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The human B12 trafficking chaperone hCblC is well conserved inmammals and non-mammalian eukaryotes. However, the C-terminal∼40 amino acids of hCblC vary significantly and arepredicted to be deleted by alternative splicing of the encodinggene. In this study, we examined the thermostability of the bovineCblC truncated at the C-terminal variable region (t-bCblC) and itsregulation by glutathione. t-bCblC is highly thermolabile (Tm =∼42oC) similar to the full-length protein (f-bCblC). However,t-bCblC is stabilized to a greater extent than f-bCblC by binding ofreduced glutathione (GSH) with increased sensitivity to GSH. Inaddition, binding of oxidized glutathione (GSSG) destabilizest-bCblC to a greater extent and with increased sensitivity ascompared to f-bCblC. These results indicate that t-bCblC is a moresensitive form to be regulated by glutathione than the full-lengthform of the protein. [BMB Reports 2013; 46(3): 169-174]

Highlights: [•] A bovine B12 trafficking chaperone bCblC catalyzes deglutathionylation of GSCbl by using GSH. [•] Deglutathionylation of GSCbl generates, via cob(I)alamin, cob(II)alamin with GSSG. [•] Deglutathionylation of GSCbl is highly efficient in terms of the catalytic turnover number. [•] GSCbl is a precursor of enzyme cofactors more effective than other cobalamins. [ABSTRACT FROM AUTHOR]

Abstract: The protein, bCblCpro, is a bovine B12 trafficking chaperone involved in intracellular B12 metabolism. bCblCpro is highly thermolabile (T m =∼42°C) and the reduced form of glutathione, GSH, has been found to stabilize bCblCpro as a positive regulator. In this study, we discovered that the oxidized form of glutathione, GSSG, destabilizes bCblCpro, which is derived from changes in the conformation of the protein upon GSSG binding. The binding affinity for GSSG was determined to be similar with the affinity for GSH. The AC50 =2.8±0.4mM of GSSG for destabilization of bCblCpro was consistently similar with the AC50 =2.1±0.5mM of GSH for stabilization of the protein. These results suggest that GSSG is a negative regulator of bCblCpro and that the molar ratio of [GSH]/[GSSG] in cells may determine the stability of the B12 trafficking chaperone. [Copyright &y& Elsevier]