Purification, characterization and crystallization of human placental estrone/dehydroepiandrosterone sulfatase, a membrane-bound enzyme of the endoplasmic reticulum.
Estrone (E1)/dehydroepiandrosterone (DHEA) sulfatase (ES/DHEAS) catalyzes the hydrolysis of E1 and DHEA-sulfates releasing unconjugated steroids. ES is a component of the three-enzyme system that has been implicated in intracrine biosynthesis of estradiol, hence, proliferation of hormone dependent breast tumors. ES is bound to the membrane of the endoplasmic reticulum, presumably through multiple transmembrane and other membrane anchoring segments. The highly hydrophobic nature of the enzyme has so far prevented its purification to homogeneity in quantities sufficient for crystallization. We report here the purification, biochemical characterization and crystallization of the full-length, active form of the enzyme from the membrane bound fraction of human placenta. Our results demonstrate that the key to successful purification and growth of diffraction quality crystals of this difficult membrane bound enzyme is the exploitation of optimal solubilization and detergent conditions to protect the structural and functional integrity of the molecule, thereby preventing nonspecific aggregation and other instabilities. This work paves the way for the first structural study of a membrane bound human sulfatase and subsequent rational design of inhibitors for use as anti-tumor agents.
Hernandez-Guzman
F G
FG
Department of Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Higashiyama
T
T
Osawa
Y
Y
Ghosh
D
D
D000595:Amino Acid Sequence; D001192:Arylsulfatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D019314:Dehydroepiandrosterone Sulfate; D004591:Electrophoresis, Polyacrylamide Gel; D004967:Estrogens / Q000096:biosynthesis; D004970:Estrone; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D020836:Protein Structure, Quaternary; D012995:Solubility; D043266:Steryl-Sulfatase; D013379:Substrate Specificity; D013429:Sulfatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D004967:Estrogens; D004970:Estrone; D019314:Dehydroepiandrosterone Sulfate; D013429:Sulfatases; C020121:estrone sulfatase; D001192:Arylsulfatases; D043266:Steryl-Sulfatase