Abstract
Myocilin is an eye protein found in the trabecular extracellular matrix (TEM), within the anatomic region that controls fluid flow. Variants of myocilin, localized to its olfactomedin (OLF) domain, have been linked to inherited forms of glaucoma, a disease associated with elevated intraocular pressure. OLF domains have also been implicated in psychiatric diseases and cancers by their involvement in signaling, neuronal growth, and development. However, molecular characterization of OLFs has been hampered by challenges in recombinant expression, a hurdle we have recently overcome for the myocilin OLF domain (myoc-OLF). Here, we report the first detailed solution biophysical characterization of myoc-OLF to gain insight into its structure and function. Myoc-OLF is stable in the presence of glycosaminoglycans, as well as in a wide pH range in buffers with functional groups reminiscent of such glycosaminoglycans. Circular dichroism (CD) reveals significant β-sheet and β-turn secondary structure. Unexpectedly, the CD signature is reminiscent of α-chymotrypsin as well as another ocular protein family, the βγ-crystallins. At neutral pH, intrinsic tryptophan fluorescence and CD melts indicate a highly cooperative transition with a melting temperature of ∼55°C. Limited proteolysis combined with mass spectrometry reveals that the compact core structural domain of OLF consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. The data presented here inform new testable hypotheses for interactions with specific TEM components, and will assist in design of therapeutic agents for myocilin glaucoma.
Highlights
The protein most closely associated with inherited forms of open angle glaucoma (OAG) through genetic linkage studies, is a,57 kDa glycoprotein composed of a secretion signal sequence, coiled-coil region, and a,30 kDa olfactomedin (OLF) domain (Figure 1), which harbors 90% of all reported pathogenic lesions [1]
Results pH stability profile of myoc-OLF We investigated the pH stability profile of myoc-OLF to elucidate any preferences of this domain as well as to gain insight into its resilience under different pH environments, such as low pH, that might be encountered under a known age-related trabecular meshwork extracellular matrix (TEM) stressor, oxidative stress [21]
There was no difference in melting temperature (Tm) between the maltose binding protein (MBP)-OLF and cleaved myoc-OLF among the pHs tested (Table S1), reinforcing the independence of the domains in the fusion protein
Summary
The protein most closely associated with inherited forms of open angle glaucoma (OAG) through genetic linkage studies, is a ,57 kDa glycoprotein composed of a secretion signal sequence, coiled-coil region, and a ,30 kDa olfactomedin (OLF) domain (Figure 1), which harbors 90% of all reported pathogenic lesions [1]. Wild-type (WT) myocilin is secreted from human trabecular meshwork (HTM) cells to the trabecular meshwork extracellular matrix (TEM) [2,3,4], the anatomical region believed to regulate intraocular pressure [4,5]. A gain-of-toxic-function is thought to underlie the pathophysiology of myocilin glaucoma [7,8]. Temperature-sensitive secretion of some myocilin variants [9,10] indicates that when protein production is slowed, some mutant proteins appear native-like and competent for trafficking out of the cell and to the TEM. The toxicity of mutant myocilins can be reduced by the addition of certain chemical chaperones [11,12], and in vitro, the compromised stability of mutant myoc-OLFs can be restored with some of the same compounds [13]
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