low cost of production, possibility of intracellular expression, and potency of blood-brain barrier passage. We have selected VHH from llama phage display libraries that specifically target the N-terminal domain of the huntingtin protein. Our VHH are capable of binding wild-type and mutant human huntingtin under native and denatured conditions and can be used in Huntington disease studies as a novel antibody that is easy to produce and manipulate.

SUBMITTER: Schut MH

PROVIDER: S-EPMC4341019 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Selection and characterization of llama single domain antibodies against N-terminal huntingtin.

Schut Menno H MH Pepers Barry A BA Klooster Rinse R van der Maarel Silvère M SM El Khatabi Mohamed M Verrips Theo T den Dunnen Johan T JT van Ommen Gert-Jan B GJ van Roon-Mom Willeke M C WM

Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology 20141008 3

Huntington disease is caused by expansion of a CAG repeat in the huntingtin gene that is translated into an elongated polyglutamine stretch within the N-terminal domain of the huntingtin protein. The mutation is thought to introduce a gain-of-toxic function in the mutant huntingtin protein, and blocking this toxicity by antibody binding could alleviate Huntington disease pathology. Llama single domain antibodies (VHH) directed against mutant huntingtin are interesting candidates as therapeutic a ...[more]

PMID: 25294428

Similar Datasets

Project description:A key advantage of recombinant antibody technology is the ability to optimize and tailor reagents. Single domain antibodies (sdAbs), the recombinantly produced variable domains derived from camelid and shark heavy chain antibodies, provide advantages of stability and solubility and can be further engineered to enhance their properties. In this study, we generated sdAbs specific for Ebola virus envelope glycoprotein (GP) and increased their stability to expand their utility for use in austere locals. Ebola virus is extremely virulent and causes fatal hemorrhagic fever in ~ 50 percent of the cases. The viral GP binds to host cell receptors to facilitate viral entry and thus plays a critical role in pathogenicity.An immune phage display library containing more than 107 unique clones was developed from a llama immunized with a combination of killed Ebola virus and recombinantly produced GP. We panned the library to obtain GP binding sdAbs and isolated sdAbs from 5 distinct sequence families. Three GP binders with dissociation constants ranging from ~ 2 to 20 nM, and melting temperatures from ~ 57 to 72 °C were selected for protein engineering in order to increase their stability through a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond. These changes served to increase the melting temperatures of the sdAbs by 15-17 °C. In addition, fusion of a short positively charged tail to the C-terminus which provided ideal sites for the chemical modification of these sdAbs resulted in improved limits of detection of GP and Ebola virus like particles while serving as tracer antibodies.SdAbs specific for Ebola GP were selected and their stability and functionality were improved utilizing protein engineering. Thermal stability of antibody reagents may be of particular importance when operating in austere locations that lack reliable refrigeration. Future efforts can evaluate the potential of these isolated sdAbs as candidates for diagnostic or therapeutic applications for Ebola.

Project description:Marburg virus (MARV) is a highly lethal hemorrhagic fever virus that is increasingly re-emerging in Africa, has been imported to both Europe and the US, and is also a Tier 1 bioterror threat. As a negative sense RNA virus, MARV has error prone replication which can yield progeny capable of evading countermeasures. To evaluate this vulnerability, we sought to determine the epitopes of 4 llama single-domain antibodies (sdAbs or VHH) specific for nucleoprotein (NP), each capable of forming MARV monoclonal affinity reagent sandwich assays. Here, we show that all sdAb bound the C-terminal region of NP, which was produced recombinantly to derive X-ray crystal structures of the three best performing antibody-antigen complexes. The common epitope is a trio of alpha helices that form a novel asymmetric basin-like depression that accommodates each sdAb paratope via substantial complementarity-determining region (CDR) restructuring. Shared core contacts were complemented by unique accessory contacts on the sides and overlooks of the basin yielding very different approach routes for each sdAb to bind the antigen. The C-terminal region of MARV NP was unable to be crystallized alone and required engagement with sdAb to form crystals suggesting the antibodies acted as crystallization chaperones. While gross structural homology is apparent between the two most conserved helices of MARV and Ebolavirus, the positions and morphologies of the resulting basins were markedly different. Naturally occurring amino acid variations occurring in bat and human Marburgvirus strains all mapped to surfaces distant from the predicted sdAb contacts suggesting a vital role for the NP interface in virus replication. As an essential internal structural component potentially interfacing with a partner protein it is likely the C-terminal epitope remains hidden or "cryptic" until virion disruption occurs. Conservation of this epitope over 50?years of Marburgvirus evolution should make these sdAb useful foundations for diagnostics and therapeutics resistant to drift.

Dataset Information

Selection and characterization of llama single domain antibodies against N-terminal huntingtin.

Publications

Selection and characterization of llama single domain antibodies against N-terminal huntingtin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets