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Abstract

Glycosylation is a ubiquitous post-translational modification essential for protein folding, stability, and function. Defects in glycosylation pathways cause congenital disorders of glycosylation (CDGs), multisystem diseases with diverse symptoms. Among these, ocular defects are particularly pronounced, often resulting in photoreceptor degeneration and retinopathies. In a model for ALG2-CDG (Alpha-1,3/1,6-mannosyltransferase-CDG) in medaka (Oryzias latipes) it was observed that photoreceptors are especially sensitive to impaired glycosylation. Therefore, I hypothesize that specific glycoproteins are critical for maintaining photoreceptor stability, and that proper N-glycosylation is required for their folding, function, and the preservation of retinal structure and visual performance. To address this, I developed a multilayer workflow to identify candidate genes with retina-specific expression and potential N-glycosylation dependence. Candidates were prioritized through comparative database analysis and CRISPR-based functional analysis, and precise interventions at predicted glycosylation sites were designed. This strategy allowed to study how specific molecular perturbations affect protein function and retinal development. The analyses revealed that perturbation of potential N-glycosylation sequons can substantially alter protein conformation and photoreceptor organization. Even single amino acid substitutions propagated structural changes that influenced photoreceptor differentiation and outer nuclear layer organization. Computational simulations using AlphaFold and GlycoShape provided structural insight, showing how Asn→Gln substitutions can affect folding, polarity, and potential protein interactions, providing a first insight into the observed developmental defects. These findings highlight the essential role of N-glycosylation in photoreceptor homeostasis and demonstrate the utility of medaka as a versatile in vivo model for studying post-translational modifications. By integrating computational and experimental approaches this work provides a framework to link subtle molecular changes to structural and functional outcomes in the retina, offering insight into the mechanisms underlying retinal development and disease.