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Cataract is one of the most prevalent protein aggregation disorders and still the most common cause of vision loss worldwide. The metabolically quiescent core region of the human lens lacks cellular or protein turnover; it has therefore evolved remarkable mechanisms to resist light-scattering protein aggregation for a lifetime. We now report that one such mechanism involves an unusually abundant lens metabolite, myo-inositol, suppressing aggregation of lens crystallins. We quantified aggregation suppression using our previously well-characterized in vitro aggregation assays of oxidation-mimicking human γD-crystallin variants and investigated myo-inositol’s molecular mechanism of action using solution NMR, negative-stain TEM, differential scanning fluorometry, thermal scanning Raman spectroscopy, turbidimetry in redox buffers, and free thiol quantitation. Unlike many known chemical chaperones, myo-inositol’s primary target was neither the native nor the unfolded state of the protein, nor the final aggregated state, but rather the rate-limiting bimolecular step on the aggregation pathway. Given recent metabolomic evidence that it is severely depleted in human cataractous lenses compared to age-matched controls, we suggest that maintaining or restoring healthy levels of myo-inositol in the lens may be a simple, safe, and globally accessible strategy to prevent or delay lens opacification due to age-onset cataract.

Age-related cataract is a major cause of blindness worldwide. Yet, the molecular mechanisms whereby large, light scattering aggregates form is poorly understood, because of the complexity of the aggregates isolated from human lenses. The predominant proteins in the lens are structural proteins called crystallins. The γS-crystallin is heavily modified in cataractous lenses by deamidation, which introduces a negative charge at labile asparagine residues. The effects of deamidation at asparagines, N14, N76, and N143, were mimicked by replacing the asparagine with aspartate using site-directed mutagenesis. The effects of these surface deamidations on the stability, unfolding, and aggregation properties of γS were determined using dynamic light scattering, chemical and thermal-denaturation, and hydrogen-deuterium exchange with mass spectrometry. We found that a small population of all the deamidation mimics aggregated directly into large light scattering bodies with a radius greater than 10 nm that contributed 14-60% of the total scattering intensity compared to 7% for WT under the same conditions. A possible mechanism was identified under partially denaturing conditions, where deamidation led to significantly more rapid unfolding and aggregation particularly for N76D compared to WT. The triple mutant was further destabilized, reflecting the enhanced aggregation properties of N14D and N143D. Thus, the effects of deamidation were both site-specific and cumulative. αA-crystallin was ineffective at acting as a chaperone to prevent the aggregation of destabilized, deamidated γS. It is concluded that surface deamidations, while causing minimal structural disruption individually, progressively destabilize crystallin proteins, leading to their unfolding and precipitation in aged and cataractous lenses.