Abstract
The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC3), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC3 to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC3 or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC3 and MT (0.7 µM) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an EC 50 of 18.6 ± 12.2 nM was determined for binding of Alexa 488-PC3 to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC3/MT/Tf (0.7 µM). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC3 and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.
Highlights
Little is known about the transepithelial transport and absorption of proteins in the intestine
We have previously demonstrated that MT and Tf are ligands of rodent 24p3-R (r24p3-R)/human NGAL-R (hNGAL-R) [24], we investigated whether hNGAL-R-mediated uptake of PC3, MT and Tf is associated with transcytosis of these proteins in colon-like Caco-2 BBE cells
In rat and mouse intestine, expression of r24p3-R increased aborally (Figures 1 and 2) with a predominant localization at the luminal cell side of the enterocytes in the ileal and colonic mucosa. This was further supported by the surface expression of hNGAL-R in human colon (Figure 3) and in colonic-like Caco-2 BBE cells whereas duodenum-like Caco-2 BBE cells were devoid of hNGAL-R (Figure 4)
Summary
Little is known about the transepithelial transport and absorption of proteins in the intestine. Neonates have the ability to absorb immunoglobulins from the intestine as a means of passive immunization [1,2]. Viruses, such as HIV, may infect the host by transcytosis across the intestinal mucosa [3]. Non-digested dietary components, such as plant components, can be degraded in the ileum and large intestine by microbial fermentation and serve as a source of energy and nutrients for host metabolism [5,6]. Once the complex carbohydrates of the plant wall have been broken down by the intestinal microbiota, released plant proteins may be reabsorbed or undergo proteolysis by the large intestine microbiota [7]. A significant part of plant-derived toxic cadmium-bound phytochelatins (PCs) and metallothioneins (MTs) are absorbed intact by enterocytes and are found subsequently in the kidney [8,9]
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