The Role of the C1q Domain of Zebrafish Otolin 1a in Otolith Morphogenesis

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 comprises 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, Ca 2+ and CO 3 2– ions bind during otolith morphogenesis, with C1q trimers 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. 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, thioredoxin 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 complexes. Experiments are underway to characterize the various complexes in more detail and to test whether the C1q domain is necessary to trimerize of full‐length Otolin1a. (Supported by NIH R01DC006962)