The purification and application of biologically active recombinant steroid cytochrome P450 21-hydroxylase: The major autoantigen in autoimmune Addison's disease

Bratland, Eirik; Bredholt, Geir; Mellgren, Gunnar; Knappskog, Per M.; Mozes, Edna; Husebye, Eystein S.

doi:10.1016/j.jaut.2009.02.018

Cited by 8 publications

(6 citation statements)

References 39 publications

(48 reference statements)

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“…Although the k cat values were similar with both substrates, the 7-fold lower K m value with the substrate progesterone resulted in much higher catalytic efficiency than with the substrate 17␣-OH-progesterone. Compared with other previous studies (11,30), the significantly higher enzymatic activities of human P450 21A2 reported herein are probably due to the fact that the purified enzyme was utilized in the assays rather than whole cells or cell lysates containing expressed human P450 21A2.…”

Section: Purification Of Human P450 21a2 and Catalytic Properties-contrasting

confidence: 76%

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Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase

Pallan

Wang

Lei

et al. 2015

Journal of Biological Chemistry

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Background: P450 21A2 catalyzes 21-hydroxylation of both progesterone and 17␣-hydroxyprogesterone, an important step in adrenal steroidogenesis. Results: A crystal structure of human P450 21A2 with progesterone can explain many functional variants. Conclusion: High kinetic deuterium isotope effects show the importance of a closely spaced site for C21 hydrogen abstraction. Significance: The structure provides insight into enzyme deficiencies in congenital adrenal hyperplasia.

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Section: Purification Of Human P450 21a2 and Catalytic Properties-contrasting

confidence: 76%

“…That structure has been used to model some of the possible effects of variants observed in clinics (6,7). Human P450 21A2 has been heterologously expressed in yeast microsomes and utilized in mutagenesis and basic kinetic isotope effect (KIE) studies (13) and also expressed in baculovirus (11) and bacterial systems (16).…”

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confidence: 99%

Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase

Pallan

Wang

Lei

et al. 2015

Journal of Biological Chemistry

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“…Additionally, using the entire 21OH protein instead of peptide pools in the ELISPOT assay would probably facilitate the identification of autoreactive CD4 T cells [34]. However, purification of the correctly-folded entire 21OH was recently reported as quite challenging [35], which may explain the lack of prior identification of 21OH-specific CD4 T cells. Indeed, using this purified protein and a few 21OH peptides selected from data in immunized mice, the same group identified an immunodominant peptide (residues 342-361) that induced proliferation and/or IFNg production in HLA-DRB1*0404-DQ8 patients.…”

Section: Tetramer Analysis Confirms the Presence Of Hla-b8-restrictedmentioning

confidence: 99%

21-Hydroxylase epitopes are targeted by CD8 T cells in autoimmune Addison’s disease

Rottembourg

Deal

Lambert

et al. 2010

Journal of Autoimmunity

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“…The enzyme reactions were set up mainly as described previously (13). Briefly, from each 21OH construct, 50 μl of the in vitro expression product were mixed with 15 pmol of recombinant human cytochrome P450 oxidoreductase (CYPOR, Sigma–Aldrich) and 25 μl of 300 μM 17OHP (in 10% methanol) on ice.…”

Section: Methodsmentioning

confidence: 99%

Functional studies of novel CYP21A2 mutations detected in Norwegian patients with congenital adrenal hyperplasia

Brønstad

Breivik

Methlie

et al. 2014

Endocrine Connections

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In about 95% of cases, congenital adrenal hyperplasia (CAH) is caused by mutations in CYP21A2 gene encoding steroid 21-hydroxylase (21OH). Recently, we have reported four novel CYP21A2 variants in the Norwegian population of patients with CAH, of which p.L388R and p.E140K were associated with salt wasting (SW), p.P45L with simple virilising (SV) and p.V211M+p.V281L with SV to non-classical (NC) phenotypes. We aimed to characterise the novel variants functionally utilising a newly designed in vitro assay of 21OH enzyme activity and structural simulations and compare the results with clinical phenotypes. CYP21A2 mutations and variants were expressed in vitro. Enzyme activity was assayed by assessing the conversion of 17-hydroxyprogesterone to 11-deoxycortisol by liquid chromatography tandem mass spectroscopy. PyMOL 1.3 was used for structural simulations, and PolyPhen2 and PROVEAN for predicting the severity of the mutants. The CYP21A2 mutants, p.L388R and p.E140K, exhibited 1.1 and 11.3% of wt 21OH enzyme activity, respectively, in vitro. We could not detect any functional deficiency of the p.P45L variant in vitro; although prediction tools suggest p.P45L to be pathogenic. p.V211M displayed enzyme activity equivalent to the wt in vitro, which was supported by in silico analyses. We found good correlations between phenotype and the in vitro enzyme activities of the SW mutants, but not for the SV p.P45L variant. p.V211M might have a synergistic effect together with p.V281L, explaining a phenotype between SV and NC CAH.

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The purification and application of biologically active recombinant steroid cytochrome P450 21-hydroxylase: The major autoantigen in autoimmune Addison's disease

Cited by 8 publications

References 39 publications

Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase

Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase

21-Hydroxylase epitopes are targeted by CD8 T cells in autoimmune Addison’s disease

Functional studies of novel CYP21A2 mutations detected in Norwegian patients with congenital adrenal hyperplasia

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