Cyclic stretch increases transglutaminase 2-mediated serotonylation of extracellular fibronectin in cultured optic nerve head astrocytes: a pilot study

Background: Glaucoma is a leading cause of blindness characterized by progressive degeneration of the optic nerve. Elevated intraocular pressure, typically associated with glaucoma, exerts biomechanical strain on the optic nerve head, where mechanosensitive astrocytes contribute to profibrotic extracellular matrix remodeling. Cross-linking enzymes such as transglutaminase 2 play a role in this process. A unique function of transglutaminase 2 is its ability to catalyze the post-translational modification of proteins through the transamidation of serotonin onto glutamine residues ("serotonylation"). The serotonylation of extracellular matrix proteins, such as fibronectin, could plausibly be involved in extracellular matrix remodeling. Here, we investigated whether optic nerve head astrocytes could serotonylate extracellular proteins in response to a biomechanical insult. Results: Primary human optic nerve head astrocytes subjected to 0-12% cyclic stretch for 24 h exhibited increased extracellular levels of transglutaminase 2 and serotonin. Correspondingly, serotonylation of immunoprecipitated fibronectin samples, detected by immunoblotting, was 2.5-fold higher in stretched cells. Serotonylation of specific glutaminyl residues of fibronectin due to the catalytic effect of transglutaminase 2 was also proven though an in vitro experiment using nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS). Conclusions: These findings demonstrate that fibronectin can be serotonylated by optic nerve head astrocytes and that this modification is enhanced by biomechanical stress. While transglutaminase 2 is classically associated with extracellular matrix cross-linking, these results suggest that serotonylation may represent an additional and unexplored mechanism by which transglutaminase 2 contributes to extracellular matrix remodeling in the glaucomatous optic nerve head.