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27-Oxygenation of C27-sterols and 25-hydroxylation of vitamin D3 in kidney: cloning, structure and expression of pig kidney CYP27A.

ABSTRACT: This paper describes the molecular cloning of a cytochrome P450 enzyme in pig kidney that catalyses the hydroxylations of vitamin D(3) (cholecalciferol) and C(27)-sterols. DNA sequence analysis of the cDNA revealed that the enzyme belongs to the CYP27 family. The first 36 amino acids have many hallmarks of a mitochondrial signal sequence. The mature pig kidney CYP27 protein contains 498 amino acids. The M(r) of the mature protein was calculated to be 56607. The structure of pig kidney CYP27, as deduced by DNA sequence analysis, shows 77-83% identity with CYP27A in rat, rabbit and human liver. Transfection of the renal CYP27A cDNA into simian COS cells resulted in the synthesis of an enzyme that catalysed the 25-hydroxylation of vitamin D(3) and the 27-hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol, and the further oxidation of the product into the corresponding C(27)-acid 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid. As part of these studies, the enzymic activities of cultured human embryonic kidney cells were examined using vitamin D(3) and C(27)-sterols as substrates. The cells were found to express CYP27A mRNA and to convert the respective substrates into the same products as recombinantly expressed CYP27A, i.e. 25-hydroxyvitamin D(3) and 27-oxygenated C(27)-sterols. The results of the present study describing the structure and expression of CYP27A in kidney suggest that this enzyme is involved in the renal metabolism of vitamin D(3) and that the kidney plays a role in the metabolism of cholesterol and other C(27)-sterols.

SUBMITTER: Postlind H

PROVIDER: S-EPMC1220965 | biostudies-other | 2000 Apr

REPOSITORIES: biostudies-other

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Project description:The properties of cytochrome P-450 from pig kidney mitochondria, catalysing 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids [Postlind & Wikvall (1989) Biochem. Biophys. Res. Commun. 159, 1135-1140; Postlind (1990) Biochem. Biophys. Res. Commun. 168, 261-266], were compared with those of a 26-hydroxylating cytochrome P-450 from pig liver mitochondria. The liver enzyme was purified to a cytochrome P-450 content of 7.4 nmol/mg of protein and showed only one protein band with an apparent Mr of 53,000 upon SDS/PAGE. The cytochrome P-450 catalysed 26-hydroxylation of 25-hydroxyvitamin D3, cholesterol and 5 beta-cholestane-3 alpha, 7 alpha-diol at rates of 361, 1090 and 2065 pmol/min per nmol of cytochrome P-450. A monoclonal antibody against the purified liver mitochondrial cytochrome P-450 26-hydroxylase (cytochrome P-450(26] was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(26) from liver as well as from kidney mitochondria and to immunoprecipitate the 26-hydroxylase activity towards 25-hydroxyvitamin D3 and cholesterol when assayed in a reconstituted system. After SDS/PAGE and immunoblotting with the antibody, the cytochrome P-450(26) was detected in the purified liver and kidney preparations. These results indicate that similar species of cytochrome P-450 catalyse 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids in liver and kidney mitochondria. The results with the monoclonal antibody together with the finding that cholesterol competitively inhibits the 26-hydroxylation of 25-hydroxyvitamin D3 further indicate that 26-hydroxylation of 25-hydroxyvitamin D3 and cholesterol is catalysed by the same species of cytochrome P-450 in each tissue. The N-terminal amino acid sequence of cytochrome P-450(26) in kidney mitochondria resembled that of pig kidney microsomal 25-hydroxylase active in 25-hydroxylation of vitamin D3 and C27 steroids, whereas the sequence of pig liver mitochondrial cytochrome P-450(26) differed from those of rabbit and rat liver mitochondrial 26-hydroxylases as well as from those of other hitherto isolated mammalian cytochromes P-450.

Project description:RationalePatients with chronic rhinosinusitis with nasal polyps (CRSwNP) have been shown to be vitamin D3 (VD3) deficient, which is associated with more severe disease and increased polyp size. To gain mechanistic insights into these observational studies, we examined the impact of VD3 deficiency on inflammation and VD3 metabolism in an Aspergillus fumigatus (Af) mouse model of chronic rhinosinusitis (Af-CRS).MethodsBalb/c mice were fed control or VD3 deficient diet for 4 weeks. Mice were then sensitized with intraperitoneal Af, and one week later given Af intranasally every three days for four weeks while being maintained on control or VD3 deficient diet. Airway function, sinonasal immune cell infiltrate and sinonasal VD3 metabolism profiles were then examined.ResultsMice with VD3 deficiency had increased Penh and sRaw values as compared to controls as well as exacerbated changes in sRaw when coupled with Af-CRS. As compared to controls, VD3 deficient and Af-CRS mice had reduced sinonasal 1α-hydroxylase and the active VD3 metabolite, 1,25(OH)2D3. Differential analysis of nasal lavage samples showed that VD3 deficiency alone and in combination with Af-CRS profoundly upregulated eosinophil, neutrophil and lymphocyte numbers. VD3 deficiency exacerbated increases in monocyte-derived dendritic cell (DC) associated with Af-CRS. Conversely, T-regulatory cells were decreased in both Af-CRS mice and VD3 deficient mice, though coupling VD3 deficiency with Af-CRS did not exacerbate CD4 or T-regulatory cells numbers. Lastly, VD3 deficiency had a modifying or exacerbating impact on nasal lavage levels of IFN-γ, IL-6, IL-10 and TNF-α, but had no impact on IL-17A.ConclusionsVD3 deficiency causes changes in sinonasal immunity, which in many ways mirrors the changes observed in Af-CRS mice, while selectively exacerbating inflammation. Furthermore, both VD3 deficiency and Af-CRS were associated with altered sinonasal VD3 metabolism causing reductions in local levels of the active VD3 metabolite, 1,25(OH)2D3, even with adequate circulating levels.

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27-Oxygenation of C27-sterols and 25-hydroxylation of vitamin D3 in kidney: cloning, structure and expression of pig kidney CYP27A.

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