Complement C1q is hydroxylated by collagen prolyl 4 hydroxylase, and is sensitive to off target inhibition by prolyl hydroxylase domain inhibitors that stabilise Hypoxia Inducible Factor

C1q is part of the C1 macromolecular complex that mediates the classical complement activation pathway, a major arm of innate immune defence. C1q is composed of A, B, and C chains that require post-translational prolyl-4-hydroxylation of their N-terminal collagen-like domain to enable formation of the functional triple helical multimers. The prolyl-4-hydroxylase(s) that hydroxylate C1q have not previously been identified. Recognised prolyl-4-hydroxylases include collagen prolyl- 4-hydroxylases (CP4H) and the more recently described prolyl hydroxylase domain (PHD) enzymes that act as oxygen sensors regulating hypoxia-inducible factor (HIF). We show that several small molecule prolyl-hydroxylase inhibitors that activate HIF also potently suppress C1q secretion by human macrophages. However, reducing oxygenation to a level that activates HIF does not compromise C1q hydroxylation. In vitro studies showed that a C1q A chain peptide is not a substrate for PHD2, but is a substrate for CP4H1. Circulating levels of C1q did not differ between wild type mice and mice with genetic deficits in PHD enzymes, but were reduced by administration of the prolyl-hydroxylase inhibitors. Our findings imply that C1q is hydroxylated by CP4H, but not the structurally related PHD hydroxylases. A reduction in C1q levels may be an important side-effect of small molecule PHD inhibitors developed as treatments for renal anemia, as an “off-target” consequence of inhibiting CP4H