Identification of galectin-3 as a high-affinity binding protein for advanced glycation end products (AGE): a new member of the AGE-receptor complex.
BACKGROUND
Advanced glycation end products (AGE), the reactive derivatives of nonenzymatic glucose-protein condensation reactions, are implicated in the multiorgan complications of diabetes and aging. An AGE-specific cellular receptor complex (AGE-R) mediating AGE removal as well as multiple biological responses has been identified. By screening an expression library using antibody against a previously identified component of the AGE-R complex p90, a known partial cDNA clone was isolated with homology to galectin-3, a protein of diverse identity, and member of the galectin family.


METHODS
To explore this unexpected finding, the nature of the interactions between galectin-3 and AGE was studied using intact macrophage-like RAW 264.7 cells, membrane-associated and recombinant galectin-1 through -4, and model AGE-ligands (AGE-BSA, FFI-BSA).


RESULTS
Among the members of this family (galectin-1 through 4), recombinant rat galectin-3 was found to exhibit high-affinity 125I-AGE-BSA binding with saturable kinetics (kD 3.5 x 10(7) M-1) that was fully blocked by excess unlabeled naturally formed AGE-BSA or synthetic FFI-BSA, but only weakly inhibited by several known galectin-3 ligands, such as lactose. In addition to the p90, immunoprecipitation with anti-galectin-3, followed by 125I-AGE-BSA ligand blot analysis of RAW 264.7 cell extracts, revealed galectin-3 (28 and 32 kD), as well as galectin-3-associated proteins (40 and 50 kD) with AGE-binding activity. Interaction of galectin-3 with AGE-BSA or FFI-BSA resulted in formation of SDS-, and beta-mercaptoethanol-insoluble, but hydroxylamine-sensitive high-molecular weight complexes between AGE-ligand, galectin-3, and other membrane components.


CONCLUSIONS
The findings point toward a mechanism by which galectin-3 may serve in the assembly of AGE-R components and in the efficient cell surface attachment and endocytosis by macrophages of a heterogenous pool of AGE moieties with diverse affinities, thus contributing to the elimination of these pathogenic substances.
Picower Institute for Medical Research, Manhasset, New York 11030, USA.
H
H
Vlassara
Y M
YM
Li
F
F
Imani
D
D
Wojciechowicz
Z
Z
Yang
F T
FT
Liu
A
A
Cerami
D000818:Animals; D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D037502:Galectin 3; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008264:Macrophages; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D011869:Radioligand Assay; D051381:Rats; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D002352:Carrier Proteins; D037502:Galectin 3; D017127:Glycation End Products, Advanced; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D011994:Recombinant Proteins