Comparison On The Immunobiological Activities Of Native And Recombinant Calreticulin

Calreticulin (CRT) is a multi-functional calcium binding protein predominantlyobserved in the endoplasmic reticulum (ER), it was first identified and purified from theER of striated skeletal muscle, the sarcophasmic reticulum, in1974. Full length CRThas416amino acids, which is a46KDa protein with N, P, C, three domains. The N-Pconcatenate region of the protein owns chaperone function, and the C domain isresponsible for Ca2+storage. Recent research indicates that the CRT also present out ofER, and play an important biological role, such as antigen processing and presentationfor the adaptive immune response, the uptake of CRT-expressing cancer cells bydendritic cells, phagocytosis of apoptotic cells, cell adhesion and migration, cellproliferation, activation and so on.In order to further study the biological activity of CRT, we acquired the freshmouse liver to isolate the native calreticulin (N-CRT), and utilized ammonium sulfate toprecipitate contaminant, at last used DEAE anion chromatography linear elute theN-CRT. We also expressed and purified recombinant calreticulin (rCRT) in E.colisystem. Because the rCRT have abundant polymers, gel filtration chromatography wasused to isolate the polymers and monomers. As a result, we obtained three, rCRTpolymers (PrCRT), rCRT MonomersⅠ (MrCRTⅠ) and rCRT MonomersⅡ (MrCRTⅡ).Experiments suggested that the peritoneal macrophage cells (PMCs) cultured withthe CRT have more proinflammatory cytokines (TNF-α, IL6) and NO production. We observed an amusing difference that the rCRT and PrCRT stimulated the PMCs secretedthe cytokine stronger than N-CRT, MrCRT Ⅰ and MrCRT Ⅱ. Following thisphenomenon, we speculated that whether the endotoxin from the E.colin systeminduced the PrCRT to represent more activity, hence we decided to investigate thepossibility that the interaction of N-CRT with LPS augments the biological activity ofLPS. We observed no difference that stimulated PMCs with a low concentration of LPSin the presence or absence of N-CRT.To investigate the immunogenicity of CRT, we immunized the C57BL/6mice withdifferent components of CRT. Our data indicated that the PrCRT plays a major role inspecific antibody production, and the N-CRT or MrCRTs almost cannot induce theantibody production.Taken together, we found that the CRT have the activity to stimulate the PMCs toproduct cytokine, for example TNF-α，IL-6. In addition, we noticed that the PrCRT haveincredible activity to PMCs, and this function is not associated with LPS. And theantibody assay also proved this phenomenon. These information is helpful for us tofurther investigate the biological functions of CRT.