Anti-PolyQ Antibodies Recognize a Short PolyQ Stretch in Both Normal and Mutant Huntingtin Exon 1

• We assessed binding of MW1 and 3B5H10 anti-polyQ monoclonal antibodies and Fabs to normal and expanded huntingtin exon 1 polyQ repeats.
• Western and dot blots revealed binding of MW1 and 3B5H10 IgGs to both short and expanded polyQ tracts in huntingtin exon 1 fusion proteins.
• Equilibrium gel-filtration studies showed that multiple MW1 or 3B5H10 Fabs bound to a huntingtin exon 1 fusion protein with 39 glutamines.
• Selectivity of antibodies for specific conformations of polyQ to distinguish species of huntingtin exon 1 fusion protein was not observed, and no evidence for a conformational transition between soluble wild type and mutant huntingtin exon 1 was found.

Huntington's disease is caused by expansion of a polyglutamine (polyQ) repeat in the huntingtin protein. A structural basis for the apparent transition between normal and disease-causing expanded polyQ repeats of huntingtin is unknown. The “linear lattice” model proposed random-coil structures for both normal and expanded polyQ in the preaggregation state. Consistent with this model, the affinity and stoichiometry of the anti-polyQ antibody MW1 increased with the number of glutamines. An opposing “structural toxic threshold” model proposed a conformational change above the pathogenic polyQ threshold resulting in a specific toxic conformation for expanded polyQ. Support for this model was provided by the anti-polyQ antibody 3B5H10, which was reported to specifically recognize a distinct pathologic conformation of soluble expanded polyQ. To distinguish between these models, we directly compared binding of MW1 and 3B5H10 to normal and expanded polyQ repeats within huntingtin exon 1 fusion proteins. We found similar binding characteristics for both antibodies. First, both antibodies bound to normal, as well as expanded, polyQ in huntingtin exon 1 fusion proteins. Second, an expanded polyQ tract contained multiple epitopes for fragments antigen-binding (Fabs) of both antibodies, demonstrating that 3B5H10 does not recognize a single epitope specific to expanded polyQ. Finally, small-angle X-ray scattering and dynamic light scattering revealed similar binding modes for MW1 and 3B5H10 Fab–huntingtin exon 1 complexes. Together, these results support the linear lattice model for polyQ binding proteins, suggesting that the hypothesized pathologic conformation of soluble expanded polyQ is not a valid target for drug design.

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