Human 11β-Hydroxysteroid Dehydrogenase Type 1 Is Enzymatically Active in Its Nonglycosylated Form

Blum, Andreas; Martin, H.; Maser, Edmund

doi:10.1006/bbrc.2000.3491

Cited by 31 publications

(22 citation statements)

References 33 publications

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“…Western blot analysis revealed double bands of 32 and 34 kDa molecular weight, possibly corresponding to different glycosylated forms of 11 -HSD1 proteins as previously described (Blum et al 2000). Expression of the 34 kDa glycosylated form of the enzyme is believed to reflect a more mature, physiologically active form, whereas the 32 kDa non-glycosylated form is proposed to be immature and to require further processing before being rendered functionally active.…”

Section: Discussionsupporting

confidence: 73%

“…Expression of the 34 kDa glycosylated form of the enzyme is believed to reflect a more mature, physiologically active form, whereas the 32 kDa non-glycosylated form is proposed to be immature and to require further processing before being rendered functionally active. However, Blum et al (2000) have reported that human 11 -HSD1 is enzymatically active regardless of its degree of glycosylation. Expression of the immature 32 kDa form remained relatively constant, with increased cortisol in the presence or absence of increased oestradiol concentrations.…”

Section: Discussionmentioning

confidence: 99%

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Effects of cortisol and oestradiol on hepatic 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor proteins in late-gestation sheep fetus

Gupta¹,

Alfaidy²,

Holloway³

et al. 2003

Journal of Endocrinology

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In the late-gestation sheep, increased fetal plasma cortisol concentration and placental oestradiol (E 2 ) output contribute to fetal organ maturation, in addition to the onset of parturition. Both cortisol and E 2 are believed to regulate the enzyme 11 -hydroxysteroid dehydrogenase type 1 (11 -HSD1), which interconverts bioactive 11-hydroxy glucocorticoids and their inactive 11-keto metabolites. 11 -HSD1, abundantly expressed in fetal liver, operates primarily as a reductase enzyme to produce bioactive cortisol and thus regulates local hepatic glucocorticoid concentrations. Cortisol acts through the glucocorticoid receptor (GR) present in the liver. In this study, we examined the effects of cortisol and E 2 on hepatic 11 -HSD1 and GR in the liver of chronically catheterized sheep fetuses treated with saline (n=5), cortisol (1·35 mg/ h; n=5), saline+4-hydroxyandrostendione, a P450 aromatase inhibitor (4-OHA; 1·44 mg/h; n=5), or cortisol+4-OHA (n=5). Cortisol infusion resulted in increased plasma concentrations of fetal cortisol and E 2 ; concurrent administration of 4-OHA attenuated the increase in plasma E 2 concentrations. Using immunohistochemistry, we showed that fetal hepatocytes expressed both 11 -HSD1 and GR proteins. Cortisol treatment increased GR in both cytosol and nuclei of hepatocytes; concurrent administration of 4-OHA was associated with distinct nuclear GR staining. Western blot revealed that cortisol, in the absence of increased E 2 concentrations, significantly increased concentrations of 11 -HSD1 (34 kDa) and GR (95 kDa) proteins. 11 -HSD1 enzyme activity was measured in the liver microsomal fraction in the presence of [ + (dehydrogenase activity) respectively. 11 -HSD1 reductase activity was significantly greater in the presence of cortisol. In summary, we found that, in sheep during late gestation, cortisol increased both 11 -HSD1 and GR in the fetal liver, and these effects were accentuated in the absence of increased E 2 .

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Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Effects of cortisol and oestradiol on hepatic 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor proteins in late-gestation sheep fetus

Gupta¹,

Alfaidy²,

Holloway³

et al. 2003

Journal of Endocrinology

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“…The catalytic domain is glycosylated (13)(14)(15), which is in agreement with a lumenal orientation. Experiments to resolve the importance of glycosylation have also yielded varying results.…”

supporting

confidence: 66%

“…Experiments to resolve the importance of glycosylation have also yielded varying results. Enzymatic deglycosylation of rabbit (9) and human (13) 11␤-HSD1 has indicated that glycosylation is not important for enzyme activity. However, partial inhibition of glycosylation of the rat enzyme by tunicamycin decreased dehydrogenase activity but not reductase activity (14), and mutation of the rat (15) and human (13) sequences at putative N-glycosylation sites resulted in reduced or abolished activity.…”

mentioning

confidence: 99%

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Functional Expression, Characterization, and Purification of the Catalytic Domain of Human 11-β-Hydroxysteroid Dehydrogenase Type 1

Walker¹,

Clark

Hewison³

et al. 2001

Journal of Biological Chemistry

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11-␤-hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders including insulin resistance and obesity. The enzyme is a glycosylated membrane-bound protein that has proved difficult to purify in an active state. Extracted enzyme typically loses the reductase properties seen in intact cells and shows principally dehydrogenase activity. The C-terminal catalytic domain is known to contain a disulfide bond and is located within the lumen of the endoplasmic reticulum, anchored to the membrane by a single N-terminal transmembrane domain. We report here the functional expression of the catalytic domain of the human enzyme, without the transmembrane domain and the extreme N terminus, in Escherichia coli. Moderate levels of soluble active protein were obtained using an N-terminal fusion with thioredoxin and a 6xHis tag. In contrast, the inclusion of a 6xHis tag at the C terminus adversely affected protein solubility and activity. However, the highest levels of active protein were obtained using a construct expressing the untagged catalytic domain. Nonreducing electrophoresis revealed the presence of both monomeric and dimeric disulfide bonded forms; however, mutation of a nonconserved cysteine residue resulted in a recombinant protein with no intermolecular disulfide bonds but full enzymatic activity. Using the optimal combination of plasmid construct and E. coli host strain, the recombinant protein was purified to apparent homogeneity by single step affinity chromatography. The purified protein possessed both dehydrogenase and reductase activities with a K m of 1.4 M for cortisol and 9.5 M for cortisone. This study indicates that glycosylation, the N-terminal region including the transmembrane helix, and intermolecular disulfide bonds are not essential for enzyme activity and that expression in bacteria can provide active recombinant protein for future structural and functional studies.

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11β‐Hydroxysteroid Dehydrogenase Type 1 as a Therapeutic Target for Type 2 Diabetes

Hale

Jean

2011

Metabolic Syndrome

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Human 11β-Hydroxysteroid Dehydrogenase Type 1 Is Enzymatically Active in Its Nonglycosylated Form

Cited by 31 publications

References 33 publications

Effects of cortisol and oestradiol on hepatic 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor proteins in late-gestation sheep fetus

Effects of cortisol and oestradiol on hepatic 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor proteins in late-gestation sheep fetus

Functional Expression, Characterization, and Purification of the Catalytic Domain of Human 11-β-Hydroxysteroid Dehydrogenase Type 1

11β‐Hydroxysteroid Dehydrogenase Type 1 as a Therapeutic Target for Type 2 Diabetes

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