Expression and Function of the Lipocalin-2 (24p3/NGAL) Receptor in Rodent and Human Intestinal Epithelia

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.

Cadmium Handling, Toxicity and Molecular Targets Involved during Pregnancy: Lessons from Experimental Models

“…Placenta has been showed to highly express megalin in cytotrophoblasts during first and third trimester [ 65 ]: megalin is a transmembrane receptor which mediates uptake and trafficking of many ligands including MT in kidney and other tissues [ 66 , 67 , 68 ]. However, other transporters that participate in receptor-mediated endocytosis of MT could be also involved [ 69 , 70 ]. These findings can be relevant for women in reproductive age who have been exposed chronically to Cd because this behavior increases the risk of Cd-induced toxicity in the kidneys and placenta during gestation.…”

Expression and localisation of brain-type organic cation transporter (BOCT/24p3R/LCN2R) in the normal rat hippocampus and after kainate-induced excitotoxicity

“…Interaction between LCN2 and BOCT has been proposed (Devireddy et al, 2005, Fang et al, 2007. Recent reports characterised BOCT as a promiscuous high-affinity, multi-ligand receptor for internalisation of proteins and peptides in the distal nephron and intestinal epithelium (Langelueddecke et al, 2012, Langelueddecke et al, 2013. However, the possibility of interaction between LCN2 and BOCT is not without controversy.…”

Biochemical and Structural Characterization of the Interaction between the Siderocalin NGAL/LCN2 (Neutrophil Gelatinase-associated Lipocalin/Lipocalin 2) and the N-terminal Domain of Its Endocytic Receptor SLC22A17

“…Microscale thermophoresis measurements ( 39 , 40 ) were performed on a NanoTemper Monolith NT.115 instrument (NanoTemper Technologies GmbH, Munich, Germany), as previously described ( 19 , 20 ). For the experiment purified proteins were diluted in 20 m m Tris/HCl, pH 7.4, combined at a fixed concentration of GFP-hLCN2-R-NTD (500 n m ) and varying concentrations of NGAL or NGAL/[Fe III (Ent)] 3− (0–300 μ m ), allowed to reach steady state, and loaded into standard treated capillaries (catalog #K002) by capillary action.…”

“…Despite the important body of literature establishing the involvement of NGAL and LCN2-R in inflammation and cancer, little is known about the biochemistry of the interaction between NGAL and its cellular receptor. It has been shown that 24p3-R binds NGAL with a very high affinity ( K D ≈ 90 p m ) ( 16 ) and that 24p3-R also exhibits high avidities for various ligands (such as transferrin, albumin, or metallothionein) ( 17 – 20 ). However, in a recent paper, Correnti et al ( 21 ) questioned whether NGAL is truly able to shuttle iron in and out of the cell based on 1) their observation that gentisic acid (the putative mammalian siderophore) could not form a stable ternary complex with NGAL and iron and 2) their inability to demonstrate any physical interaction between NGAL and mLCN2-R.…”