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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 alpha-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 alpha-chymotrypsin as before. Twenty-eight HPLC fragments were collected. These purified peaks were then reduced with beta-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.

Gaucher disease is an inherited lysosomal storage disorder caused by a deficiency of human acid beta-glucosidase (glucocerebrosidase). This enzyme is inactivated by the specific, mechanism-based enzyme inactivator 2-deoxy-2-fluoro-beta-D-glucopyranosyl fluoride, which functions by forming a stable 2-deoxy-2-fluoro-alpha-D-gluco-pyranosyl-enzyme intermediate. The key nucleophilic amino acid residue involved in formation of this intermediate was conclusively identified by tandem mass spectrometry as Glu340, and not Asp443 as thought previously. This was confirmed by site-directed mutagenesis. Identification, and mass determination, of the labeled peptide in a proteolytic hydrolysate involved detection of a collision-induced fragmentation reaction specific to the sugar-peptide linkage. Confirmation of the identity of the labeled peptide was obtained both by tandem mass spectrometric sequencing and by chemical degradation of the purified peptide. This method allowed the rapid, sensitive, and non-isotopic determination of an essential amino acid residue in a clinically important enzyme.

Important therapeutic principles were established in developing effective enzyme replacement therapy for patients with Gaucher disease. The background and sequence of the investigations that led to effective delivery of exogenous glucocerebrosidase to the lipid-storing macrophages in patients with Gaucher disease are described. The principle of targeting the intravenously injected enzyme to the mannose lectin on the surface of these cells by engineering the glycoform of the enzyme is a useful model of an essential requirement for effective enzyme therapy. Similar strategies are expected to be effective for the treatment of a number of hereditary metabolic disorders of humans.

Glucocerebrosidase was purified from human cultured dermal fibroblasts more than 2200-fold to apparent homogeneity using high performance Alkyl-Superose HR 5/5 hydrophobic interaction and Bio-Sil TSK-250 gel permeation column chromatography. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis and protein staining of the catalytically active and concentrated enzyme fractions from the gel permeation columns revealed the presence of one band of Mr 64,000. The glucocerebrosidase preparation purified to homogeneity was digested with peptide N-glycosidase F that cleaves N-linked oligosaccharide structures from glycoproteins. The molecular weight of glucocerebrosidase after digestion with peptide N-glycosidase F was reduced to Mr 57,000, suggesting that the mature enzyme is a glycoprotein and that N-linked oligosaccharide constitutes a minimum of about 10% of the total molecular weight of the polypeptide. These findings are compatible with the hypothesis that glucocerebrosidase was initially synthesized as a precursor polypeptide which was subsequently glycosylated to become the mature enzyme.