Formation of an intramolecular disulfide bond of glycolipid transfer protein

Functional and structural differences between the two forms of glycolipid transfer protein (GLTP) with (the faster component) and without (the slower component) an intramolecular disulfide bond were studied. GLTP treated with N-ethylmaleimide (NEM) and Na2S4O6 had a transfer activity of about 70% and 55%, respectively, of the control GLTP. No significant decrease was found in the binding affinity of NEM-treated GLTP to pyrene-labeled galactosylceramide (PyrGalCer). A small decrease in the binding affinity was found in the Na2S4O6-treated GLTP. Oxidation of NEM-treated and Na2S4O6-treated GLTP catalyzed by CuSO4 resulted in a stoichiometric conversion of the slower component to the faster component. The faster component thus formed was quantitatively reduced back to the slower component by treatment with 2-mercaptoethanol in the presence of 1% SDS. These results provided strong evidence for the conversion of the slower component to the faster component as a result of the formation of an intramolecular disulfide bond. The transfer activity of the NEM-treated and oxidized GLTP (the faster component) was 1.7-fold higher than that of the original GLTP and 2.4-fold higher than that of the NEM-treated GLTP. The transfer activity of the Na2S4O6-treated and oxidized GLTP was 2-fold higher than that of the original GLTP and 3.6-fold higher than that of the Na2S4O6-treated GLTP. The binding affinity of the faster components, produced from both the NEM-treated GLTP and the Na2S4O6-treated GLTP, to PyrGalCer was found to be twice that of the respective modified GLTPs before oxidation. By circular dichroism measurements, it was found that a small decrease in the magnitude of mean residue ellipticity but no significant change in ellipticity spectrum was brought about either upon the modification of GLTP by Na2S4O6 or upon the formation of an intramolecular disulfide bond in GLTP. The results suggest that the formation of an intramolecular disulfide bond results in only a small change in the secondary and tertiary structure of GLTP. The results presented in this paper suggest that the transfer activity of GLTP may be regulated by the redox state of sulfhydryl groups present in GLTP.