Deorphanizing Enzymes: Characterization of NXPE1 and Its Role in Mucin Glycosylation and Ulcerative Colitis Pathogenesis

Abstract

Orphan enzymes represent a largely unexplored frontier in molecular biology, with their unknown substrates and functions posing significant challenges and opportunities for understanding human health and disease. This thesis investigates the biochemical role of NXPE1, a gene implicated in ulcerative colitis (UC), to uncover its enzymatic function and physiological relevance. NXPE1 encodes an O-acetyltransferase that selectively modifies the sialic acid N-acetylneuraminic acid (Neu5Ac) at the 9-OH position, producing Neu5,9Ac2. This acetylation process, localized to the Golgi apparatus of colonic epithelial cells, is critical for modulating the biophysical properties of mucus glycoproteins. Using a multidisciplinary approach that integrates in silico modeling, activity-based protein profiling (ABPP), and organoid-based in vitro validation, this study demonstrates that the UC-protective variant NXPE1 G353R disrupts enzyme stability and acetylation activity. Key findings reveal that NXPE1-mediated sialic acid acetylation is essential for maintaining the structural and functional integrity of the colonic mucosal barrier. The absence of NXPE1 or loss of function due to genetic variation correlates with increased mucus viscosity and altered mucosal sialoglycome, which may protect individuals from inflammation and dysbiosis. These findings not only elucidate a mechanistic basis for the protective effect of NXPE1 variants in UC but also highlight the broader significance of sialic acid modifications in health and disease. By deorphanizing NXPE1, this thesis advances our understanding of enzyme function and its implications for therapeutic strategies targeting inflammatory bowel diseases.