Glycosylation of lysosomal membrane protein plays a key role in colitis and pancreatitis
Postdoc fellow Janaki Sudhakar and research associate Hsueh-Han Lu, led by assistant research fellow Dr. Jr-Wen Shui at Institute of Biomedical Sciences, address those questions with animal disease models and publish in Nature Communications on Aug 27.2020. The group shows lack of glycosylation of lysosomal membrane protein or loss of glycan-binding galectin is pathologically associated with colitis and pancreatitis.
Lectins are carbohydrate-binding proteins that can recognize specific glycans for binding. Among them, galectin specifically binds to beta-galactosides through a conserved Carbohydrate Recognition Domain (CRD). Galectins are highly conserved throughout evolution and play key roles in cell function and human health.
Galectins can be secreted extracellularly to mediate important protein-protein interactions that contribute to cell functionality. Intracellular galectins are enriched in the cytosol but at the steady state the cytosol is mostly devoid of complicated glycans. These so-called cytosolic galectins are well studied to sense damaged organelles such as lysosomes and are rapidly recruited to lysosome membranes to bind to exposed glycans of lysosomal membrane proteins. The question is that , in the physiological conditions, it is unclear whether galectins reside in lysosomes, bind to glycosylated proteins, and regulate lysosome functions.
Galectin-9 binds to glycans that are specifically added to Asn(N) residues (N-linked glycans) on a protein. Galectin-9 was originally identified in a genomic screen as an autophagy-sensitive regulator that inhibits intracellular growth of Mycobacterium tuberculosis. The key function of galectin-9 in intracellular host defense likely supports why it was later reported as a risk factor for inflammatory Crohn’s disease in humans. However, how galectin-9 is involved in inflammation is unclear.
Here using genetic tools such as gene-knockout cells and mice, we reveal in gut epithelial cells, galectin-9 is indeed enriched in lysosomes at the steady state and predominantly binds to lysosome-associated membrane protein 2 (Lamp2) in a Asn(N)-glycan dependent manner.
At the cellular level, we found the galectin-9-Lamp2 interaction stabilizes lysosomes, promotes autophagy, and protects cells from endoplasmic reticulum (ER) stress-associated LMP (lysosomal membrane permeabilization) and cell apoptosis, especially in autophagy-active secretory cells, such as intestinal Paneth cells and pancreatic acinar cells.
At the molecular level, we identified N-glycosylated M6PR (mannose-6-phosphate receptor) and Lamp2 are major lysosomal binding proteins of galectin-9 in gut epithelial cells. We found the galectin-9-Lamp2 interaction contributes to endolysosome/lysosome function and cargo degradation. We also reveal that binding of Gal-9 to N-glycosylated Asn175 in Lamp2 is critical for functionality of autophagy at he physiological conditions without endolysosomal/lysosomal damage. We also provide evidence that Gal-9-mediated autophagy is indeed through its binding to poly-LacNAc chains in N-glycosylated targets.
At the global level in mice, we found the main targets of galectin-9 is intestinal Paneth cells and pancreatic acinar cells, both are autophagy-active secretory cells. While conditional deletion of galectin-9 in Paneth cells renders mice more susceptible to DSS-induced colitis, loss of galectin-9 in mice leads to spontanenous pancreatitis and fibrosis. This indicates lysosome dysfunction, due to loss of galectin-9, could serve as a shared cell-intrinsic defect in the intestine and pancreas that renders mice more susceptible to stress or organ pathogenesis.
Therefore, highly secretory cells are homeostatically being protected from ER stress and apoptosis via galectin-9-mediated autophagy that otherwise could lead to tissue inflammation or injury. Our study not only provides clues why galectin-9, via targeting and regulating autophagy in Paneth cells, is a contributing risk factor for Crohn’s disease, but also supports that IBD and extraintestinal manifestations (EIMs), such as pancreatitis, are pathologically linked.