Functional role of proteolytic processing of recombinant myocilin in self-aggregation.

Myocilin is an extracellular glycoprotein present in muscular and ocular tissues such as iris, ciliary body (CB), and trabecular meshwork (TM).1–6 It forms large molecular aggregates in the aqueous humor (AH).7–9 Mutations in the myocilin (MYOC) gene cause autosomal dominant juvenile glaucoma, and they are also present in a reduced proportion (3%–5%) of adult-onset primary open-angle glaucoma (POAG) in different populations.10–12 POAG is a heterogeneous disease originated by the progressive apoptosis of the optic nerve,13 which constitutes a leading cause of blindness in developed countries. Myocilin presents a modular design composed of the N-terminal region (amino acids 1-202), the central linker domain (amino acids 202-244),14,15 and the compact16 C-terminal olfactomedin-like domain (amino acids 244-504).3 The N-terminal domain contains two coiled-coil (CC) domains17 and a leucine zipper-like motif3 in the second CC. These three modules are encoded by exons 1, 2, and 3 and coincide approximately with three independent folding domains.17 We have reported that recombinant myocilin undergoes an intracellular endoproteolytic cleavage by calpain II in the central linker domain of the polypeptide chain.15 The processing takes place in the endoplasmic reticulum and releases two fragments that contain the N- and C-terminal structural domains. The C-terminal fragment has been detected in both human and bovine CB and the AH, indicating that the proteolytic processing also occurs in ocular tissues.15 Of note, photomedin-1 and gliomedin, two olfactomedin-like domain–containing proteins, are cleaved in a similar fashion.18,19 Pathogenic mutations located in the olfactomedin-like domain reduce the specific cleavage of the recombinant protein.15 Despite many efforts having being made since the discovery of MYOC as a glaucoma gene in 1997,10 the function of this protein in normal and glaucomatous eyes remains poorly understood. Similarly, the functional meaning of the proteolytic processing of myocilin is currently unknown, although it has been suggested to contribute to the modulation of myocilin interactions.15 In the present study, the specific proteolytic cleavage of recombinant myocilin reduced its extracellular covalent aggregates. In addition, the results revealed the existence of noncovalent interactions between myocilin aggregates, which may play an important role in the extracellular function of the protein.