Disulfide Connectivity in Cerebroside Sulfate Activator Is Not Necessary for Biological Activity or α-Helical Content but Is Necessary for Trypsin Resistance and Strong Ligand Binding

Abstract

Cerebroside sulfate activator (CSAct) protein is exceptionally resistant to heat denaturation and proteolytic digestion. Although water soluble the protein binds membrane-associated lipids. Its biological role is thought to be to transfer certain lipids between membranes and to facilitate their catabolism in the lysosomes. An example of the latter is the removal of the sulfate group from cerebroside sulfate by arylsulfatase A. The mechanism of lipid sequestration from membranes and presentation of the lipid–protein complex to catabolic enzymes is a crucial aspect of the function of this protein. The widespread occurrence of the protein class of which CSAct is one of the best known members underscores the significance of this protein. The preparation, purification and chemical and biological properties of a stable disulfide blocked derivative of CSAct is described. The pyridoethylated protein was susceptible to tryptic attack and devoid of a significant population of solvent-protected exchange resistant protons. It apparantly formed a CS complex. However, unlike the complex with the native protein, this was not sufficiently stable to remain intact during size exclusion chromatography. The disulfide-blocked protein had a similar CD spectrum as native protein, indicating similar α-helical content. Unexpectedly, the activities of disulfide-blocked protein in the arylsulfatse A catalyzed sulfate hydrolysis from cerebroside sulfate were substantial. Hitherto, it had been assumed that the disulfide connectivities were essential for the protein to maintain a correctly folded configuration to bind lipid ligands and potentiate their hydrolysis. Some revision of our thoughts on the importance of the disulfide connectivities in the structure and function of the protein are necessary.