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.

Although macrophage phagocytoses modified low-density lipoprotein (LDL), excessive accumulation of modified LDL induces macrophage foam cell formation, which is a feature of atherosclerotic plaque. Thus, the identification of scavenger receptor for modified LDL will provide better understanding of an atherosclerotic event. We recently showed that nucleolin expressed on macrophages acts as a scavenger receptor for various endogenous discarded products. Here, we investigated whether or not nucleolin is involved in the uptake of acetylated LDL (AcLDL). In contrast to normal LDL, AcLDL directly bound to immobilized nucleolin. AcLDL exhibited a higher affinity for macrophages than normal LDL. This binding of AcLDL was inhibited by anti-nucleolin antibody and antineoplastic guanine-rich oligonucleotide (AGRO), a nucleolinspecific oligonucleotide aptamer. In addition, AcLDL exhibited a higher affinity for HEK cells transfected with nucleolin than those without. Further, intracellular accumulation of AcLDL was also inhibited by antinucleolin antibody. The results of this study suggest that nucleolin expressed on macrophages is a receptor for AcLDL.Key words nucleolin; macrophage; acetylated low-density lipoprotein (AcLDL); scavenger receptor Low-density lipoprotein (LDL) elevates cholesterol deposits in macrophage foam cells, which are involved in the development of atherosclerotic lesions.1) As the uptake of normal LDL via its receptor is controlled by feedback regulation, excess levels of LDL-derived cholesterol in macrophages might be attributed to modifications of LDL such as oxidation, acetylation, and aggregation.2) Scavenger receptors (SRs) and other unknown receptors account for up to 95% of the uptake of modified LDL.