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Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center.

ABSTRACT: Collagen is the most abundant protein in animals. The posttranslational hydroxylation of proline residues in collagen contributes greatly to its conformational stability. Deficient hydroxylation is associated with a variety of disease states, including scurvy. The hydroxylation of proline residues in collagen is catalyzed by an Fe(II)- and ?-ketoglutarate-dependent dioxygenase, collagen prolyl 4-hydroxylase (CP4H). CP4H has long been known to suffer oxidative inactivation during catalysis, and the cofactor ascorbate (vitamin C) is required to reactivate the enzyme by reducing its iron center from Fe(III) to Fe(II). Herein, we report on the discovery of the first synthetic activators of CP4H. Specifically, we find that 2,2'-bipyridine-4-carboxylate and 2,2'-bipyridine-5-carboxylate serve as ligands for the iron center in human CP4H that enhance the rate of ascorbate-dependent reactivation. This new mode of CP4H activation is available to other biheteroaryl compounds but does not necessarily extend to other prolyl 4-hydroxylases. As collagen is weakened in many indications, analogous activators of CP4H could have therapeutic benefits.

SUBMITTER: Vasta JD

PROVIDER: S-EPMC5141248 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center.

Vasta James D JD Raines Ronald T RT

Biochemistry 20160531 23

Collagen is the most abundant protein in animals. The posttranslational hydroxylation of proline residues in collagen contributes greatly to its conformational stability. Deficient hydroxylation is associated with a variety of disease states, including scurvy. The hydroxylation of proline residues in collagen is catalyzed by an Fe(II)- and α-ketoglutarate-dependent dioxygenase, collagen prolyl 4-hydroxylase (CP4H). CP4H has long been known to suffer oxidative inactivation during catalysis, and t ...[more]

PMID: 27183028

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Project description:BackgroundWe describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H alpha2beta2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-alpha subunit antibody magnetic beads and an anti-beta subunit antibody binds to the PDI/beta subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.ResultsWe applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield.ConclusionsHere we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits.

Dataset Information

Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center.

Publications

Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center.

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