Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease of complex genetic aetiology. A rare de novo mutation in the C1q and TNF related 4 gene (C1QTNF4) was recently discovered through exome sequencing of SLE parent-affected-offspring trios. C1QTNF4 is a member of the C1QTNF family, a family of secreted proteins that were identified as paralogs of adiponectin and have structural homology with both complement C1q and the TNF superfamily. C1QTNF4 is structurally unique, being formed of two C1q-like domains and lacking a collagen-like region. Little is known about the function of C1QTNF4, and its role in immunity and inflammation is poorly defined. The aim of my thesis was to gain an understanding of the immune-related functions of C1QTNF4 by identifying its cell surface receptor. Here I recombinantly produced human C1QTNF4 in mammalian cells and bacteria and assessed oligomerisation state by size exclusion chromatography. I used a flow cytometry-based cell-binding assay to identify target cells of C1QTNF4 and immunoprecipitation followed by mass spectrometry to identify a cell surface receptor. I performed knockdown experiments of the candidate receptor using siRNA, as well as competition experiments with soluble nucleolin. Finally, I set up crystallisation trials to determine the structure of the first C1q-like domain of C1QTNF4 by X-ray crystallography. I found that recombinant C1QTNF4 produced in mammalian cells uniformly formed very high molecular weight clusters, whereas bacterially produced C1QTNF4 was present in monomeric and oligomeric complexes. I showed that the cell surface receptor of C1QTNF4 was present on ex vivo monocytes and to a lesser extent on B and T cell subsets. The receptor was furthermore abundant on neuroblastoma cell lines and human embryonic kidney cells. I identified nucleolin as a potential cell surface binding partner of C1QTNF4 and showed that knockdown and competition with soluble nucleolin reduced binding of C1QTNF4 to cells. Both oligomerisation and receptor-binding were mediated by the second C1q-like domain of C1QTNF4. My results suggest that myeloid cells likely mediate the immune-related functions of C1QTNF4 and are thus being prioritised for further study. Nucleolin may serve as a docking molecule for C1QTNF4 and act in a context-dependent manner through co-receptors. Having identified nucleolin as a receptor is the first step to understanding the function of C1QTNF4 in the healthy immune system and how dysfunction may contribute to the development of SLE.
Investigating C1QTNF4 in health and systemic lupus erythematosus
Vester, S. (Author). 1 Feb 2021
Student thesis: Doctoral Thesis › Doctor of Philosophy