Mechanisms of NO-Mediated Protein S-Nitrosylation in the Lens-Induced Myopia

Myopia is a chronic ocular disease, emerging as the most common type of refractive error. This study intends to preliminarily explore the roles of protein S-nitrosylation of nitric oxide (NO) in the regulation of myopia by detecting the expression of neuronal nitric oxide synthase (nNOS) and downstream S-nitrosylation, using the animal model of lens-induced myopia (LIM) in mice.

The 3-week-old C57BL/6 J mice were divided into three groups: group I, lens-induced 0-week group (take eyeballs at the age of 3 weeks); group II, self-control eyes of experimental group (take eyeballs at the age of 7 weeks); and group III, lens-induced 4-week group (take eyeballs at the age of 7 weeks). The diopter and axial length of each group were measured by streak retinoscopes and optical coherence tomography (OCT) before and after model establishment. The protein expressions and locations of nNOS and S-nitrosylated proteins (PSNOs) were measured by western blot and immunofluorescence staining. Site-specific proteomic for protein S-nitrolysation was used to detect the existence and location of S-nitrosylation proteins in the retina of myopic and nonmyopic mice. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and motif enrichment analyses were performed. The differential sites were analyzed by GO, KEGG, and motif. Irreversible biotinylation procedure combined with protein purification and western blot was used to detect the protein expression of α-enolase (ENO1), a key player in the hypoxia-related signal pathway.

The expressions of nNOS and PSNOs were significantly lower in the retina of experimental eyes than that in self-control eyes and 3-week-old baseline group. A total of 595 S-nitrosylated proteins, 709 S-nitrosylated peptides, and 708 S-nitrosylated sites were identified by site-specific S-nitrolysation proteomics in the retina of myopic and control eyes. A total of 19 differentiation loci were screened, of which 13 sites were downregulated and 6 sites were upregulated in experimental eyes compared with the self-control group. Specifically, the expression of SNO-ENO1 was significantly lower in the retina of experimental eyes than that in self-control eyes and 3-week-old baseline group.

LIM induces the decrease of nNOS and PSNO protein levels in the retina of myopic mice. NO-mediated nonclassical protein S-nitrosylation modification may play an important role in the regulation of lens-induced myopia. ENO1 may be a key factor in the regulation of S-nitrosylation modification of myopia.