# Epitope-specific antibodies can distinguish between soluble huntingtin exon-1 and its diverse cellular aggregates[image]

## TL;DR

This paper explores how specific antibodies can distinguish between different forms of a protein linked to Huntington’s disease, offering insights for better diagnostics or treatments.

## Contribution

The study identifies epitopes on huntingtin exon-1 that allow antibodies to selectively bind monomers or aggregates, revealing new principles for antibody design.

## Key findings

- PHP5 and PHP6 bind monomers via an N-terminal α-helix epitope, avoiding fibrils.
- PHP7–PHP9 bind aggregates via proline-rich domains, independent of polyglutamine core regions.
- PRD-binding antibodies show varied recognition of different aggregate regions and types.

## Abstract

Misfolding and aggregation of huntingtin exon-1 (Httex1) with an expanded polyglutamine region is a key pathological hallmark of Huntington’s disease, making conformationally specific Httex1 binders potentially valuable diagnostic or therapeutic tools. To define epitopes, which might confer conformationally specific Httex1 binding, we characterized five newly developed huntingtin antibodies (PHP5–PHP9). Binding to recombinant proteins as well as staining of human embryonic kidney 293 cells and R6/1 mice shows that PHP5 and PHP6 preferentially bind monomers over fibrils. Using electron paramagnetic resonance, peptide arrays, and deletion mutants, we mapped binding of PHP5 and PHP6 to the hydrophobic surface of an N-terminal α-helix spanning residues 4 to 18 of Httex1. In contrast, PHP7, PHP8, and PHP9, raised against protofibrils, recognize proline repeats within the C-terminal proline-rich domain (PRD). These antibodies showed a preference for aggregates in cells, but neither the N-terminal N17 region nor the polyglutamine fibril–forming core region was required. Similar fibril binding was also observed with an α-synuclein–PRD chimera, where the PRD was fused to the fibril-forming core of α-synuclein. Thus, a high density of PRD regions, rather than fibril core features, is needed for fibril binding. Interestingly, all PRD-binding antibodies, including PHP1 and P90, preferentially bound aggregates, but recognition of different cellular aggregates varied, revealing heterogeneity both within aggregates (rim versus interior) and between aggregates. Together, the binding principles uncovered here could serve as a basis for the design and optimization of binders with potential diagnostic or therapeutic relevance.