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 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.

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.

Figure 1

A schematic representation of the two constructs used in this study. LB, left border of Agrobacterium T-DNA; T35S, CaMV 35S terminator; bar, bialaphos resistant coding sequence; P, CaMV 35S promoter; PIN II, potato protease inhibitor II gene terminator; CIα1, human collagen I α1 chain coding sequence; BAASS, barley alpha amylase signal sequence; P4Hα, prolyl 4-hydroxylase α subunit; P4Hβ, prolyl 4-hydroxylase β subunit; Pglb, maize globulin-1 promoter; Pubi, maize ubiquitin promoter; RB, right border of Agrobacterium T-DNA.

Figure 2

Analysis of electrophoretic mobility difference of rCIα1 in the CGB and CGD line. Equal volumes of total protein extracts from seeds of CGB, CGD (10 × concentrated by volume) and mixture of CGB+CGD extracts were loaded on the gel. The rCIα1 from CGB and CGD lines were detected by western blot using anti-foldon antibody. Pichia-derived rCIα1 (FE291) and rCIα1-OH (FE285) were included as controls and detected by Coomassie Brilliant Blue staining. Open arrows, rCIα1 from CGB or FE291; solid arrows, OH-rCIα1 from CGD or FE285. M, molecular weight marker.

Figure 3

Western blot analysis of the rP4Hβ using anti-P4Hβ antibody. Equal volume of total protein extraction from seeds of CGB, CGD and non-transgenic control maize (WT) extracts was loaded on the gel. Open triangle, rP4Hβ. M: molecular weight marker.

Figure 4

LC-MS/MS analysis of the rCIα1. Full length peptide sequences of 1057 amino acid are listed. Pichia rCIα1, Pichia-derived rCIα1 from strain FE291; Maize rCIα1, maize-derived rCIα1 from line CGB; Human CIα1, gel-isolated CIα1 fragment from commercial collagen (CalBiochem Inc); Maize rCIα1-OH, maize-derived rCIα1 from line CGD; Pichia rCIα1-OH, Pichia-derived rCIα1 from strain FE285. Yellow-highlighted letters: amino acid sequences identified by the Orbitrap; green-highlighted letters: Hyp residues identified by the Orbitrap; red boxes: peptide regions identified in all five samples by the Orbitrap. Black boxes: collagenous triplets Xaa-Pro-Gly with Pro changed to Hyp in Maize rCIα1-OH but not in Maize rCIα1; single underlines: triplets with Pro unchanged in both maize lines; double underlines: triplets with Pro changed to Hyp in both maize lines.

Figure 5

Thermal stability analysis of the rCIα1 from maize, Pichia and human. (A) Western blot results of the maize-derived rCIα1 (CGB), rCIα1-OH (CGD), Pichia-derived rCIα1 (FE291), and rCIα1-OH (FE285) after 4°C incubation and pepsin treatment, using anti-foldon antibody. (B) Western blot results of the maize-derived rCIα1 (CGB), rCIα1-OH (CGD), and human CIα1 (HuC) after heat treatments under various temperatures and pepsin treatments as indicated, using both anti-foldon and anti-25 kD collagen antibody. M: molecular weight marker.