Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase

Purified human glucocerebrosidase isolated from placenta was modified with [¹⁴C]-iodoacetic acid without reduction and digested with both protease-V8 at pH 4.0 followed by-chymotrypsin at pH 7.5. The majority of radioactivity was found in a peptide that contained the [¹⁴C]-carboxymethylated-cysteine identified as CM-Cys₁₈. Direct sequencing of the N-terminus of the intact labeled protein confirmed the modification of Cys₁₈. For identification of disulfide bond-containing peptides, another portion of glucocerebrosidase was alkylated with nonlabeled iodoacetic acid and then digested with protease V8 and-chymotrypsin as before. Twenty-eight HPLC fragments were collected. These purified peaks were then reduced with-mercaptoethanol followed by S-carboxymethylation with [¹⁴C]-iodoacetic acid. Three peptides among these 28 peptides generated two radioactive daughter peptides. These peptides were sequenced and the position of the radioactive CM-cysteines identified. The locations of these disulfides are Cys₄-Cys₁₆, Cys₂₃-Cys₃₄₂, and Cys₁₂₆-Cys₂₄₈. Attempts to reproduce the free sulfhydryl labeling experiments using the glucocerebrosidase isolated from Ceredase proved unsuccessful. No label was incorporated by this enzyme prior to reduction. This result suggests that the form of the protein used in the clinic differs from the native protein.     

Identification of the binding and activating sites of the sphingolipid activator protein, saposin C, with glucocerebrosidase.

Saposin C is a sphingolipid activator protein of 8.5 kDa that activates lysosomal glucocerebrosidase. Previously, we synthesized and characterized a synthetic full-length human saposin C protein that displays 85% of the activity of the native saposin C. In this study we use shorter synthetic peptides derived from the saposin C sequence to map binding and activation sites. By determining the activity and kinetic constant (Kact) values of these peptides, we have identified two functional domains, each comprising a binding site adjacent to or partially overlapping with an activation site. Domains 1 and 2 are located within amino acid positions 6-34 and 41-60, respectively. The activation sites span residues 27-34 and 41-49, whereas binding sites encompass residues 6-27 and 45-60. Peptides containing the sequences of either domain displayed 90% of the activity of the full-length synthetic saposin C. Domain 2, however, bound to glucocerebrosidase by at least an order of magnitude more strongly than domain 1. Binding sites within these domains contain sequences that are excellent candidates for forming amphipathic helical structures. Competition assays demonstrated that the binding of one domain to glucocerebrosidase prevents binding of the other domain, and that saposin A and saposin C bind to the same sites on glucocerebrosidase. A model predicting a saposin C:glucocerebrosidase complex with a stoichiometry of 4:2, respectively, is presented.