Glutathione and vitamin B₁₂ cooperate in stabilization of a B₁₂ trafficking chaperone protein.
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.
School of Biotechnology, Yeungnam University, Gyeongsan, Republic of Korea.
Jihyun
J
Park
Jihoe
J
Kim
D000818:Animals; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002417:Cattle; D005470:Fluorometry / Q000379:methods; D005978:Glutathione / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008024:Ligands; D018832:Molecular Chaperones / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D059748:Proteolysis; D011994:Recombinant Proteins / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014805:Vitamin B 12 / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D014357:Trypsin; D005978:Glutathione; D014805:Vitamin B 12