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Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase.

ABSTRACT: BACKGROUND: Collagens require the hydroxylation of proline (Pro) residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H) as a posttranslational processing step. RESULTS: A recombinant human collagen type I ?-1 (rCI?1) with high percentage of hydroxylated prolines (Hyp) was produced in transgenic maize seeds when co-expressed with both the ?- and ?- subunits of a recombinant human P4H (rP4H). Germ-specific expression of rCI?1 using maize globulin-1 gene promoter resulted in an average yield of 12 mg/kg seed for the full-length rCI?1 in seeds without co-expression of rP4H and 4 mg/kg seed for the rCI?1 (rCI?1-OH) in seeds with co-expression of rP4H. High-resolution mass spectrometry (HRMS) analysis revealed that nearly half of the collagenous repeating triplets in rCI?1 isolated from rP4H co-expressing maize line had the Pro residues changed to Hyp residues. The HRMS analysis determined the Hyp content of maize-derived rCI?1-OH as 18.11%, which is comparable to the Hyp level of yeast-derived rCI?1-OH (17.47%) and the native human CIa1 (14.59%), respectively. The increased Hyp percentage was correlated with a markedly enhanced thermal stability of maize-derived rCI?1-OH when compared to the non-hydroxylated rCI?1. CONCLUSIONS: This work shows that maize has potential to produce adequately modified exogenous proteins with mammalian-like post-translational modifications that may be require for their use as pharmaceutical and industrial products.

SUBMITTER: Xu X

PROVIDER: S-EPMC3151215 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase.

Xu Xing X Gan Qinglei Q Clough Richard C RC Pappu Kameshwari M KM Howard John A JA Baez Julio A JA Wang Kan K

BMC biotechnology 20110624

<h4>Background</h4>Collagens require the hydroxylation of proline (Pro) residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H) as a posttranslational processing step.<h4>Results</h4>A recombinant human collagen type I α-1 (rCIα1) with high percentage of hydr ...[more]

PMID: 21702901

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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.

Project description:Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1(H662A) ). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity versus complete ablation of the prolyl 3-hydroxylation complex.

Dataset Information

Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase.

Publications

Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase.

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