Abstract
BackgroundCollagens 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.ResultsA recombinant human collagen type I α-1 (rCIα1) with high percentage of hydroxylated prolines (Hyp) was produced in transgenic maize seeds when co-expressed with both the α- and β- subunits of a recombinant human P4H (rP4H). Germ-specific expression of rCIα1 using maize globulin-1 gene promoter resulted in an average yield of 12 mg/kg seed for the full-length rCIα1 in seeds without co-expression of rP4H and 4 mg/kg seed for the rCIα1 (rCIα1-OH) in seeds with co-expression of rP4H. High-resolution mass spectrometry (HRMS) analysis revealed that nearly half of the collagenous repeating triplets in rCIα1 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 rCIα1-OH as 18.11%, which is comparable to the Hyp level of yeast-derived rCIα1-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 rCIα1-OH when compared to the non-hydroxylated rCIα1.ConclusionsThis 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.
Highlights
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
To avoid undesired DNA rearrangement caused by using identical sequences, we chose to use the maize ubiquitin promoter (Pubi, [24]) to drive the expression of a and b subunits of recombinant human prolyl 4-hydroxylases (P4Hs) (rP4H)
This result indicates that the co-expression of rP4H in maize can greatly enhance the hydroxylation of prolines on collagen molecules
Summary
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. Collagen is the most abundant protein found in animals. Human type I collagen is the most abundant collagen type in the human body and is one of the most studied collagen types. It is a heterotrimer composed of two a1 (CIa1) and one a2 (CIa2) chains with the helical region composed by a repeating composition of Xaa-Yaa-Gly, where X and Y are typically proline (Pro) and hydroxyproline (Hyp) [3]. The production of plant derived recombinant collagen I a-1 (rCIa1) was reported in 2000 using tobacco [10] and tobacco cell culture [2]
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