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 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.
Lee
Y
Y
Department of Pediatrics, University of Southern California Medical School, Childrens Hospital of Los Angeles, California 90027, USA.
Kinoshita
H
H
Radke
G
G
Weiler
S
S
Barranger
J A
JA
Tomich
J M
JM
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry* / Q000302:isolation & purification; D004795:Enzyme Stability; D005962:Glucosylceramidase / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D008264:Macrophages / Q000201:enzymology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010449:Peptide Mapping; D010920:Placenta / Q000201:enzymology; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000302:isolation & purification
D004220:Disulfides; D013438:Sulfhydryl Compounds; D005962:Glucosylceramidase