Collagen prolyl 4-hydroxylases. The roles of P4HA3 in collagen synthesis and 4Hyp in the quality control of collagen secretion

Collagens are the most common proteins in the body. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen biosynthesis. They catalyze the formation of 4-hydroxyproline (4Hyp), an abundant modification needed for collagen triple helix folding and thermal stability. C-P4Hs, α2β2 tetramers, have three known isoforms of the catalytic α subunit (encoded by P4HA1, P4HA2 and P4HA3), while protein disulphide isomerase (PDI) serves as the β subunit. C-P4H is responsible for hydroxylation of prolyl residues in (-X-Pro-Gly-) triplets to 4Hyp in collagens and collagenous proteins in a process that requires Fe2+, 2-oxoglutarate, ascorbate and O2. C-P4Hs are promising targets for treating diseases involving excess collagen production and accumulation. The roles of P4HA1 and P4HA2 have been studied extensively, but very little is currently known about the roles of P4HA3 in the collagen synthesis.

To study the role of P4HA3, we first investigated its expression during murine development. Our results suggest that the major function of P4ha3 could be during the embryonal development and early postnatal developmental phases.

To study the role of P4HA3 in cellulo, we sequentially inactivated C-P4Hs in cells by the CRISPR/Cas9 technology and produced cells that lack P4HA3 and cells that have only P4HA3. This thesis provides new evidence that P4HA3 differs significantly from P4HA1 and P4HA2 by having a different expression pattern and possibly a different β subunit. The data also strongly suggests that P4HA3 does not hydroxylate collagen, at least not type I collagen, and its substrate could also be a non-collagenous protein that is yet to be identified.

In the second part of the thesis, we produced cells without any C-P4H isoform. The biosynthesis of collagen molecules is a strictly controlled process and underhydroxylated collagen polypeptides are thought to be retained in the endoplasmic reticulum (ER) causing ER stress, and eventually degraded. Surprisingly, our data showed that the collagen polypeptides completely lacking 4Hyp did not accumulate in the cell and did not initiate unfolded protein response but were simply secreted in a non-triple-helical conformation without harmful cellular effects. This thesis reveals novel aspects in the collagen synthesis, its quality control and how cells process abnormal collagen.