Glutathione and Vitamin B12 Cooperate in Stabilization of a B12 Trafficking Chaperone Protein
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The protein bCblC (bCblCpro) is a bovine homolog of a human B12 trafficking chaperone that is responsible for the processing of vitamin B12 and its escorted delivery in intracellular B12 metabolism. In this study, we found that bCblCpro is highly thermolabile with a T m = 42.0 ± 0.2 °C as shown for the human homolog, suggesting thermal regulation of these proteins. Binding of the reduced form of glutathione (GSH) that is a predominant cellular thiol increased the T m of bCblCpro from 42 °C to ~45 °C (ΔT m max = 3.1 ± 0.2 °C and AC50 = 2.1 ± 0.5 mM). Binding of vitamin B12 and its derivatives also stabilized bCblCpro increasing the T m to a different extent and vitamin B12 (cyanocobalamin, CNCbl) was the least efficient (ΔT m max = 4.3 ± 0.3 °C and AC50 = 291 ± 36 μM). However, the stabilizing effect of CNCbl was significantly greater for GSH-bound bCblCpro (ΔT m max = 12.8 ± 0.6 °C and AC50 = 9.3 ± 1.6 μM) than for GSH-free bCblCpro. In addition, the stabilizing effect of GSH was also greater for CNCbl-bound bCblCpro (ΔT m max = 9.3 ± 0.3 °C and AC50 = 57.0 ± 6.8 μM). Limited proteolysis revealed that thermal stabilization of bCblCpro is derived from conformational changes of the protein induced by binding of the ligands. The results in this study indicate that GSH cooperates with vitamin B12 in thermal stabilization of bCblCpro and is a positive regulator of the protein.
KeywordsGlutathione Vitamin B12 Trafficking chaperone Thermostability
Vitamin B12 derivatives
Bovine CblC protein
Cyanocobalamin or vitamin B12
Reduced form of glutathione
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0021596), and Yeungnam University research grant in 2009.
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