ABSTRACT
Extracellular heat shock protein 70 (Hsp70) is an adjuvant molecule that stimulates the immune system. The C-terminal domain of Hsp70 (C70), without the ATPase domain, is sufficient for antigen cross-presentation. However, the mechanism by which the receptor mediates the uptake of C70–peptide complex remains unclear. We therefore aimed to determine the process by which the receptor mediates the uptake of antigenic peptide-bound C70. Methodology: Hsp70 and C70 individually cloned into pET28a were expressed in Escherichia coli BL21 (DE3) and were purified on Ni-NTA agarose and MonoQ HR5/5. Hsp70 and C70 were labeled with Alexa 555 and Alexa 633, respectively, to detect cellular binding. HEK293 cells stably expressing lectin-like oxidized LDL receptor-1 (LOX 1) and KG-1 human dendritic-like cells were incubated with Alexa-labeled Hsp70 and C70 individually or with C70 and antigenic complexes and were observed using fluorescence microscopy. The affinity of LOX-1 toward Hsp70 and C70 was analyzed by chip assay using surface plasmon resonance, which immobilized LOX-1 ligand recognition domain. Results: HEK293 cells stably expressing LOX-1 and KG-1 cells accepted the C70– peptide and Hsp70–peptide complexes. Anti-LOX-1-neutralizing antibody inhibited the uptake of the C70–peptide complexes by KG-1 cells. The dissociation constant (KD) of C70 toward the LOX-1 extracellular domain, measured by surface plasmon resonance, was 4.02 × 10−7 M and that of the C70–peptide complex was 6.6 × 10−8 M. C70 increased the LOX-1 affinity by forming a complex with the antigen peptide. Conclusion: Our findings suggest that LOX-1 is the primary receptor for the C70–peptide and the Hsp70–peptide complexes. C70 is a promising adjuvant molecule that is internalized via LOX-1. In addition, it is convenient to prepare C70 using an E. coli expression system and C70 is more stable than full-length Hsp70.