The glaucoma-associated olfactomedin domain of myocilin is a novel calcium binding protein - PubMed (original) (raw)

The glaucoma-associated olfactomedin domain of myocilin is a novel calcium binding protein

Rebecca K Donegan et al. J Biol Chem. 2012.

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.

PubMed Disclaimer

Figures

FIGURE 1.

FIGURE 1.

PROMALS (30) sequence alignment of the OLF domain of myocilins from human (gi_3065674), zebrafish (gi_62632725), cow (gi_74356501), pig (gi_47522798), mouse (gi_15077142), and rat (gi_3845607), as well as (non-myocilin) amassin (gi_28453877) from sea urchin. Consensus secondary structure (SS) is depicted above the alignment. e, β-strand; h, helix. Asterisk indicates location of glaucoma-causing mutations examined in this study. Red residues are identical; blue residues are similar. Highlighted residues are conserved aspartate/glutamate positions subjected to site-directed mutagenesis in this study.

FIGURE 2.

FIGURE 2.

Ca2+ binding assay for myoc-OLF and myoc-OLF(D380A). Fluorescence (in a.f.u., arbitrary fluorescence units) of chelator Quin-2 due to Ca2+ binding is measured under native and denaturing conditions.

FIGURE 3.

FIGURE 3.

Structural and stability comparison of myoc-OLF and myoc-OLF(D380A). Shown is a comparison of secondary structure from far-UV spectra (a) and tertiary structure from near-UV spectra (b) among wild-type myoc-OLF at pH 7.2, wild-type myoc-OLF at pH 4.6, and myoc-OLF(D380A) at pH 7.2. c, CD thermal melts for myoc-OLF and myoc-OLF(D380A) in MES pH 6.0, monitored at 215 nm in the presence and absence of exogenous Ca2+.

Similar articles

Cited by

References

    1. Evenäs J., Malmendal A., Forsén S. (1998) Calcium. Curr. Opin. Chem. Biol. 2, 293–302 - PubMed
    1. Maurer P., Hohenester E. (1997) Structural and functional aspects of calcium binding in extracellular matrix proteins. Matrix Biol. 15, 569–580 - PubMed
    1. Zhang K., Zhang L., Weinreb R. N. (2012) Ophthalmic drug discovery: novel targets and mechanisms for retinal diseases and glaucoma. Nat. Rev. Drug Discov. 11, 541–559 - PubMed
    1. McLaughlin C. W., Karl M. O., Zellhuber-McMillan S., Wang Z., Do C. W., Leung C. T., Li A., Stone R. A., Macknight A. D., Civan M. M. (2008) Electron probe X-ray microanalysis of intact pathway for human aqueous humor outflow. Am. J. Physiol. Cell Physiol. 295, C1083–1091 - PMC - PubMed
    1. Polansky J. R., Fauss D. J., Chen P., Chen H., Lütjen-Drecoll E., Johnson D., Kurtz R. M., Ma Z. D., Bloom E., Nguyen T. D. (1997) Cellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Ophthalmologica 211, 126–139 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources