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.Key words advanced glycosylation end-product (AGE); glyceraldehyde; glycolaldehyde; macrophage; nucleolin; toxic AGE 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 crosslinking, rearrangement, and condensation.

Nucleolin Acts as a Scavenger Receptor for Acetylated Low-Density Lipoprotein on Macrophages

“…We recently reported that nucleolin is a phagocytic receptor that initiates the removal of a variety of discarded elements. [10][11][12][13] These previous observations and the present study support our hypothesis that cell surface-expressed nucleolin on phagocytes has a general scavenger-like ability.…”

“…12) Taken together, these findings suggest that the polyanionic nature of AcLDL might play a major role in its binding to nucleolin. However, as the binding of rNUC284 and AcLDL did not dissociate with NaCl treatment in SPR analysis (data not shown), factors other than negative charge might also contribute to the binding affinity of nucleolin for AcLDL.…”

“…Thioglycollate-elicited murine macrophages were isolated from male ddY mice (Japan SLC, Inc., Shizuoka, Japan), as previously described. 10,12,20) Macrophages were suspended at 5×10 5 cells/mL in RPMI-1640 medium buffered with 20 mM HEPES (pH 7.2) and then incubated with 0 to 10 µg/mL DiI-labeled normal LDL or AcLDL in the presence or absence of anti-nucleolin antibody, control rabbit IgG, AGRO or CRO at 37°C for 2 h. After 2 h, cells were incubated with Alexa Fluor 488-labeled anti-CD11b antibody for a further 30 min on ice and then washed. Cells were 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. 10,12,20) Binding of Normal LDL and AcLDL to NucleolinTransfected HEK Cells Human nucleolin cDNA was transfected into HEK 293 cells (HEK cells; Health Science Research Bank, Osaka, Japan) as previously described. 10) After transfection, adherent HEK cells were detached from the bottom of the culture well by incubation with Puck's EDTA solution (5 mM HEPES, 0.1 M NaCl, 5 mM potassium chloride (KCl), 4 mM sodium bicarbonate (NaHCO 3 ), 1 mM EDTA, and 5.6 mM glucose) for 10 min and gentle pipetting.…”

“…7) Nucleolin is a multifunctional shuttling protein that is present in the nucleus, cytoplasm, and on the surface of macrophages. 8,9) We previously identified nucleolin as a scavenger receptor for apoptotic cells 10) and modified proteins such as fibril amyloid-β, 11) advanced glycosylation end products, 12) and maleylated-proteins. 13) In addition, nucleolin is a scavenger receptor for microbes and their substituents, including lipopolysaccharide, 14) enterohemorrhagic Escherichia coli (E. coli) O157 : H7, 15) and human parainfluenza virus type 3.…”