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Calcium dysregulation potentiates wild-type myocilin misfolding: implications for glaucoma pathogenesis

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Abstract

Myocilin is secreted from trabecular meshwork cells to an eponymous extracellular matrix that is critical for maintaining intraocular pressure. Missense mutations found in the myocilin olfactomedin domain (OLF) lead to intracellular myocilin misfolding and are causative for the heritable form of early-onset glaucoma. The OLF domain contains a unique internal, hetero-dinuclear calcium site. Here, we tested the hypothesis that calcium dysregulation causes wild-type (WT) myocilin misfolding reminiscent of that observed for disease variants. Using two cellular models expressing WT myocilin, we show that the Ca2+ ATPase channel blocker thapsigargin inhibits WT myocilin secretion. Intracellular WT myocilin is at least partly insoluble and aggregated in the endoplasmic reticulum (ER), and stains positively with an amyloid dye. By comparing the effect of thapsigargin on WT myocilin to that on a de novo secretion-competent Ca2+-free variant D478S, we discern that non-secretion of WT myocilin is due initially to calcium dysregulation, and is potentiated further by resultant ER stress. In E. coli, depletion of calcium leads to recombinant expression of misfolded isolated WT OLF but the D478S variant is still produced as a folded monomer. Treatment of cells expressing a double mutant composed of D478S and either disease variants P370L or Y437H with thapsigargin promotes its misfolding and aggregation, demonstrating the limits of D478S to correct secretion defects. Taken together, the heterodinuclear calcium site is a liability for proper folding of myocilin. Our study suggests a molecular mechanism by which WT myocilin misfolding may contribute broadly to glaucoma-associated ER stress.

Graphical abstract

This study explores the effect of calcium depletion on myocilin olfactomedin domain folding.

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Acknowledgements

We thank Sara Bahri for technical assistance. This study was supported by NIH R01EY021205 and R21EY031093. EGS was supported in part by GAANN P200A210014, MDM was supported in part by 3R01EY021205-10S1, and MTM was supported by T32EY007092.

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Author notes

  1. Emily G. Saccuzzo, Mackenzie D. Martin and Kamisha R. Hill contributed equally.

Authors and Affiliations

  1. School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Dr. NW, Atlanta, GA, 30332-0400, USA

    Emily G. Saccuzzo, Mackenzie D. Martin, Kamisha R. Hill, Minh Thu Ma, Yemo Ku & Raquel L. Lieberman

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  1. Emily G. Saccuzzo
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  2. Mackenzie D. Martin
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Correspondence to Raquel L. Lieberman.

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Saccuzzo, E.G., Martin, M.D., Hill, K.R. et al. Calcium dysregulation potentiates wild-type myocilin misfolding: implications for glaucoma pathogenesis. J Biol Inorg Chem 27, 553–564 (2022). https://doi.org/10.1007/s00775-022-01946-3

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