Generation of camelid single-domain antibody fragments raised against proteins containing polyglutamine expansions

Nine progressive neurodegenerative diseases are associated with the expansion of a polyglutamine (polyQ) tract above a threshold size (~ 35-45 residues) into nine different proteins [1]. These proteins with expanded polyQ repeats have been found to form intranuclear amyloid-like aggregates, and the formation of these aggregates could play an important role in the pathogenesis [2-4]. The polyQ expansion is the only common feature among the proteins involved, suggesting it may be responsible for the aggregation phenomenon. Understanding the molecular mechanism by which the polyQ expansions promote aggregation is therefore crucial for the development of therapeutic strategies.
The nine proteins associated with polyQ diseases are difficult to express recombinantly due to their big size and/or their insoluble character. In order to get further insights into the mechanism by which polyQ tracts promote aggregation, we have therefore decided to insert polyQ sequences into a well studied protein, the b-lactamase BlaP from B. licheniformis [5-6]. We have created chimeras containing 23, 30, 55, and 79 glutamines and we have investigated the effects of the insertions on the activity, the structure, the stability of BlaP as well as on its aggregating properties. Preliminary results indicate that BlaP is a good framework to study the molecular mechanism of aggregation associated with expanded polyglutamine tracts.
On another hand, our previous work on the amyloidogenic variants of human lysozyme has shown that camelid single domain antibody fragments are very powerful structural probes to understand, at the molecular level, the mechanism of amyloid fibril formation [7]. Moreover, a recent study has suggested that expanded polyQ strectches adopt multiple conformations in solution that can be readily distinguished by monoclonal antibodies [8]. Altogether these results have encouraged us to generate VHHs against our different chimeras and we present here our preliminary results.

References
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[3] Paulson HL (2000) Brain Pathol 10, 293-299.
[4] Sanchez I. et al. (2003) Nature 421, 373-379.
[5] Scarafone N. (2008) Mémoire de DEA en Sciences. Université de Liège.
[6] Pain C. (2009) Mémoire de Master en Biochimie. Université de Liège.
[7] Dumoulin et al. (2003) Nature 424, 783-788.
[8] Legleiter J. et al. (2009) J Biol Chem 284, 21647-21648.