Investigating the Molecular Mechanisms of Protein Absorption in the Vertebrate Gut by Lysosome Rich Enterocytes

The guts of neonatal mammals and stomachless fish have a limited capacity for luminal protein digestion, which allows oral acquisition of antibodies and antigens. However, this poses a challenge for dietary protein digestion and absorption during this critical developmental stage. Interestingly, it has been reported that there exist specialized intestinal cells in the ileum of suckling mammals, which are highly endocytic and possess a large lysosomal vacuole. The vacuolated enterocytes have been suggested to be responsible for protein digestion and absorption during the suckling stage. However, the molecular machinery mediating the function of the vacuolated enterocytes remained unknown. Here, we show that the vacuolated enterocytes are conserved in zebrafish as well as in suckling mammals and name them lysosome-rich enterocytes (LREs). We utilized zebrafish to investigate the molecular machinery mediating endocytic events in LREs. Using oral gavage technique and live confocal imaging, we show that LREs mediate the internalization of proteins and fluid-phase cargoes. Then, we performed RNAseq analysis on LREs and other intestine cells isolated via fluorescence-activated cell sorting (FACS). This led us to identify a conserved endocytic machinery in LREs, composed of the scavenger receptor complex Cubilin (Cubn)/Amnionless (Amn) and Dab2, that are highly enriched in LREs. By generating CRISPR/Cas9 genetic mutants of cubn, amn and dab2, we show that the Cubn/Amn receptor complex mediates protein uptake in LREs and that Dab2 mediates uptake of fluid-phase cargo as well as protein in LREs. By subjecting these genetic mutants to feeding experiments with custom diets, we show that impaired LRE uptake leads to compromised growth and survival of larval zebrafish. Moreover, impairing LRE function in suckling mice, via conditional deletion of Dab2, leads to stunted growth and severe protein malnutrition reminiscent of kwashiorkor, a devastating human malnutrition syndrome. These findings identify digestive functions and conserved molecular mechanisms in LREs that are crucial for vertebrate growth and survival.