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Editors Selection IGR 13-2

Basic Reseach: Myocillin

Comment by Daniel Stamer on:

12150 Myocilin mutations causing glaucoma inhibit the intracellular endoproteolytic cleavage of myocilin between amino acids Arg²²⁶ and Ile²²⁷, Aroca-Aguilar JD; Sanchez-Sanchez F; Ghosh S et al., Journal of Biological Chemistry, 2005; 280: 21043-21051

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Aroca-Aguilar et al. (450) characterize a proteolytically sensitive site in myocilin, the protein product of GLC1A that results in two peptide fragments of 20 and 35 kDa. In human tissues and aqueous humor samples the authors show that a small portion of endogenous/full-length myocilin (~1-15%) is found as a 35 kDa fragment. Using recombinant myocilin transiently expressed by different cell lines, a cleavage site between amino acids 226 and 227 was identified. This location corresponds to an exposed stretch of amino acids between two major folding domains of myocilin, a coiled-coil and an olfactomedin module (35 kDa fragment). When a deletion mutant was created to eliminate the site, myocilin was no longer susceptible to digestion.

The authors hypothesize that regulated intracellular cleavage of myocilin impacts extracellular function. The purpose of their study was to test whether myocilin was processed intracellularly in a specific manner and executed an experimental strategy designed to distinguish between artifactual proteolysis (as previously suggested) and site-specific processing. Since proteolysis can occur rapidly for susceptible proteins during sample preparation, specific controls are necessary. In response, a spectrum of protease inhibitors was included in the culture medium as well as in cell lysis buffers. Upon inhibition of secretory pathways of mammalian cells proportionally more of the 35 kDa fragment appeared intracellularly. The authors interpreted these data to mean that the fragment originates intracellularly. In agreement with this idea, ex pression of recombinant myocilin containing disease-causing mutations was protected from digestion, associated with the cytoskeleton (triton-insoluble) and was not secreted efficiently as described before. The authors suggest that misfolding or aberrant binding inhibited access of proteases to mutant myocilin.

The study by Aroca-Aguilar et al. opens up new ways to think about myocilin and its role in glaucoma previously not considered. For example, why is only a small proportion of myocilin processed? Why is there proportionally more myocilin fragments compared to full-length found extracellularly? Since perfusion of the olfactomedin module (35 kDa fragment) into the conventional pathway does not alter outflow facility by itself, what role does it have in outflow function?

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