Purification, characterization and crystallization of human placental estrone/dehydroepiandrosterone sulfatase, a membrane-bound enzyme of the endoplasmic reticulum

• Published in: The Journal of steroid biochemistry and molecular biology Vol. 78; no. 5; pp. 441 - 450
• Main Authors: Hernandez-Guzman, Francisco G, Higashiyama, Tadayoshi, Osawa, Yoshio, Ghosh, Debashis
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.11.2001; Elsevier Science
• Subjects: Amino Acid Sequence; Arylsulfatases - chemistry; Arylsulfatases - genetics; Arylsulfatases - isolation & purification; Arylsulfatases - metabolism; Biological and medical sciences; Crystallization; Crystallography, X-Ray; Dehydroepiandrosterone Sulfate; DHEA sulfate; Electrophoresis, Polyacrylamide Gel; Estrogen biosynthesis; Estrogens - biosynthesis; Estrone; Estrone sulfate; Female; Fundamental and applied biological sciences. Psychology; Humans; In Vitro Techniques; Membrane protein crystallization; Membranes - enzymology; Molecular Sequence Data; Placenta - enzymology; Pregnancy; Protein Structure, Quaternary; Solubility; Solubilization of microsomal enzymes; Steroid hormones. Cholecalciferol derivatives; Steroid sulfatase; Steryl-Sulfatase; Substrate Specificity; Sulfatases - chemistry; Sulfatases - genetics; Sulfatases - isolation & purification; Sulfatases - metabolism; Vertebrates: endocrinology; 17β-HSD1, 17β-hydroxysteroid dehydrogenase type 1; Estrone sulfate; P450arom, cytochrome P450 aromatase; Solubilization of microsomal enzymes; Dpm, radioactive disintegrations per minute; BME, β-mercaptoethanol; E1-S, estrone sulfate; HA, hydroxylapatite; SDS, sodium dodecyl sulfate; Membrane protein crystallization; Steroid sulfatase; PAGE, polyacrylamide gel electrophoresis; Estrogen biosynthesis; E2, 17β-estradiol; ES, estrone sulfatase; BOG, n-octyl-β- d-glucopyranoside; DHEA-S, DHEA sulfate; E1, estrone; DHEAS, DHEA sulfatase; DHEA sulfate; DHEA, dehydroepiandrosterone; MPD, methylpentanediol; Human; EC 3.1.6.1; Purification; Enzyme; Estrogen; 17β-Estradiol; Esterases; Biosynthesis; Ovarian hormone; Characterization; Dehydroepiandrosterone sulfate; Arylsulfatase; Hydrolases; Estrone; Sulfuric ester hydrolases; Endoplasmic reticulum
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/S0960-0760(01)00119-4

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.