Abstract
Accumulating evidence suggests an important role for the Disrupted-in-Schizophrenia 1 (DISC1) protein in neurodevelopment and chronic mental illness. In particular, the C-terminal 300 amino acids of DISC1 have been found to mediate important protein-protein interactions and to harbor functionally important phosphorylation sites and disease-associated polymorphisms. However, long disordered regions and oligomer-forming subdomains have so far impeded structural analysis. VHH domains derived from camelid heavy chain only antibodies are minimal antigen binding modules with appreciable solubility and stability, which makes them well suited for the stabilizing proteins prior to structural investigation. Here, we report on the generation of a VHH domain derived from an immunized Lama glama, displaying high affinity for the human DISC1 C region (aa 691–836), and its characterization by surface plasmon resonance, size exclusion chromatography and immunological techniques. The VHH-DISC1 (C region) complex was also used for structural investigation by small angle X-ray scattering analysis. In combination with molecular modeling, these data support predictions regarding the three-dimensional fold of this DISC1 segment as well as its steric arrangement in complex with our VHH antibody.
Highlights
An anti-Disrupted- in-schizophrenia 1 (DISC1) camelid VHH phage library was generated using mRNA obtained from a llama that had been immunized with an insoluble fraction of recombinant human DISC1598-785
The phage library generated was subjected to three rounds of panning by phage display in order to select the strongest binding clone against the DISC1598-785 protein
The camelid VHH antibody was overexpressed as a recombinant protein in the periplasm of E. coli, providing an oxidizing environment for proper folding, and was purified by Ni2+-NTA affinity chromatography, followed by size exclusion chromatography (SEC)
Summary
Disrupted- in-schizophrenia 1 (DISC1) was discovered as the gene disrupted by a balanced chromosomal translocation (1; 11) (q42.1; q14.3), which is strongly linked with. The C-terminal region comprising amino acids 598–854 of the human DISC1 protein (UniProtKB Q9NRI5) contains interaction sites for several binding partners, such as nuclear distribution element 1 (NDE1), NDE-like 1 (NDEL1), and lissencephaly 1 (LIS1, encoded by the PAFAH1B1 gene), which are important for brain development and neuronal migration [10, 11] It harbors functionally important sites such as the mental illness associated S704C polymorphism [12,13,14] and a phosphorylation site at S713, which was found to coordinate the switch of neuronal progenitor cell proliferation to migration during corticogenesis [15]. These results provide insight into the binding mode of our VHH antibody to the DISC1 protein
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