Abstract
Complement C1q is part of the C1 macromolecular complex that mediates the classical complement activation pathway: a major arm of innate immune defense. 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 the formation of the functional triple helical multimers. The prolyl 4-hydroxylase(s) that hydroxylate C1q have not previously been identified. Recognized 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 or mice with genetic deficits in PHD enzymes, but were reduced by prolyl hydroxylase inhibitors. Thus, C1q is hydroxylated by CP4H, but not the structurally related PHD hydroxylases. Hence, reduction of C1q levels may be an important off-target side effect of small molecule PHD inhibitors developed as treatments for renal anemia.
Highlights
Complement C1q is part of the C1 macromolecular complex that mediates the classical complement activation pathway: a major arm of innate immune defense
We examined 3 other small molecules that have been shown to inhibit prolyl hydroxylase domain (PHD) enzymes and stabilize hypoxiainducible factor (HIF)-1a: L-mimosine, an iron chelator,[17,18] and two compounds structurally related to N-oxalylglycine, roxadustat (FG4592)[19,20] and FG0041.21 All 3 molecules decreased C1q secretion by macrophages in a concentration-dependent manner, and C1q levels were significantly reduced at doses that were lower than (L-mimosine and FG0041) or similar to those required to stabilize HIF-1a efficiently (Figure 1c–e)
FG0041, DMOG and L-mimosine are all effective inhibitors of collagen synthesis,[26] which is entirely consistent with our observation that they are effective inhibitors of C1q secretion and our conclusion that collagen prolyl-4-hydroxylases (CP4H) catalyzes C1q modification
Summary
Complement C1q is part of the C1 macromolecular complex that mediates the classical complement activation pathway: a major arm of innate immune defense. The enzymes responsible were subsequently identified as collagen prolyl-4-hydroxylases (CP4Hs),[1,2] which are members of the superfamily of 2oxoglutarate (2-OG)–dependent dioxygenases that have an Fe(II) atom at the active site and require oxygen and 2-OG as cosubstrates. Another important group of prolyl 4-hydroxylases has more recently been identified: prolyl hydroxylase domain (PHD) enzymes. These are structurally related to CP4Hs and belong to the superfamily of 2-OGÀdependent dioxygenases. S Kiriakidis et al.: Inhibition of C1q by HIF activators basic research marrow–derived cells.[11,12,13] The importance of C1q is underlined by the fact that C1q deficiency causes susceptibility to infections and the autoimmune disease systemic lupus erythematosus.[14]
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