The enzymatic activity of recombinant sponge P H was

The enzymatic activity of recombinant sponge P4H was assayed in vitro both at 37°C, the standard enzymatic kinetics temperature, and at 15°C, the typical sea temperature of C. reniformis marine environment (Fig. 2C). Surprisingly the data obtained indicate that the sponge enzyme has a lower activity at its natural environment temperature. As a matter of fact, the proline hydroxylation rate on collagen chains is minimal in the heterothermal animals living in cold marine environments, and conversely its hydroxylation is enhanced in animals living in warmer seas to increase collagen thermal stability (Kimura et al., 1988). Thus, the behavior of the recombinant sponge P4H suggests that the enzyme can be able to modulate its activity allowing the animal to respond to sea temperature alterations by adjusting the levels of collagen hydroxylation to the necessities of the environmental changes. Western blot analysis of αβH4 strain lysate after methanol induction could confirm that also in the recombinant yeast system, α and β polypeptides are assembled as tetramers (Fig. 3B) as already observed for the wild type protein analyzed in C. reniformis cell extracts (Pozzolini et al., 2015). Finally, the enzymatically best performing strain αβH4 was transformed with the conotoxin vector pPICZ\\colCH containing a C. reniformis non fibrillar collagen gene, producing the colCH4 strain. This strain shows an enhanced recombinant P4H enzymatic activity with respect to the αβH4 lacking the collagenous sequence (Fig. 4D). These data seem to confirm results obtained by others (Vuorela et al., 1997) the explanation of which was ascribed at a higher stability of the P4H tetramer in the presence of its natural substrate in the recombinant system. But surprisingly, when we analyzed the levels of each recombinant transcript in our system we found that, differently from previous work, the enhanced enzymatic activity seems to be mainly linked to an enhanced transcription of the α and β P4H recombinant genes in the presence of the ColCH gene as displayed in Fig. 4A and B. Indeed, it has been reported that genes with promoters susceptible of activation by the same transcription factors tend to localize in close proximity in the nucleoplasmic space likely in the same interchromatin compartment to be optimally transcribed by a single transcription factory (Larkin et al., 2013). Thus, in this case the introduction of further copies of another recombinant gene (ColCH) in the αβH4 strain may have the effect to promote a more efficient transcription also of the other two inserted genes (α and β P4H) localized on different chromosomes but sharing the same promoter sequence. This phenomenon, which surely needs further investigation, could be of importance when setting up the production of multiple recombinant proteins in yeast since, surprisingly, it seems to suggest that the more proteins are introduced under the same promoter the more efficient becomes their production. 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.