Expression and characterization of recombinant C-terminal biotinylated extracellular domain of human receptor for advanced glycation end products (hsRAGE) in Escherichia coli.

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the development of diabetic complications. Using an Escherichia coli expression system, we have successfully expressed and purified the C-terminal biotinylated extracellular domain of human RAGE (hsRAGE), which consists of three immunoglobulin-like domains carrying three putative disulfide bonds. Over 90% of hsRAGE was expressed in soluble form in trxB and gor mutant E. coli strain Origami (DE3). Most hsRAGE was biotinylated with a C-terminal AviTag, and stably immobilized onto matrix via streptavidin without any treatment. Immobilized hsRAGE without glycosylation recognized its ligands, such as AGEs. Biotinylated hsRAGE was also able to apply in the detection of AGEs on microtitre wells like antibodies used in enzyme-linked immunoassay. SPR analysis demonstrated that the dissociation constant (K(d)) of RAGE for AGE-BSA was 23.1 nM with the two-state reaction model, and 13.5 nM with the 1:1 binding model, comparable to those of RAGEs on cell surface. These results indicate that biotinylated hsRAGE must be useful not only in analysing RAGE-ligand interactions but also detect AGEs.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) functions as an oligomer and oligomerization is dependent on receptor density.

Lectin-like oxidized low-density lipoprotein (LDL) receptor (LOX-1) exists as a homodimer formed by an intermolecular disulfide bond. Although the dimer is the minimum structural unit of LOX-1 on cell membranes, LOX-1 can form larger noncovalent oligomeric complexes. But, the functional unit of LOX-1 is not known. We quantitatively analyzed the correlation between cyan fluorescent protein-tagged LOX-1 expression and the fluorescence-labeled ligand (DiD-AcLDL) binding ability on each cell. The results clearly indicate that there is a threshold level of expression that enables LOX-1 to bind ligand. Above this threshold level, the ability of LOX-1 to bind ligand was proportional to its level of expression. Using the membrane impermeable crosslinker BS(3), we detected oligomers (primarily hexamers) only on the cell lines that stably expressed LOX-1 above the threshold level. In contrast, little oligomer or ligand binding was detected in cell lines expressing LOX-1 below the threshold level. Moreover, oligomerization was independent of ligand binding. These results indicate that the functional unit of LOX-1 is an oligomer and that oligomerization of LOX-1 is dependent on the receptor density on the plasma membrane.