Finally the presence of the recombinant hydroxylated

Finally, the presence of the recombinant hydroxylated sponge collagen polypeptide was detected in the colCH4 strain by MS-analysis using a TripleTOF instrument and allowing to confirm the ability of the recombinant sponge P4H to display its catalytic activity inside the yeast cell on its natural substrate (Fig. 5). Overall the percentage of identified hydroxylated prolines in the sponge recombinant collagen was of 36.3% with respect to all prolines present in the sequence. In particular 13.6% of prolines were hydroxylated in X position and 20.5% in Y position in the Xaa-Yaa-Gly triplets. Thus it seems that the sponge enzyme has the ability to hydroxylate proline in both X and Y positions, with the latter slightly preferred, differently from its human counterpart that acts almost only on the Y position, and demonstrates a rate of collagen hydroxylation (36.3% of all prolines) comparable to other recombinant eukaryotic P4H proteins (Xu et al., 2011). In conclusion, this paper describes the creation of the first Pichia pastoris strain able to produce an active sponge P4H tetramer that is suitable of insertion of further hydroxylation transgene targets of biotechnological interest. Other than sponge collagen, other marine collagens from a variety of species and conotoxins may be considered favorite targets of this new hydoxylating system, and indeed their high pharmacological interest justifies the current efforts towards their recombinant production in transgenic organisms able to apply the correct post-translational modifications.
Acknowledgement This work was supported by grant form EU (FP7 grant agreement no.: 266033 SPonge Enzyme End Cell for Innovative AppLication-SPECIAL).
Introduction Prolyl 4-hydroxylases (P4Hs) have central roles in the synthesis of collagens and the regulation of oxygen Palomid 529 [1]. The posttranslational modification, the 4 hydroxylation of a proline residue, carried out by P4Hs, is important for the stability of the collagen triple helix and the degradation of the alpha subunit of hypoxia-inducible transcription factor (HIF-α) [1]. Vertebrate collagen prolyl-4-hydroxylases (CPHs) are alpha2beta2 tetramers with three isoenzymes differing in their catalytic alpha subunits [2]. Another P4H family, HIF-P4Hs (HPHs), hydroxylate specific prolines in oxygen degradation domain of HIF-α and regulate von Hippel-Lindau (VHL)-dependent degradation of hydroxylated HIF-α in an oxygen-dependent manner [3], [4], [5]. Although CPHs and HPHs catalyze prolyl 4-hydroxylation of different substrates, collagen and HIF-α, respectively, they are mechanistically identical enzymes with a conserved active site. All P4Hs require Fe2+, 2-oxoglutarate, O2, and ascorbate to hydroxylate proline(s) in their substrates [1]. While CPHs are regarded as attractive targets for pharmacological inhibition to control excessive collagen accumulation in fibrotic diseases and severe scarring, HPH inhibitors are believed to have beneficial effects in the treatment of diseases such as myocardial infarction, stroke, peripheral vascular disease, diabetes, and severe anemias [1], [6]. Although selective modulation of activity of each enzyme holds therapeutic potential for treatment of a spectrum of pathological conditions, agents that selectively inhibit one of the P4Hs have not so far been established. As mentioned above, CPHs and HPHs require iron as a cofactor, 2-ketoglutarate and oxygen as substrates and ascorbate as an iron-reducing agent, to hydroxylate proline residues in its respective substrate, collagen and HIF-α. HPHs and CPHs have similar Km values for 2-ketoglutarate and a 2-ketoglutarate mimicking agent such as dimethyloxallyl glycine does not exhibit selective inhibition of different prolyl-4 hydroxylases [7]. On the contrary, HPHs have much greater Km values for another substrate, dioxygen (230–250μM) than do CPHs (40μM) [8], suggesting that a dioxygen-mimicking agent is likely to distinguish the two enzymes.