Abstract
Myocilin is a protein found in the trabecular meshwork extracellular matrix tissue of the eye that plays a role in regulating intraocular pressure. Both wild-type and certain myocilin variants containing mutations in the olfactomedin (OLF) domain are linked to the optic neuropathy glaucoma. Because calcium ions are important biological cofactors that play numerous roles in extracellular matrix proteins, we examined the calcium binding properties of the myocilin OLF domain (myoc-OLF). Our study reveals an unprecedented high affinity calcium binding site within myoc-OLF. The calcium ion remains bound to wild-type OLF at neutral and acidic pH. A glaucoma-causing OLF variant, myoc-OLF(D380A), is calcium-depleted. Key differences in secondary and tertiary structure between myoc-OLF(D380A) and wild-type myoc-OLF, as well as limited access to chelators, indicate that the calcium binding site is largely buried in the interior of the protein. Analysis of six conserved aspartate or glutamate residues and an additional 18 disease-causing variants revealed two other candidate residues that may be involved in calcium coordination. Our finding expands our knowledge of calcium binding in extracellular matrix proteins; provides new clues into domain structure, function, and pathogenesis for myocilin; and offers insights into highly conserved, biomedically relevant OLF domains.
Highlights
Myocilin is an extracellular protein linked to glaucoma but is of unknown structure and function
Because calcium ions are important biological cofactors that play numerous roles in extracellular matrix proteins, we examined the calcium binding properties of the myocilin OLF domain
Initial Identification of Ca2ϩ in myoc-OLF—We previously examined the effects of metal ions on the stability of myoc-OLF, but aspects of experimental design excluded calcium ions [27]
Summary
Myocilin is an extracellular protein linked to glaucoma but is of unknown structure and function. Myocilin is a protein found in the trabecular meshwork extracellular matrix tissue of the eye that plays a role in regulating intraocular pressure Both wild-type and certain myocilin variants containing mutations in the olfactomedin (OLF) domain are linked to the optic neuropathy glaucoma. Because calcium ions are important biological cofactors that play numerous roles in extracellular matrix proteins, we examined the calcium binding properties of the myocilin OLF domain (myoc-OLF). Myocilin-associated inherited glaucoma falls into a protein conformational disorder; mutant myocilin aggregation may be a function of the fact that disease-causing myoc-OLF variants are less stable than their wild-type counterpart [9] and/or their ability to form amyloid fibrils [10]. Our results suggest new roles for myocilin in the TM and possible contribution to the pathogenesis of glaucoma
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