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J Am Soc Nephrol 14:1123-1131, 2003
© 2003 American Society of Nephrology

Role of Megalin in Endocytosis of Advanced Glycation End Products: Implications for a Novel Protein Binding to Both Megalin and Advanced Glycation End Products

Akihiko Saito*, Ryoji Nagai{dagger}, Atsuhito Tanuma*, Hitomi Hama*, Kenji Cho*, Tetsuro Takeda*, Yutaka Yoshida{ddagger}, Tosifusa Toda§, Fujio Shimizu, Seikoh Horiuchi{dagger} and Fumitake Gejyo*

*Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; {dagger}Department of Biochemistry, Kumamoto University School of Medicine, Kumamoto, Japan; {ddagger}Department of Renal Pathology, Institute of Nephrology, Faculty of Medicine, Niigata University, Niigata, Japan; §Department of Gene Regulation and Protein Function, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; and Department of Cell Biology, Institute of Nephrology, Faculty of Medicine, Niigata University, Niigata, Japan.

Correspondence to Dr. Akihiko Saito, Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata, 951-8510, Japan. Phone: 81-25-227-2200; Fax: 81-25-227-0775;


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Advanced glycation end products (AGE) are filtered by glomeruli and reabsorbed and metabolized by proximal tubule cells (PTC). In renal failure, decreased renal AGE metabolism likely accounts for the accumulation in serum that is related to uremic complications. In diabetes, AGE generation is increased, and the handling mechanisms in PTC are likely associated with the pathogenesis of tubulointerstitial injury. It is therefore important to clarify the mechanisms of the AGE metabolism to develop a strategy for removing AGE in uremia and to elucidate the pathogenesis of diabetic nephropathy. To this end, this study focused on the molecular analysis of megalin, a multi-ligand endocytic receptor, in PTC. AGE uptake analysis was performed using the rat yolk sac-derived L2 cell line system established for the analysis of megalin’s endocytic functions. The cells mediated specific internalization and degradation of AGE, which were significantly blocked by anti-megalin IgG, indicating that megalin is involved in the cellular processes. However, cell surface AGE-binding assays and ligand blot analysis revealed no evidence that megalin is a direct AGE receptor. Affinity chromatography and ligand blot analysis originally revealed that 200-kD and 400-kD proteins in the cells bind to AGE and the 200-kD protein to megalin in a Ca2+-dependent manner. The binding of megalin with the 200-kD protein was suppressed by receptor-associated protein (RAP), a ligand for megalin. In conclusion, megalin functions for endocytosis of AGE via an indirect mechanism. L2 cells express novel AGE-binding proteins, one of which may interact with megalin. E-mail: akisaito@med.niigata-u.ac.jp


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Megalin was originally identified as a target antigen of rat Heymann nephritis, an experimental model of membranous nephropathy (1). It is a large (approximately 600 kD) glycoprotein belonging to the LDL receptor gene family (2). Megalin is located at the clathrin-coated pits, internalizes the ligands into the endocytic compartments, and is recycled to the cell surface (3,4). It is expressed abundantly at the apical membranes of proximal tubule cells (PTC) that normally reabsorb and metabolize low–molecular weight proteins (LMWP) filtered by glomeruli (3). Megalin is known to serve as a major receptor for endocytosis of multiple LMWP, including transcobalamin-B12 (5), vitamin D-binding protein (6), retinol-binding protein (7), parathyroid hormone (8), insulin, {beta}2-microglobulin ({beta}2-m), epidermal growth factor, prolactin, lysozyme, cytochrome c (9), {alpha}1-microglobulin, PAP-1, odorant-binding protein (10), and transthyretin (11). In renal failure, LMWP accumulate in serum and tissues; some of them, such as {beta}2-m causing dialysis-related amyloidosis (DRA) (12), act as uremic toxin proteins associated with complications in patients. Parathyroid hormone is also recognized as a uremic toxin (13). Megalin thus appears to be a useful therapeutic molecular tool to remove such uremic toxin proteins in uremia, and we recently developed a novel cell therapy model by subcutaneous implantation of megalin-expressing cells to metabolize {beta}2-m in renal failure (14).

Advanced glycation end products (AGE), involved in the pathogenesis of diabetic complications (15), are also filtered by glomeruli and reabsorbed and metabolized by PTC (16,17). In renal failure, AGE increase in serum and are associated with the pathophysiology of uremic complications such as DRA (18) and atherosclerosis (19), suggesting that they also represent a uremic toxin (20,21). Decreased renal metabolism of AGE is likely to be associated with the serum accumulation in uremia. Elucidation of the mechanisms of the cellular endocytosis of AGE is also useful to establish a strategy for removing AGE in uremia, as we suggested for metabolizing {beta}2-m using megalin-expressing cells (14). We therefore designed this study to investigate the role of megalin in the endocytosis of AGE.

AGE are involved in the pathogenesis of diabetic glomerulopathy (22,23) as well as tubulopathy (24–27). Recently, AGE were demonstrated to mediate proximal tubule epithelial-myofibroblast transdifferentiation via the receptor for AGE (RAGE) (28), a cell-surface signaling receptor (29). However, the molecular mechanisms of AGE endocytosis by PTC, which should regulate cellular AGE uptake and determine the amounts of AGE available for stimulation of the cell surface receptor, have not been elucidated. Including RAGE, several AGE-binding proteins have been identified, such as galectin-3 (30), macrophage scavenger receptor class A types I and II (SR-A) (31,32), CD36 (33), and scavenger receptor class B type I (SR-BI) (34), although they have not been reported to be expressed by PTC.

In this study, we used a rat yolk sac-derived L2 cell culture system, which has been well characterized for studies of endocytic functions of megalin (4,35,36). We show that the cells specifically take up and degrade AGE and that megalin is involved in these processes. In addition, we demonstrate that the cells express novel AGE-binding proteins and that one of them binds to megalin.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Na 125I (IODINE-125, 3.7 GB/ml), redivue Pro-mix L-[35S] in vitro cell labeling mix (530 MBq/ml), CNBr-activated Sepharose 4B, and Hyperfilm MP were obtained from Amersham Pharmacia Biotech UK Limited (Little Chalfont, Buckinghamshire, England). Iodo-Beads were purchased from Pierce (Rockford, IL). Dulbecco modified Eagle medium (DMEM) (high glucose) and fetal calf serum (FCS) were obtained from Life Technologies BRL (Rockville, MD). Bovine serum albumin (BSA, Fraction V), chloroquine, and leupeptin were from Sigma (St. Louis, MO). Centriplus-100 was purchased from Millipore (Bedford, MA). Ready Gel J, Immun-Blot polyvinylidene difluoride (PVDF) membrane, Coomassie Stain Solution, and Coomassie R-250 Destain Solution were obtained from Bio-Rad Laboratories (Hercules, CA).

Protein Purification
AGE-BSA was prepared as described previously (31–34,37). Recombinant rat receptor–associated protein (RAP) was prepared using a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST) as described previously (38). Rat megalin was prepared from renal microvillar membranes by affinity chromatography using monoclonal antibody 20B as described previously (39). Anti-rat megalin rabbit sera were raised as described previously (35), and protein A-purified IgG was prepared as described previously (40). Nonimmune rabbit IgG was also prepared.

Radioiodination
Proteins (100 µg) were radioiodinated using 1 mCi Na 125I and one Iodo-Bead according to the manufacturer’s instructions. The specific activities of 125I-AGE-BSA, 125I-RAP, and 125I- megalin were 2.9 x 103, 4.0 x 103, and 4.5 x 103 cpm/ng protein, respectively.

Ligand Uptake Analysis
Rat yolk sac tumor-derived L2 cells (41) were grown (37°C; 5% CO2) to confluence (1 x 105 cells/well) in DMEM supplemented with 10% FCS on 12-well tissue culture plates coated with 0.1% gelatin. The cells were washed with DMEM and incubated in DMEM containing 2% BSA with 125I-AGE-BSA (1.5 µg/ml). The cell incubation was carried out at 37°C in 5% CO2 in the absence or presence of competitors: unlabeled AGE-BSA, anti-rat megalin rabbit IgG, rabbit nonimmune IgG, RAP, and GST. The cells were also incubated in the presence of chloroquine or leupeptin, inhibitors of lysosomal enzyme activity (42). The media containing 125I-AGE-BSA were also incubated on cell-free gelatin-uncoated plates to measure the spontaneous degradation of the radiolabeled protein. After incubation, the culture media were precipitated with 15% TCA in the presence of 1% BSA and the radioactivity levels of the degradation products in the supernatants were counted. Cell-mediated degradation of 125I-AGE-BSA was determined by subtracting degradation in the absence of cells. The cells were washed with ice-cold PBS, and cell-associated radioactivity was measured following solubilization of the cells with 1 N NaOH. Statistical analyses were carried out using the unpaired t test.

Cell Surface Ligand Binding Analysis
L2 cells were grown to confluence as above on 12-well tissue culture plates coated with 0.1% gelatin. The cells were washed twice with the binding buffer (10 mM HEPES, 150 mM NaCl, 2 mM CaCl2, pH 7.4, at 25°C) and incubated in the buffer containing 2% BSA at 37°C in 5% CO2 for 20 min. The cells were then washed twice with the ice-cold binding buffer containing 2% BSA. The cells were incubated with 1.5 µg/ml 125I-AGE-BSA or 125I-RAP at 4°C for 4 h in the buffer containing 2% BSA with or without anti-rat megalin rabbit IgG (200 µg/ml) or rabbit nonimmune IgG (200 µg/ml). After incubation, the cells were washed twice with the ice-cold binding buffer and solubilized with 1 N NaOH for radioactivity counting. Statistical analyses were carried out using the unpaired t test.

Preparation of Membrane and Cytosolic Proteins of L2 Cells
Cultured L2 cells were homogenized in ice-cold binding buffer containing 0.2 mM phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin, and centrifuged at 600 x g for 5 min to prepare postnuclear supernatants (PNS) (43). The PNS were centrifuged at 100,000 x g to prepare membrane (pellet) and cytosolic (supernatant) fractions. The membrane proteins were solubilized with the binding buffer containing 1% Triton X-100. 35S-labeled L2 cell membrane and cytosolic proteins were prepared using the cells cultured in the medium containing 2.1 MBq/ml redivue Pro-mix L-[35S] in vitro cell labeling mix.

AGE-BSA-Sepharose Affinity Chromatography
CNBr-activated Sepharose 4B was used to conjugate BSA-AGE according to the manufacturer’s protocol. Fifty microliters of the AGE-BSA Sepharose 4B was washed in an Eppendorf tube twice with 1 ml of the binding buffer containing 1% Triton X-100. The Sepharose was used for incubation with the 35S-labeled L2 cell membrane and cytosolic proteins (5 mg/ml each) separately in the same Sepharose/protein ratio in the binding buffer containing 1% Triton X-100 and 2% BSA at 4°C for 14 h with gentle shaking. The Sepharose was washed twice with the binding buffer containing 1% Triton X-100 and equally aliquoted in Eppendorf tubes. An aliquot of the Sepharose was incubated in 2 x Laemmli SDS-PAGE sample buffer containing 4% {beta}-mercaptoethanol to elute the proteins bound to the Sepharose both specifically and nonspecifically. The elution was mixed with the same volume of the binding buffer before heating at 95°C for 5 min and applying onto SDS-PAGE. The other aliquots were used for elution with 0.5 mg/ml AGE-BSA, 0.5 mg/ml BSA, respectively, in the binding buffer or the binding buffer containing 20 mM EDTA. The eluted samples were mixed with the same volume of 2 x Laemmli SDS-PAGE sample buffer containing 4% {beta}-mercaptoethanol, heated at 95°C for 5 min, and used for SDS-PAGE.

Preparation of the AGE-Binding Proteins
To prepare the AGE-binding proteins, the PNS of L2 cells was used for binding to the AGE-BSA Sepharose 4B at 4°C for 14 h in the binding buffer containing 1% Triton X-100 and 2% BSA. The proteins bound to the Sepharose were eluted with 100 mM Tris-HCl and 20 mM EDTA (pH 7.4 at 25°C). The eluted solution was concentrated using Centriplus-100 (Millipore, Bedford, MA). The Sepharose was washed with the binding buffer and stored at 4°C in the presence of 0.02% NaN3.

Ligand Blot Analysis
Megalin (10 µg) was prepared in Laemmli sample buffer containing no {beta}-mercaptoethanol and resolved by 4% SDS-PAGE. The protein was then transferred to PVDF membranes. Nonspecific sites on membranes were blocked by incubation in the binding buffer containing 0.2% Tween 20 (buffer A) and 3% BSA. The membranes were then incubated with 125I-AGE-BSA or 125I-RAP (1 x 106 cpm/ml) in buffer A with 3% BSA for 2 h at 25°C, washed with buffer A (4 times, 15 min each), air dried, and exposed to Hyper film at -80°C with an intensifier screen.

The proteins (30 µg) specifically eluted from AGE-BSA Sepharose 4B with EDTA were also dissolved in the Laemmli sample buffer supplemented with 4% {beta}-mercaptoethanol, heated at 95°C for 5 min, resolved by 4% SDS-PAGE, and transferred to PVDF membranes. The protein blotting was confirmed by staining the membranes with Coomassie Stain Solution and washing with Coomassie R-250 Destain Solution. The membranes were then incubated with 125I-AGE-BSA (1 x 106 cpm/ml) in the presence or absence of unlabeled AGE-BSA (35 µg/ml) or EDTA (20 mM), washed, and subjected to autoradiography as described above. Also, the membranes were used for ligand blot analysis by incubation with 125I-megalin (1 x 106 cpm/ml) in the presence or absence of RAP (300 nM) or EDTA (20 mM).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Megalin-Dependent Cellular Uptake and Metabolism of AGE
In this study using megalin-expressing L2 cells, we used nonspecifically modified AGE-BSA for the following reasons. First, BSA per se is not significantly taken up by L2 cells (data not shown). Therefore, BSA-AGE is an appropriate tool to determine the specific effect of AGE-modification on the cellular uptake and metabolism in the cells. Second, nonspecifically modified AGE-BSA is the most general material available for cellular AGE receptor analysis.

To investigate whether megalin, a multi-ligand endocytic receptor, is involved in the AGE uptake and metabolism in the cells, we performed 125I-AGE-BSA uptake assays using anti-megalin IgG as a specific competitor (Figure 1, A and B). Cell association and degradation of 125I-AGE-BSA were significantly suppressed by the addition of anti-megalin IgG to the culture medium compared with the addition of nonimmune IgG, demonstrating that megalin is involved in the uptake and metabolism of AGE in L2 cells. However, the addition of recombinant RAP, an inhibitor of megalin’s binding to its ligands, decreased the degradation but increased the cell-association, compared with the addition of GST (Figure 1, A and B). This result suggests that blocking megalin with RAP may inhibit the cellular internalization or intracellular metabolism of AGE. It also suggests that the involvement of megalin in the cellular uptake and metabolism of AGE differs from the processes for the ligands that directly bind to megalin at its RAP-binding sites.



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  		Figure 1. L2 cell uptake analysis for endocytosis of 125I-AGE-BSA. Cultured L2 cells were incubated with 125I-AGE-BSA (1.5 µg/ml) at 37°C in DMEM containing 2% bovine serum albumin (BSA) for 2 h for degradation (A) and cell association (B) assays in the absence (control) or presence of competitors (300 µg/ml each) as indicated. The addition of anti-megalin IgG to the culture medium inhibited degradation and cell association of 125I-AGE-BSA, compared with the addition of nonimmune IgG, indicating that megalin is involved in the cellular uptake and metabolism of advanced glycation end products (AGE). The addition of receptor-associated protein (RAP; 300 µg/ml) suppressed degradation but increased cell association, compared with the addition of glutathione S-transferase (GST), suggesting that RAP inhibits the cellular internalization or intracellular metabolism of AGE. *P < 0.01. (C) The cells were incubated as well with 125I-AGE-BSA (1.5 µg/ml) for 4 h in the presence of chloroquine or leupeptin (100 µM each), inhibitors of lysosomal activities. Both chloroquine and leupeptin suppressed the AGE degradation ({blacksquare}) while the latter increased the cell association ({square}) (*P < 0.01 versus control), confirming that the degraded products were the results of receptor-mediated endocytosis and lysosomal degradation. Values (means ± SD, n = 4) are expressed relative to the control.

 
Receptor-mediated endocytosis and lysosomal degradation of AGE-BSA in L2 cells were tested by the ligand uptake analysis using chloroquine, a membrane-diffusible base which raises pH in intracellular compartments, and leupeptin, a membrane-diffusible proteinase inhibitor (42) (Figure 1C). Both reagents significantly suppressed 125I-AGE-BSA degradation, while only the latter increased the cell association. These results are consistent with the previous report (42), and they confirm the endocytosis and lysosomal degradation of AGE-BSA because chloroquine is supposed to inhibit endosomal and lysosomal activities and leupeptin to accumulate endocytosed, undegraded proteins in lysosomes.

Megalin Is Not Identified as a Direct Cell Surface Receptor for AGE-BSA
To investigate whether megalin is a direct AGE receptor, cell surface ligand binding analysis was carried out using L2 cells incubated at 4°C with 125I-AGE-BSA in the presence of 2% BSA (Figure 2A). First, the addition of unlabeled AGE-BSA to the incubation buffer was shown to significantly suppress the 125I-AGE-BSA cell surface binding, indicating the presence of a specific L2 cell surface receptor for AGE. However, the addition of anti-megalin IgG to the incubation buffer failed to suppress the specific binding. As a control, 125I-RAP was shown to specifically bind to L2 cells at 4°C, and the binding was significantly suppressed by the addition of anti-megalin IgG (Figure 2B). In addition, the ligand blot analysis revealed that 125I-AGE-BSA did not bind to megalin, whereas 125I-RAP bound to it under the same conditions (Figure 3). We also performed surface plasmon resonance analysis to investigate whether megalin binds to AGE-BSA, but we did not find evidence of direct binding (data not shown). These findings indicate that megalin is not identified as a direct cell surface receptor for AGE-BSA.



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  		Figure 2. L2 cell surface AGE-binding analysis. (A) Cultured L2 cells were incubated at 4°C for 4 h with 125I-AGE-BSA (1.5 µg/ml) for cell surface binding in the binding buffer (10 mM HEPES, 150 mM NaCl, 2 mM CaCl2, pH 7.4, at 25°C) containing 2% BSA. Addition of unlabeled AGE-BSA (150 µg/ml) into the incubation buffer suppressed the binding (*P < 0.01 versus control), indicating the presence of a specific L2 cell surface receptor for AGE. However, the anti-megalin IgG (200 µg/ml) did not inhibit the binding, suggesting that megalin is not involved in the cell surface binding of AGE. (B) As a control, the cell surface binding of 125I-RAP (1.5 µg/ml) was specifically inhibited by unlabeled RAP (150 µg/ml) (*P < 0.01 versus control), and the specific binding was significantly inhibited by anti-megalin IgG (200 µg/ml) compared with nonimmune IgG (200 µg/ml) (**P < 0.01). Values (means ± SD, n = 4) are expressed relative to the control.

 


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  		Figure 3. Ligand blot analysis of megalin with 125I-AGE-BSA and 125I-RAP. Megalin was resolved by 4% SDS-PAGE under nonreducing conditions and blotted to polyvinylidene difluoride (PVDF) membranes, followed by binding with 125I-AGE-BSA and 125I-RAP, respectively (1 x 106 cpm/ml), in the binding buffer containing 0.2% Tween 20 and 3% BSA. Megalin was bound with 125I-RAP but not with 125I-AGE-BSA.

 
Identification of Ca2+-Dependent AGE-Binding Proteins in L2 Cells
To identify the AGE-binding receptors in L2 cells that may cooperate with megalin, affinity chromatography was carried out using AGE-BSA-conjugated Sepharose 4B to find L2 cell proteins that bind to AGE (Figure 4A). The Sepharose was incubated with 35S-labeled L2 cell membrane and cytosolic proteins for 14 h at 4°C in the binding buffer (10 mM HEPES, 150 mM NaCl, 2 mM CaCl2, pH 7.4, at 25°C) containing 1% Triton X-100 and 2% BSA. After washing the Sepharose with the buffer, AGE-binding proteins were specifically eluted from the Sepharose with AGE-BSA but not with BSA. The eluted proteins included three large-molecular weight ones (190, 200, and 400 kD, respectively, shown under the reducing condition in 4% SDS-PAGE). These proteins were also eluted from the Sepharose with EDTA, indicating that the AGE binding is Ca2+-dependent. In non-reducing 4% SDS-PAGE, the three proteins were stacked at the gel top (Figure 4B), suggesting that they may form a complex.



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  		Figure 4. Identification of three proteins in L2 cells by AGE-BSA affinity chromatography. (A) 35S-labeled L2 cell membrane (lane 1) and cytosolic (lane 6) proteins were incubated with AGE-BSA-Sepharose 4B for 14 h at 4°C in the binding buffer containing 1% Triton X-100 and 2% BSA. The proteins were eluted from the Sepharose with Laemmli sample buffer containing {beta}-mercaptoethanol (lanes 2 and 7); and the binding buffer containing 0.5 mg/ml AGE-BSA (lanes 3 and 8), 20 mM EDTA (lanes 4 and 9), and 0.5 mg/ml BSA (lanes 5 and 10) for gel loading. Three proteins (190-kD, 200-kD, and 400-kD) were specifically eluted from the Sepharose with AGE-BSA but not with BSA, indicating that the proteins have AGE-binding properties. The three proteins were also eluted with EDTA, suggesting that the AGE-binding is Ca2+-dependent. (5 to 20% SDS-PAGE under reducing conditions followed by autoradiography.) (B) The three proteins bound to the Sepharose were eluted with EDTA and resolved by 4% SDS-PAGE under reducing (lane 1, arrows) and nonreducing (lane 2, arrowhead) conditions. The proteins were separated under the reducing condition, but stacked at the gel top under the nonreducing condition, suggesting that they may form a complex.

 
To examine whether each of the three proteins directly binds to AGE, we performed ligand blot analysis (Figure 5A): The proteins were reduced with 4% {beta}-mercaptoethanol, separated by SDS-PAGE, and blotted to PVDF membranes. After blocking with the binding buffer containing 0.2% Tween 20 and 3% BSA, the membranes were incubated with 125I-AGE-BSA in the same buffer in the presence or absence of unlabeled AGE-BSA or EDTA. The ligand blot analysis showed that 125I-AGE-BSA specifically bound to the 200-kD and 400-kD proteins but not to the 190-kD protein, and the binding was blocked by EDTA, indicating that the AGE-binding is Ca2+-dependent. The 190-kD protein did not appear to be directly bound by AGE, suggesting that it may be associated with either the 200-kD or 400-kD proteins. The 200-kD and 400-kD proteins are very likely novel AGE-binding proteins; to our knowledge, there have been no reports of AGE-binding proteins with such molecular weights and properties.



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  		Figure 5. Ligand binding analysis of the AGE-binding proteins. The 190-kD, 200-kD, and 400-kD proteins bound to AGE-BSA-Sepharose 4B were eluted with EDTA, resolved by 4% SDS-PAGE under reducing conditions, and blotted to PVDF membranes (arrows). The protein blotting was confirmed by Coomassie staining (*). (A) The membranes were used for binding with 125I-AGE-BSA (1 x 106 cpm/ml) in the binding buffer containing 0.2% Tween 20 and 3% BSA in the absence or presence of unlabeled AGE-BSA (35 µg/ml) or EDTA (20 mM). The 200-kD and 400-kD proteins were bound with 125I-AGE-BSA, which was suppressed by AGE-BSA and EDTA, confirming that the binding was specific and Ca2+-dependent. (B) The membranes were also bound with 125I-megalin (1 x 106 cpm/ml) in the same buffer in the absence or presence of unlabeled RAP (300 nM) and EDTA (20 mM). The 200-kD protein was only bound with 125I-megalin, which was suppressed by EDTA, indicating a Ca2+-dependent binding mechanism. The binding was also suppressed by RAP, suggesting that megalin may interact with the 200-kD protein at the RAP binding site.

 
Ca2+-Dependent Binding of Megalin to the 200-kD AGE-Binding Protein
To examine whether the AGE-binding proteins interact with megalin, ligand blot analysis was carried out using 125I- megalin (Figure 5B). The 190-kD, 200-kD, and 400-kD proteins were separated by 4% SDS-PAGE and blotted to the PVDF membranes. The analysis revealed that 125I-megalin bound to the 200-kD protein in the same conditions as described above. The binding was blocked by EDTA, indicating that the reaction is Ca2+-dependent. Also, the binding was blocked by RAP, suggesting that megalin may interact with the 200-kD protein at the site for binding with RAP.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using the L2 cell line system, we found that AGE are specifically internalized via receptor-mediated endocyotosis and degraded in lysosomes. In the cell system, we identified megalin, a multi-ligand endocytic receptor, as being involved in endocytosis of AGE, but not the direct binding receptor. We also found that the cells have novel 200-kD and 400-kD proteins that bind AGE in a Ca2+-dependent manner. Either of the proteins is likely to form a complex with a 190-kD protein that is co-purified with the AGE-binding proteins using AGE-BSA-bound Sepharose 4B chromatography. The proteins appear to be present both in the membrane and cytosolic fractions of L2 cells. Ligand blot analysis revealed that the 200-kD protein binds to megalin, suggesting an interactive function of the proteins. The binding is also Ca2+-dependent, and it is blocked by RAP, suggesting that megalin binds to the 200-kD protein at the RAP binding site. However, further studies are required to determine whether the novel AGE-binding proteins constitute a cell-surface AGE receptor and cooperate with megalin for the endocytosis of AGE.

Megalin is known to be involved in the endocytosis of multiple ligands in PTC by directly binding them or by cooperating with cubilin, another cell-surface receptor that binds its specific ligands (44). Cubilin, also known as the intestinal intrinsic factor-cobalamin receptor, is a 460-kD glycoprotein with no transmembrane domain and no known signal for endocytosis. It binds multiple ligands, including albumin, Ig light chain, HDL, and apolipoprotein A-I, and most likely is mediated for internalization by megalin at the clathrin coated-pits (45,46). Cubilin is reported to be expressed in cultured yolk sac epithelial cells (47), but our mass spectrometry analysis in progress for identifying the AGE-binding proteins has found that the proteinase-digested fragments do not match those deduced from the amino acid sequence of rat cubilin (data not shown).

Like the indirect role of megalin involved in the endocytosis of cubilin’s ligands, we speculate that a similar mechanism exists for the endocytosis of AGE in L2 cells, in which a putative AGE receptor is present at the cell surface and is mediated by megalin for internalization. The addition of RAP, an inhibitor of megalin’s binding to its ligands, to the L2 cell culture system decreased AGE-BSA degradation but increased the cell-association. This result suggests that blocking megalin with RAP may inhibit the cellular internalization of the complex of AGE and the cell-surface AGE receptor or suppress the intracellular metabolism of endocytosed AGE. The RAP-binding sites of megalin are likely involved in the molecular mechanisms of interaction or cooperation with the putative AGE receptor.

In this study, highly glucose-modified AGE-BSA was used as a ligand for binding and uptake assays. We found that mildly modified AGE-BSA, prepared by incubating 50 mg/ml BSA with 50 mM D-glucose at 37°C for 4 wk, was not specifically taken up by the cells in our assay system (data not shown). It suggests that mildly modified AGE is not be recognized by the cells or that the sensitivity of our assays is insufficient to evaluate the cellular uptake. Highly glucose-modified proteins contain various AGE structures, and it should be determined which structures are recognized by the megalin-mediated endocytosis system. We have evidence that glycoaldehyde-modified AGE structures, which are reactive intermediates of the Maillard reaction (48), may be recognized by the system (Saito et al., unpublished observation).

The mechanism of AGE metabolism associated with megalin has an important aspect regarding the pathogenesis and treatment of uremic complications. Various uremic toxin proteins are known to accumulate in serum and tissues of patients with end-stage renal disease and cause serious complications (49). {beta}2-m is a well-established 12-kD uremic toxin protein that causes DRA characterized by osteoarthropathy and failure of various organs due to the deposition of {beta}2-m–derived amyloid proteins (50). Despite the development of high-flux membrane hemodialysis devices and a direct absorbent column, {beta}2-m accumulation is inevitable in afflicted patients because the therapeutic effects are transient and insufficient. Megalin appears to be an effective molecular tool to remove LMWP in uremia, and we have developed a novel strategy to metabolize {beta}2-m by subcutaneous implantation of megalin-expressing cells in renal failure (14). The current study indicates that megalin is also expected to function in such a strategy for endocytosis of AGE that accumulate in uremia and cause serious complications (18–21). Further characterization of the AGE-binding proteins that may interact and cooperate with megalin will be important for increasing the efficiency of the strategy.

In the development of diabetic nephropathy, tubulointerstitial injury is thought to be as significant as glomerulopathy (26). A number of studies have indicated that AGE are associated with PTC injury (24,25,27). From our current work, it is very likely that megalin is also involved in the endoytosis of AGE in PTC and could thus be a therapeutic molecular target for preventing AGE accumulation in the cells. Recently, PTC were found to express RAGE (28), a cell surface AGE-binding receptor that functions for AGE signal transduction (51). It remains to be determined whether or not RAGE is also involved in the endocytosis through cooperation with endocytic receptors such as megalin. Further characterization of megalin-mediated endocytosis of AGE would be useful for elucidating the molecular mechanisms of diabetic tubulointerstitial injury and developing a strategy for its treatment.


   Acknowledgments
 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (10670989 and 14571018). We acknowledge technical support from Ms. Hiromi Takahashi.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication March 5, 2002. Accepted for publication January 26, 2003.




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