Macrophage Recognition of Toxic Advanced Glycosylation End Products through the Macrophage Surface-Receptor Nucleolin

Abstract

Advanced glycosylation end-products (AGEs) are non-enzymatically glycosylated proteins that play an important role in several diseases and aging processes, including angiopathy, renal failure, diabetic complications, and some neurodegenerative diseases. In particular, glyceraldehyde (GCA)- and glycolaldehyde (GOA)-derived AGEs are deemed toxic AGEs, due to their cytotoxicity. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether or not macrophages recognize toxic AGEs through nucleolin receptors expressed on their surface. Free amino acid groups and arginine residues found in bovine serum albumin (BSA) were time-dependently modified by incubation with GCA and GOA. In addition, average molecular size was increased by incubation with GCA and GOA. While GCA-treated BSA (GCA-BSA) and GOA-treated BSA (GOA-BSA) were recognized by thioglycollate-elicited mouse peritoneal macrophages in proportion to their respective aldehyde-modification ratios, aldehyde-untreated control-BSA was not. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with GCA-BSA and GOA-BSA, but not with control-BSA. Further, pretreating macrophages with anti-nucleolin antibody, but not control-Immunoglobulin G, inhibited recognition of GCA-BSA and GOA-BSA by macrophages. Additionally, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited recognition of GCA-BSA and GOA-BSA. Moreover, nucleolin-transfected HEK293 cells recognized more GCA-BSA and GOA-BSA than control HEK cells did. Binding of nucleolin and GCA-BSA/GOA-BSA was also blocked by anti-nucleolin antibody at molecular level. These results indicate that nucleolin is a receptor that allows macrophages to recognize toxic AGEs.

The modification, aggregation, and deposition of proteins are prominent events in many pathological processes and can play a direct role in tissue damage. Advanced glycation end-products (AGEs) are one type of post-translational modification product that form from non-enzymatic reactions between proteins and reducing sugars, dicarbonyl compounds, or reactive aldehydes such as α-hydroxyaldehyde, which are followed by several chemical modifications including cross-linking, rearrangement, and condensation.¹⁾ Interest in AGEs is increasing due to their possible correlation with several diseases and aging processes, including angiopathy, renal failure, diabetic complications, and neurodegenerative disease.²⁾ Additionally, reports have shown that AGEs are recognized by macrophages with a receptor identical or closely similar to that for aldehyde-modified proteins.³⁾ However, which AGE subtypes and which AGE receptors mediate AGE-recognition by macrophages remains unclear.

AGEs are formed by glycation, which is a non-enzymatic reaction between ketones or aldehydes and the amino groups of several proteins, resulting in browning, cross-linking, aggregation, and fluorescenation.⁴⁾ Several pathways for AGE formation have been proposed.^4,5) Takeuchi et al. classified AGEs into six types (AGEs 1–6) based on ketone or aldehyde of origin. According to this scheme, protein reactions with glucose generate AGE-1, those with high-reactivity aldehydes including α-hydroxyaldehyde generate AGE-2 and AGE-3, and those with dicarbonyl compounds including methylglyoxal, glyoxal, and 3-deoxyglucoson generate AGEs 4, 5, and 6.⁴⁾ In particular, α-hydroxyaldehyde-derived AGEs (AGE-2 and AEG-3, by glyceraldehyde [GCA] and glycolaldehyde [GOA], respectively) have high toxicity and are therefore deemed toxic AGEs. Toxic AGEs have been demonstrated to play an important role in the pathogenesis of renal failure, arteriosclerosis, angiopathy, and retinopathy in diabetic patients.^4,6,7) Several AGE receptors have been identified on vascular endothelial cells, vascular smooth muscle cells, and monocyote/macrophage membranes, including receptor for advanced glycosylation end-products (RAGE),⁸⁾ galectin-3,⁹⁾ scavenger receptor A1/A2,^10–13) CD36,¹⁴⁾ scavenger receptor B1,¹⁵⁾ lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1),¹⁶⁾ and link domain-containing scavenger receptor-1/2.¹⁶⁾ Among these receptors, RAGE, scavenger receptor A, and LOX-1 have been reported as toxic (GCA or GOA-derived) AGE receptors.^7,12,16) The abundance of receptors that recognize AGEs indicates that their clearance is an important function for macrophages. However, although macrophages recognize, phagocytose, and degrade AGEs, excessive levels of AGEs may lead to macrophage accumulation-induced foam-cell formation, resulting in arteriosclerosis.^17,18) Further research into the mechanisms by which AGEs are removed is therefore critical to improve understanding, facilitate prevention, and enable treatment of AGE-related diseases such as renal failure, arteriosclerosis, angiopathy, retinopathy in diabetic patients, neurodegenerative diseases, and aging.

Nucleolin is naturally present in the nucleus, cytoplasm, and on the surface macrophages.^19,20) Previous studies have shown that macrophages utilize nucleolin to recognize and phagocytose apoptotic and oxidized cells^21,22) as well as living cells that have deteriorated due to treatment with cytochalasin B and A23187.^23,24) Nucleolin-mediated recognition is therefore simple and powerful, as indicated by the indiscriminant removal of cells. Nucleolin is also a receptor for monomeric and fibril amyloid β1-42,²⁵⁾ lipoproteins,²⁶⁾ coxsackie B virus,²⁷⁾ human immunodeficiency virus,²⁸⁾ human parainfluenza virus type 3,²⁹⁾ and enterohemorrhagic Escherichia coli O157:H7.³⁰⁾ Further, nucleolin can bind to anionic molecules such as DNA and RNA.^20,31) Together, these observations suggest that macrophages with nucleolin on their surface may have a general scavenger-like ability, and that nucleolin might therefore also be involved in the recognition of AGEs.

Here, in order to clarify the mechanism by which macrophages clear AGEs, we investigated whether or not nucleolin is involved in the recognition of toxic (GOA and GCA derived) AGEs by macrophages.

MATERIALS AND METHODS

Materials

Bovine serum albumin (BSA) and PKH 26 red fluorescent cell-linker kits were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). GCA and GOA were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Alexa Fluor® 488 C5 maleimide was obtained from Life Technologies (Carlsbad, CA, U.S.A.). rNUC284, a truncated recombinant human nucleolin containing residues 284–710 and corresponding to the C-terminal two-thirds of the molecule, was produced in E. coli and purified as previously described.²¹⁾ Phycoerythrin-labeled anti-CD11b antibody was obtained from BD Pharmingen (Tokyo, Japan). Anti-nucleolin antibody was obtained from Bethyl Laboratories, Inc (Montgomery, AL, U.S.A.). Control-rabbit Immunoglobulin G (IgG) was obtained from Santa Cruz Biotechnology (Delaware, CA, U.S.A.).

Preparation of GCA-BSA and GOA-BSA

Toxic AGEs were prepared by long term-incubation of GCA or GOA with BSA, similar to what was described by Takeuchi et al.,⁴⁾ but with some modifications. BSA (2000 µg/mL) containing 10 mM GCA or GOA were incubated in Ca²⁺, Mg²⁺-free Dulbecco’s phosphate-buffered saline (DPBS[−]) for 0.25–32 d at 37°C, under sterile conditions. After incubation, these samples were dialysed 6 times with 100 times the amount of DPBS(−) at 4°C to remove low-molecular-weight reactants and aldehydes, and then the amount of protein was measured via the Lowry method.³²⁾

Measurement of Modified Free Amino Acid Groups in GCA-BSA and GOA-BSA

Modification of free amino acid groups in GCA-BSA and GOA-BSA preparations was measured as previously described.³³⁾ Briefly, 4% NaHCO₃ and 0.1% trinitrobenzene sulfonic acid were added to 1 mg/mL GCA-BSA, GOA-BSA, or control-BSA in DPBS(−), and incubated for 2 h at 40°C. Then, 10% sodium dodecyl sulfate and 1 M HCl were added, and absorbance was immediately measured at 335 nm using a microspectrometer (SAFIRE; Wako Pure Chemical Industries, Ltd.). Modification rate was calculated relative to the absorbance of control-BSA that was incubated without aldehydes. The absorbance of solvent without protein was also measured as a blank control.

Measurement of Arginine Residue Modification in GCA-BSA and GOA-BSA

Modification of arginine residues in GCA-BSA and GOA-BSA preparations was measured as previously described.³⁴⁾ Briefly, 150 µM 9,10-phenanthrenequinone and 2 M NaOH were added to 1 mg/mL GCA-BSA, GOA-BSA, or control-BSA in DPBS(−) and incubated for 3 h at 60°C. Then, 1.2 M HCl was added and incubated for 1 h at room temperature in the dark. Fluorescence intensity was then measured at an excitation wavelength of 312 nm and an emission wavelength of 395 nm using a microspectrometer (SAFIRE; Wako Pure Chemical Industries, Ltd.). Modification rate was calculated relative to the fluorescent intensity of control-BSA incubated without aldehydes. The fluorescent intensity of solvent without protein was also measured as a blank control.

Size Exclusion Chromatography

BSA, GCA-BSA, GOA-BSA were prepared by incubation with or without aldehydes for 16 d. Additionally, unincubated native BSA and molecular size marker (Bio-rad, Tokyo, Japan) were also prepared. DPBS(−) was used for sample dilution and analysis. Twenty microliter of each sample was applied to the analytical size exclusion chromatography system at a flow rate of 0.5 mL/min and detection UV280 nm (TSKgel G3000SWXL, 7.8 mm×30 cm [TOSO Corporation, Tokyo, Japan]). Average molecular size was calculated by rate of peak area.

Fluorescent Labeling of Control-BSA, GCA-BSA, and GOA-BSA Using Alexa Fluor® 488 C5 Maleimide

Control-BSA, GCA-BSA, and GOA-BSA were prepared by incubation with or without aldehydes for 16 d. Each BSA was incubated with 20 times the amount of Alexa Fluor® 488 C5 maleimide overnight at 4°C in DPBS(−) under nitrogen replacement and then dialysed 6 times with 100 times amount of DPBS(−). Protein amounts were measured via the Lowry method,³²⁾ and fluorescence intensities were measured using a SAFIRE microspectrometer (excitation: 492 nm, emission: 519 nm). The strengths of Alexa-BSA, Alexa-GCA-BSA, and Alexa-GOA-BSA fluorescence per unit dosage were each adjusted by adding unlabeled proteins.

Recognition of Control-BSA, GCA-BSA, and GOA-BSA by Mouse Macrophages

The protocol was approved by the committee on the Ethics of Animal Experiments of Tokyo University of Pharmacy and Life sciences (Permit Number: P13–76). All surgery was performed under diethyl ether anesthesia, and all efforts were made to minimize suffering.

Mouse macrophages were isolated from the peritoneal cavities of adult male ddy mice (Japan SLC, Inc., Shizuoka, Japan) that had been injected with 2 mL of 3% thioglycolate medium 4 d earlier; after induction, the macrophages were then harvested by lavage with Hanks’ balanced salt solution and seeded into a culture flask.²¹⁾ Macrophages that adhered to culture substrates were detached by incubation in Puck’s ethylenediaminetetraacetic acid (EDTA) solution (5 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES], 0.1 M NaCl, 5 mM KCl, 4 mM NaHCO₃, 1 mM EDTA, and 5.6 mM glucose) for 10 min and gentle pipetting,²¹⁾ suspended at 5×10⁵ cells/mL in RPMI-1640 medium buffered with 20 mM HEPES (RPMI 1640-HEPES), and then incubated with 5 µg/mL Alexa Fluor® 488-labeled control-BSA, GCA-BSA, or GOA-BSA at 37°C for 2 h. After 2 h, the cells were incubated with phycoerythrin-labeled anti-CD11b antibody for 30 min on ice, then washed, and immediately analyzed using a flow cytometer (FACS Calibur, Becton-Dickinson, Franklin Lakes, NJ, U.S.A.) with CELLQUEST software (Becton-Dickinson) and gating for forward scatter (FSC) and side scatter (SSC) regions of intact cells. CD11b-positive cells were regarded as macrophages.³⁵⁾

Confocal Images of Phagocytosed GCA-BSA and GOA-BSA

Macrophages were seeded on coverslip,²¹⁾ and incubated with or without 5 µg/mL Alexa Fluor® 488-labeled control-BSA, GCA-BSA, or GOA-BSA for 2 h, their membranes were stained with the fluorescent cell-linker compound PKH 26 red. BSAs taken up by macrophages were identified using confocal laser-scanning fluorescence microscopy (FV1000D; Olympus, Tokyo, Japan).

Surface Plasmon-Resonance (Biacore)

Binding of nucleolin to control-BSA, GCA-BSA, and GOA-BSA (each incubated for 16 d) was analyzed using a Biacore 2000X (GE Healthcare, Tokyo, Japan). This biosensor can directly measure the binding of a recombinant protein to its natural biological ligand in real time and in a quantitative and highly reproducible manner.³⁶⁾ Briefly, HBS-running buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.005% Tween 20 [pH 7.4]) was used for sample dilution and analysis. The research-grade CM5 dextran sensor-chip was activated with equal amounts of 0.2 M N-ethyl-N′-[3-diethylamino-propyl]carbodiimide and 0.05 M N-hydroxysuccinimide. rNUC284 was produced in E. coli and purified as previously described²¹⁾ at 20 µg/mL and immobilized in 10 mM sodium acetate buffer (pH 4.0) followed by 1 M ethanolamine-hydrochloride (pH 8.0) to deactivate excess N-hydroxysuccinimide-esters. This coupling resulted in approximately 10 ng protein/mm² of immobilized protein per flow-cell. To evaluate binding, each protein was diluted in HBS buffer, analyzed at different concentrations, and passed over the sensor chip at a flow rate of 20 µL/min. An activated and blocked flow-cell without immobilized ligands was used to evaluate nonspecific binding. Results were calculated using BIAevaluation 4.0 software (Biacore).

Pretreatment of Macrophages with Anti-nucleolin Antibody for the Recognition Assay

Macrophages were pre-incubated with 20 µg/mL anti-nucleolin antibody or control-rabbit IgG in RPMI1640-HEPES containing 0.2% BSA at 4°C for 30 min, washed, and subjected to a recognition assay using Alexa Fluor® 488-labeled GCA-BSA or GOA-BSA.

Recognition of GCA-BSA and GOA-BSA by Nucleolin-Transfected HEK Cells

Human nucleolin cDNA was transfected into the monolayer of HEK 293 cells (HEK cells) (Health Science Research Bank, Osaka, Japan) as previously described.²¹⁾ Sixteen hours after transfection, adherent HEK cells were detached from the bottom of the culture well by incubation with Puck’s EDTA solution for 10 min and gentle pipetting. HEK cells were then incubated with 5 µg/mL Alexa Fluor® 488-labeled GCA-BSA or GOA-BSA at 37°C for 2 h, and immediately analyzed using a flow cytometer with CELLQUEST software, gating for FSC and SSC regions of intact HEK cells.

Blocking of the Binding of Nucleolin and GCA-BSA/GOA-BSA by Anti-nucleolin Antibody at the Molecular Level

Blocking of the binding of nucleolin and GCA-BSA/GOA-BSA (each incubated for 16 d) was analyzed using a Biacore 2000X. rNUC284 was immobilized on sensor chip as described above. To evaluate blocking of the binding by antibody, rabbit polyclonal anti-nucleolin antibody (NOVUS Biologicals, CO, U.S.A.) or control-rabbit IgG were passed over the sensor chip to block rNUC284, and then GCA-BSA/GOA-BSA were passed over the sensor chip. An activated and blocked flow-cell without immobilized ligands was used to evaluate nonspecific binding. Results were calculated using BIAevaluation 4.0 software.

Statistical Analysis

Data are presented as the mean±standard deviation (S.D.) of at least three experiments and were analyzed using Student’s t-tests.

RESULTS

Time-Dependent Modification of BSA Amino Groups by Incubation with GCA or GOA

We prepared toxic AGEs (AGE-2 and AGE-3) by incubating BSA with either GCA or GOA for an extended period. As shown in Figs. 1A, and B, GCA and GOA treatments time-dependently increased the amount of modification undergone by free amino acid groups in BSA for 8 d, reaching a 70–80% plateau over the remaining days.

Fig. 1. Modification of Bovine Serum Albumin (BSA) Amino-Groups through Time-Dependent Incubation with GCA and GOA

(A, B) Percent of free amino residue modification for GCA-BSA (A) and GOA-BSA (B) over time. (C, D) Percent of ariginine residue modification for GCA-BSA (C) and GOA-BSA (D) over time. Each point represents the mean±S.D. of at least three experiments.

Albumin can undergo glycation on some of its arginine and lysine residue, and arginine side-chains were modified to a lesser extent compared to lysine side-chains.³⁴⁾ Therefore, we also confirmed the amount of amino groups of arginine-residue modification induced by GCA and GOA treatment in BSA and found that they also increased to 60% (Figs. 1C, D).

Size Exclusion Chromatography of BSA, GCA-BSA, and GOA-BSA

We next investigated that molecular size distribution of BSA, GCA-BSA, and GOA-BSA by size exclusion chromatography analysis. First, we determined analytical curve of the relation between molecular size and retension time. Analytical curve was calculated as y=4e+8e^−0.506x (R²=0.9619) (Fig. 2A). Although the molecular weight of one BSA molecule was calculated as 66463 from constructed amino acid residues, the average molecular size of native-BSA was calculated as 150146.3 by multimer form (Fig. 2B). Similar result was obtained using 16 d incubated BSA without aldehydes (Fig. 2C). In contrast, large size constructs, and therefore average molecular sizes were increased in GCA-BSA and GOA-BSA (Figs. 2D, E). Theses results indicating that toxic-AGE modification increased aggregation or cross-linking of protein molecules.

Fig. 2. Size Exclusion Chromatography of BSA, GCA-BSA, and GOA-BSA

Size exclusion chromatography profiles of BSA, GCA-BSA, and GOA-BSA. (A, left panel) Profile of molecular molecular size marker. The marker was constructed by thyroglobulin (MW; 670000), γ-globulin (MW; 158000), ovalbumin (MW; 44000), myoglobin (MW; 17000), vitamin B₁₂ (MW; 1350). (A, right panel) Analytical curve of molecular size and retension time. (B–E; left panel) Profiles of native-BSA (B), 16 d incubated BSA without aldehydes (C), 16 d incubated BSA with GCA (D), and 16 d incubated BSA with GOA (E). (B–E; right panel) Calculation of molecular sizes by detected peak area of each BSA. Average molecular sizes were also calculated by rate of peak areas.

Recognition of GCA-BSA and GOA-BSA by Macrophages

We found that while mouse macrophages did not recognize control-BSA (Fig. 3A) they did recognize GCA-BSA and GOA-BSA (Figs. 3B, C, respectively), and did so in proportion to the modification ratio (Fig. 1). Figures 3D–F indicate ingestion of toxic AGEs by macrophages. While macrophages did not phagocytose control-BSA (D), they did phagocytose GCA-BSA and GOA-BSA (E and F, respectively).

Fig. 3. Recognition of GCA-BSA and GOA-BSA by Macrophages

Recognition over time of 5 µg/mL Alexa Fluor® 488-labeled control-BSA (A), GCA-BSA (B) and GOA-BSA (C) by macrophages as measured by the mean fluorescence intensity. (D–F) Confocal images of macrophages ingesting aldehyde-modified BSAs. Control-BSA (D), GCA-BSA (E) and GOA-BSA (F) taken up by macrophages were identified using confocal laser scanning fluorescence microscopy. Green, aldehyde-modified BSAs. Red, macrophage cell-surface. A BSA-untreated macrophage is shown as a control image (G). Each point represents the mean±S.D. of at least three experiments.

Association of Nucleolin with GCA-BSA and GOA-BSA

We next investigated whether or not nucleolin is a receptor for toxic AGEs. First, we tested whether or not nucleolin binds to toxic AGEs by observing the interactions between nucleolin and GCA-BSA and GOA-BSA using surface plasmon-resonance. Control-BSA, GCA-BSA, and GOA-BSA were applied to a CM5 dextran sensor-chip, which was immobilized with rNUC284. The sensorgram demonstrated that while control BSA had no affinity for rNUC284 (Fig. 4A), GCA-BSA and GOA-BSA had a high concentration-dependent affinity and did not easily dissociate by washing (Figs. 4B, C). The ka, kd, and KD values between rNUC284 and each BSA were calculated as follows: control-BSA (MW: 150721.0 [Fig. 2C]): 2.28, 6.74×10⁻³, and 1.09×10⁻²; GCA-BSA (MW: 212837.3 [Fig. 2D]): 3.51×10³, 2.08×10⁻², and 2.18×10⁻⁵; GOA-BSA (MW: 307496.8 [Fig. 2E]): 1.49×10³, 1.87×10⁻², and 4.59×10⁻⁵). These results indicate that toxic AGEs bound to nucleolin and that non-AGE proteins did not.

Fig. 4. Association of Nucleolin with Control-BSA, GCA-BSA, and GOA-BSA

Surface plasmon-resonance analyses of control-BSA, GCA-BSA, and GOA-BSA binding to nucleolin. Each BSA was prepared by incubation with or without each aldehyde for 16 d. Control-BSA (A), GCA-BSA (B), and GOA-BSA (C) were made to flow over rNUC284-immobilized CM5 dextran-sensor chips. Control-BSA, GCA-BSA, and GOA-BSA were applied at 1 (magenta), 5 (green), 10 (blue), and 20 (red) µg/mL. An activated and blocked flow cell without immobilized ligands was used to evaluate nonspecific binding.

Involvement of Macrophage-Surface Nucleolin in the Recognition of GCA-BSA and GOA-BSA

We have previously showed that nucleolin was detected at the surface of macrophages. In addition, nucleolin-transfected HEK cells expressed nucleolin on the cell surface. The extent of nucleolin molecules expressed on the surface of these cells were theoretically examined by flow cytometry (see Hirano et al.²¹⁾). We next investigated whether or not cell surface-expressed nucleolin is a receptor for GCA-BSA and GOA-BSA. Recognition of GCA-BSA and GOA-BSA by macrophages was inhibited by anti-nucleolin antibody but not by control-rabbit IgG (Figs. 5A, B). Further, when nucleolin was blocked using AGRO, a nucleolin-specific oligonucleotide aptamer,³⁷⁾ macrophage recognition of GCA-BSA and GOA-BSA was inhibited, whereas adding CRO as a control did not (Figs. 5C, D). Additionally, GCA-BSA and GOA-BSA were bound more strongly by HEK cells that expressed recombinant nucleolin on their surfaces,²¹⁾ than by those that did not (Figs. 5E, F). These results indicate that nucleolin is a receptor for GCA-BSA and GOA-BSA.

Fig. 5. Involvement of Macrophage-Surface Nucleolin in the Recognition of GCA-BSA and GOA-BSA

Each BSA was prepared by incubation with or without each aldehyde for 16 d. (A, B) Pretreatment of anti-nucleolin antibody inhibited recognition of 5 µg/mL GCA-BSA (A) and GOA-BSA (B). (C, D) Co-incubation with 10 µM nucleolin-specific aptamer AGRO inhibits recognition of 5 µg/mL GCA-BSA (C) and GOA-BSA (D). (E, F) recognition of 5 µg/mL GCA-BSA (E) and GOA-BSA (F) by nucleolin-transfected HEK cells. Each bar represents the mean±S.D. of at least three experiments. * p<0.05; ** p<0.01; and *** p<0.001.

Blocking of the Binding of Nucleolin and GCA-BSA/GOA-BSA by Anti-nucleolin Antibody at the Molecular Level

We investigated whether or not the binding of nucleolin and GCA-BSA/GOA-BSA was blocked by anti-nucleolin antibody at the molecular level, using surface plasmon-resonance. Anti-nucleolin antibody or control-rabbit IgG were applied to a CM5 dextran sensor-chip, which was immobilized with rNUC284, and then GCA-BSA/GOA-BSA were successively applied to the sensor-chip. The sensorgram demonstrated that antibody-untreated rNUC284 showed a high affinity against GCA-BSA (Fig. 6A, red line). Anti-nucleolin antibody-treated rNUC284 was more decreased the affinity for GCA-BSA (green line) than control-IgG-treated rNUC284 (blue line). Similar results were also obtained using GOA-BSA (Fig. 6B). These results also indicate that GCA-BSA and GOA-BSA are ligands for nucleolin.

Fig. 6. Blocking of the Binding of Nucleolin and GCA-BSA/GOA-BSA by Anti-nucleolin Antibody at the Molecular Level

Surface plasmon-resonance analyses of GCA-BSA and GOA-BSA binding to anti-nucleolin antibody or control-rabbit IgG treated nucleolin. GCA-BSA and GOA-BSA were prepared by incubation with each aldehyde for 16 d. 20 µg/mL anti-nucleolin antibody (green line) or control-rabbit IgG (blue line) were made to flow over rNUC284-immobilized CM5 dextran-sensor chips, and then 20 µg/mL GCA-BSA (A) and GOA-BSA (B) were made to flow over the sensor chips. Red line; GCA-BSA or GOA-BSA was made to flow over antibody-untreated sensor chip. An activated and blocked flow cell without immobilized ligands was used to evaluate nonspecific binding.

DISCUSSION

Several receptors including RAGE, scavenger receptor A, and LOX-1 have been suggested to be receptors for toxic AGEs. Here, we showed that nucleolin, a phagocyte-receptor known to initiate removal of a variety of discarded elements,^{19–23,26–31,38)} is also a receptor for toxic AGEs when it is expressed on the surface of macrophages. These observations support the idea that phagocytes with cell-surface nucleolin have a general scavenger-like ability.

Nucleolin strongly associates with GCA and GOA modified BSA at molecular level, while nature BSA did not (Figs. 4A–C). Although a definitive answer for the binding mechanism cannot be reached at this time, we speculated as follows; Because each aldehyde modifies free amino acid groups of albumin that have a positive charge, GCA- and GOA-treated proteins become more negative than natural albumin.⁴⁾ Therefore, the polyanionic nature of AGEs may play a major role in receptor recognition. Indeed, there were several findings that involvement of scavenger receptor and poly anion. For example; scavenger receptors are a receptor for acetyl-low density lipoprotein (LDL), oxidized-LDL, and polyanions such as polysaccharides (polyvinyl sulfate, dextran sulfate and fucoidan) and polynucleotides (poly[G] and poly[C]).³⁹⁾ While identifying a common structure between AGEs and modified LDLs such as acetyl-LDL and oxidized-LDL is generally deemed quite difficult, both AGEs and acetyl-LDL themselves are polyanions in nature, and both receptors show very similar polyanion sensitivity.¹⁰⁾ Therefore, a likely interpretation is that the polyanionic nature of AGEs plays a major role as a recognition signal for scavenger receptor.¹⁰⁾

We also speculate that 3D conformation of protein is critical for binding between nucleolin and ligands. We previously demonstrated that amyloid β42 (Aβ42) peptide is bound by nucleolin, while Aβ40 peptide was not. Unlike Aβ40, which has only one hydrophobic domain, Aβ42 has three hydrophobic domains that likely allow it to aggregate and form fibrils.⁴⁰⁾ Therefore, a structure generated by the aggregation might serve as a common signal for receptor-mediated recognition of Aβ42.⁴⁰⁾ Reaction of protein amino groups with aldehydes leads to formation of Schiff base adducts and Amadori rearrangement products, followed by inter- or intra-molecular cross-linking reactions.⁴¹⁾ Indeed, present study showed that toxic AGE modification increased molecular aggregation or cross-linking (Fig. 2). Therefore, the superstructure of AGEs that is formed by cross-linking or aggregation may be necessary for binding to nucleolin.

Although the present study showed that GCA-BSA and GOA-BSA are natural ligands for macrophage cell-surface nucleolin, functional aspects of the receptor remain unknown. Reports indicate that the removal of AGEs is useful for tissues and the body. Pugliese et al. have shown that the galectin-3-regulated AGE-receptor pathway operates in vivo and protects AGE-induced tissue injury.⁹⁾ Park et al. demonstrated that intravenous administration of the soluble extracellular domain of RAGE that lacks a transmembrane domain efficiently suppressed diabetic atherosclerosis in apolipoprotein E-deficient mice.⁴²⁾ Similarly, treatment with soluble RAGE also attenuated neuronal dysfunction, renal dysfunction, ischemia/reperfusion injury, and diabetic neuropathy.⁴³⁾ Taken together, in rodent models of diabetes, chronic administration of soluble RAGE protects against macro- and microvascular complications in the great vessels, heart, kidney, retina, and peripheral nerve.⁴³⁾ Nucleolin also lacks a transmembrane domain, is detected in serum, and is reduced in quantity under conditions of neurodegenerative diseases.^19,20,44) Therefore, nucleolin operation in vivo may also be a useful means of preventing and treating toxic AGE-derived diseases.

We previously confirmed that membrane proteins of senescent erythrocytes increase prevalence of AGE modifications.⁴⁵⁾ Non-enzymatic glycation of erythrocyte-membrane protein decreases erythrocyte membrane fluidity,^46,47) and glycated erythrocytes can easily be recognized by macrophages through the modified membrane protein,^48,49) suggesting that erythrocytes functionally deteriorated by AGE-modification of membrane proteins were removed from circulation by macrophages. However, we have not clarified that how macrophages recognize the AGE-modified erythrocytes. Present study was suggesting that AGE-modified erythrocytes might be recognized by macrophages through nucleolin-mediated mechanism. Indeed, nucleolin works not only removal of molecules, but also removal of deteriorated (apoptotic) cells through aggregated polyanionic sialylpolylactosamine of CD43.²¹⁾ This finding also showed that nucleolin can bind to anionic molecules, and nucleolin on macrophages act to maintain tissue homeostasis by removing not only AGE-modified molecules but also AGE-modified cells and tissue.

Despite the benefits of AGE recognition by macrophages, excessive recognition may actually be disadvantageous for tissues and the body. GOA-pyridine has been shown to accumulate in foamed macrophages of arteriosclerotic regions, and GOA-modified protein has consequently been suggested as a cause of arteriosclerotic progression.^17,18) A likely scenario is that although a small amount of recognition may help maintain homeostasis, excessive recognition may lead to exacerbation of arteriosclerosis, in similar to macrophage-recognition of oxidized-LDL. Further study is needed to identify the actions of macrophages after nucleolin-mediated recognition of AGEs.

Here, we showed that toxic AGEs are also ligands for nucleolin. These observations support the notion that nucleolin on the surface of phagocytes has a general scavenger-like ability. Additional comprehensive evaluation of nucleolin, including the molecular composition of its ligands and the functional consequences of their composition, will further clarify our understanding of AGE-related diseases.

Acknowledgment

We thank Dr. Kazuhiro Yoshihara for technical assistance in part of this work.

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