Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue.

25-hydroxyvitamin D(3)- or 1alpha,25-dihydroxyvitamin D(3)-24R-hydroxylase (cytochromeP450C24 or CYP24) has a dual role of removing 25-OH-D(3) from circulation and excess 1,25(OH)(2)D(3) from kidney. As a result, CYP24 is an important multifunctional regulatory enzyme that maintains essential tissue-levels of Vitamin D hormone. As a part of our continuing interest in structure-function studies characterizing various binding proteins in the Vitamin D endocrine system, we targeted recombinant rat CYP24 with a radiolabeled 25-OH-D(3) affinity analog, and showed that the 25-OH-D(3)-binding site was specifically labeled by this analog. An affinity labeled sample of CYP24 was subjected to MS/MS analysis, which identified Ser57 as the only amino acid residue in the entire length of the protein that was covalently modified by this analog. Site-directed mutagenesis was conducted to validate the role of Ser57 towards substrate-binding. S57A mutant displayed significantly lower binding capacity for 25-OH-D(3) and 1,25(OH)(2)D(3). On the other hand, S57D mutant strongly enhanced binding for the substrates and conversion of 1,25(OH)(2)D(3) to calcitroic acid. The affinity probe was anchored via the 3-hydroxyl group of 25-OH-D(3). Therefore, these results suggested that the 3-hydroxyl group (of 25-OH-D(3) and 1,25(OH)(2)D(3)) in the S57D mutant could be stabilized by hydrogen bonding or a salt bridge leading to enhanced substrate affinity and metabolism.

Molecular basis for pseudo vitamin D-deficiency rickets in the Hannover pig.

The molecular basis for pseudo vitamin D deficiency rickets (PDDR) in the Hannover pig model was determined in the current study. Consistent with the inability of Hannover PDDR pigs to maintain ambient levels of 1,25-dihydroxyvitamin D (i.e., 1,25D), the bioactivation enzyme cytochrome P450C1 (or CYP27B1) was determined to contain coding-region deletions that rendered the enzyme ineffective due to frame-shift mutations and expression of a premature termination codon. Expression levels of P450C1mRNA were up-regulated in response to the low-1,25D high-parathyroid hormone state of the PDDR animals. In a complementary manner, cytochrome P450C24 mRNA was not detectable in PDDR pigs. Two different deletions were detected within the Hannover pig strain in which the P450C1 coding region contained either 173 bp or 329 bp deletions that resulted in the expression of non-sense products beginning within the I-helix region and extending through the truncated C-terminal domains. The boundaries for the deletion segments aligned with derived mRNA processing sites. This observation was consistent with an mRNA processing error as the causative factor for the coding-region deletions. Based upon the expression of a non-functional P450C1 enzyme, the Hannover pig model for PDDR was determined to be identical to the human disease in which enzyme-inhibitory mutations are the molecular basis for the calcium disorder.

Expression, structure-function, and molecular modeling of vitamin D P450s.

Although vitamin D(3) is a natural product of a sunlight-mediated process in the skin, the secosteroid's biological function is dependent upon specific cytochrome P450 enzymes that mediate the parent vitamin's bioactivation and inactivation. Cytochrome P450C1 (CYP27B1) is the regulatory rate-limiting enzyme that directs the bioactivation process through introduction of a C-1alpha hydroxyl group. The resultant 1,25-dihydroxyvitamin D(3) (1,25D) is the biologically active secosteroid hormone that directs the multitude of vitamin D-dependent actions involved with calcium homeostasis, cellular differentiation and growth, and the immune response. The circulating and cellular level of 1,25D is regulated through a coordinated process involving the hormone's synthesis and degradation. Central to the degradation and turnover of 1,25D is the regulatory multi-catalytic cytochrome P450C24 (CYP24) enzyme that directs the introduction of C-24R groups onto targeted 25-hydroxy substrates. Discussed in this article is the action of the rat CYP24 to catalyze the side-chain oxidation and cleavage of 25-hydroxylated vitamin D metabolites. Expression and characterization of purified recombinant rat CYP24 is discussed in light of mutations directed at the enzyme's active site.

The Notch ligand, Delta-1, inhibits the differentiation of monocytes into macrophages but permits their differentiation into dendritic cells.

Notch-mediated cellular interactions are known to regulate cell fate decisions in various developmental systems. A previous report indicated that monocytes express relatively high amounts of Notch-1 and Notch-2 and that the immobilized extracellular domain of the Notch ligand, Delta-1 (Delta(ext-myc)), induces apoptosis in peripheral blood monocytes cultured with macrophage colony-stimulating factor (M-CSF), but not granulocyte-macrophage CSF (GM-CSF). The present study determined the effect of Notch signaling on monocyte differentiation into macrophages and dendritic cells. Results showed that immobilized Delta(ext-myc) inhibited differentiation of monocytes into mature macrophages (CD1a+/-CD14+/- CD64+) with GM-CSF. However, Delta(ext-myc) permitted differentiation into immature dendritic cells (CD1a+CD14-CD64-) with GM-CSF and interleukin 4 (IL-4), and further differentiation into mature dendritic cells (CD1a+CD83+) with GM-CSF, IL-4, and tumor necrosis factor-alpha (TNF-alpha). Notch signaling affected the differentiation of CD1a-CD14+ macrophage/dendritic cell precursors derived in vitro from CD34+ cells. With GM-CSF and TNF-alpha, exposure to Delta(ext-myc) increased the proportion of precursors that differentiated into CD1a+CD14- dendritic cells (51% in the presence of Delta(ext-myc) versus 10% in control cultures), whereas a decreased proportion differentiated into CD1a-CD14+ macrophages (6% versus 65%). These data indicate a role for Notch signaling in regulating cell fate decisions by bipotent macrophage/dendritic precursors.

Identification of a novel parvalbumin in avian thymic tissue.

A novel calcium-binding protein has been isolated from chicken thymus tissue. Its molecular weight (approximately 11,500) and characteristic interactions with Tb3+ and Eu3+ identify the protein as a member of the parvalbumin family. Electrophoretically distinct from both chicken (muscle) parvalbumin and avian thymic hormone, it represents the third parvalbumin to be identified in avian tissues and the second to be identified in the avian thymus gland.

Metal ion-binding properties of avian thymic hormone.

Lanthanide ion luminescence studies and 45Ca2(+)-binding measurements were used to study the metal ion-binding properties of avian thymic hormone. The procedure used to isolate the protein--involving heat-treatment at 80 degrees C, trichloroacetic acid precipitation, DEAE-agarose chromatography, and gel filtration--affords material that is deemed homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as well as the absence of a detectable tryptophan signal in the fluorescence emission spectrum. Avian thymic hormone exhibits a pI = 4.35 when subjected to isoelectric focusing through polyacrylamide gels. The two ion-binding sites are indistinguishable in their interactions with Ca2+ and Mg2+, displaying KCa = 8 nM and KMg = 68 microM. The Eu3+ 7Fo----5Do excitation spectrum at pH 6 displays a peak at 5795.4 A, with a shoulder at 5792.8 A and is replaced at higher pH values by a broader spectrum with a maximum at 5784.8 A and a shoulder at 5777.1 A. The pKa governing this spectral interconversion is 8.21. All of these properties are very similar to those observed with other parvalbumins. However, polyclonal antibodies to avian thymic hormone do not cross-react with the parvalbumin from chicken leg muscle, as judged by Western blot analysis-further evidence that avian thymic hormone and the muscle-associated chicken parvalbumin are indeed distinct proteins.