Biotinylation of heat shock protein 70 induces RANTES production in HEK293 cells in a CD40-independent pathway.
Biotinylated proteins and peptides have been used as popular ligands for characterization of cell surface receptors by a variety of methods including flow cytometry. The number and the location of biotin moieties incorporated could alter the structural and physicochemical properties of ligands, although biotin is thought to be such a small molecule (244Da) that it is capable of being conjugated to most proteins without affecting their activity. Here, we demonstrate that the biotinylated HSP70 molecule via primary amines bound to epithelium-like HEK 293 cells in a saturable manner whereas the unlabeled counterparts of HSP70 other than mouse Hsp72 do not. This binding was not competed by either HSP70 or the biotin entity itself. Interestingly, the biotinylated HSP70 also elicited the production of CC-chemokine RANTES independent of CD40 signaling. This response occurred regardless of sequence diversity of HSP70 derived from different species, and neither the biotinylated ovalbumin nor the unlabeled HSP70 cross-linked with a biotinylated protein stimulated a significant level of RANTES production which was induced by biotinylated HSP70 itself. Our findings suggest that modification of HSP70 such as biotinylation may function as a biological alarm signal in the innate immune system.
Tao
Yufeng
Y
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Goka-sho, Uji, Kyoto 611-0011, Japan.
Nishikawa
Satoshi
S
Nomura
Masayo
M
Kitabatake
Naofumi
N
Tani
Fumito
F
D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation / Q000379:methods*; D019013:CD40 Antigens / Q000378:metabolism*; D002460:Cell Line; D018946:Chemokine CCL5 / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D015398:Signal Transduction / Q000502:physiology*
D019013:CD40 Antigens; D018946:Chemokine CCL5; D018840:HSP70 Heat-Shock Proteins; D001710:Biotin