The Role of the Otolin1 C1q Domain in Otolith Morphogenesis

Abstract

Otolin1 is an extracellular matrix protein of otoliths ("ear stones" of fishes), and otoconia ("ear dust" of higher vertebrates). These acellular biominerals are essential for the sense of balance; dislodging results in the most common human balance disorder, benign paroxysmal positional vertigo. Otolin1 consists of a collagen and a C1q domain, similar to atypical collagens VIII and X. We propose that Otolin1 forms a scaffold to which other otolith proteins, Ca2+ and CO2- ions bind during otolith morphogenesis. To form this scaffold, Otolin1 first forms trimers, which then interact with other trimers to build a higher order assembly in which the C1q trimers act as hubs and collagen triple-helices as spokes. To test this model, we investigated whether the Otolin1 C1q domain is necessary and sufficient to form trimers and higher complexes in vitro. The zebrafish Otolin1a C1q domain was expressed in bacteria either alone or fused to Thioredoxin (Trx). Trx was chosen, as it is a nonoligomer forming protein that also aids native folding of its fusion partner. Multimerization of affinity purified recombinant proteins was assessed by size exclusion chromatography and gel electrophoresis. Under denaturing conditions, recombinant C1q proteins formed monomers and trimers, but not dimers or higher order complexes. Under native conditions, they formed only higher-order complexes. In contrast, Trx alone only appeared as a monomer. Our results are consistent with the proposed ability of the Otolin C1q domain to form trimers that, in turn, assemble into higher-order 3-D mesh that supports controlled mineral deposition.