Functional Characterization of Fusion Proteins of the Tumor Necrosis Factor Alpha‐Induced Protein 6 (TNFAIP6) Domains

Abstract

TNFAIP6 is an extracellular protein that is essential for cumulus cell extracellular matrix formation and female fertility in mice. A mammalian expression system was used to generate recombinant proteins of the full length TNFAIP6 and its individual domains, Link and CUB. These proteins contained a short amino terminal sequence essential for catalytic function and a carboxy‐terminal Fc region of mouse IgG2a for easy detection, and were termed TNFAIP6Fc, LinkFc and CUBFc, respectively. Hyaluronan‐binding of the fusion proteins was measured with an enzyme‐linked immunosorbent assay. The catalytic activity of the proteins was assessed by their ability to remove heavy chain 1 (HC1) from inter‐alpha‐trypsin inhibitor (IαI) using western‐blot. The ability of the fusion proteins to restore cumulus cell extracellular matrix formation was tested in a cell culture assay of Tnfaip6−/− cumulus cell‐oocyte complexes (COCs). Both TNFAIP6Fc and LinkFc were able to bind hyaluronan, with LinkFc showing much stronger binding at equimolar concentrations. CUBFc showed no significant binding to hyaluronan even at high concentrations. TNFAIP6Fc catalysed the removal of HC1 from IαI in a concentration dependent manner, indicating that the Fc tag did not interfere with catalytic function. LinkFc, CUBFc, and their combination were unable to remove the HC1 from IαI at high concentrations even though they contained the essential short amino‐terminal region. TNFAIP6Fc successfully restored cumulus cell extracellular matrix formation in Tnfaip6−/− COCs, while LinkFc, CUBFc, and their combination were unable to do so. LinkFc, CUBFc, and their combination did not disrupt normal cumulus cell extracellular matrix formation of Tnfaip6+/− COCs. Our results suggest that the catalytic function of TNFAIP6 requires both of its domains in addition to the previously identified serine residue in the amino‐terminal region, because neither domain alone is able to remove HC1 from IαI or support cumulus cell extracellular matrix formation in Tnfaip6−/− COCs. Furthermore, the absence of catalytic activity of both domains when combined together could imply a pivotal conformational interaction normally achieved by their covalent attachment. The finding that the LinkFc has stronger hyaluronan binding than TNFAIP6Fc could support the presence of such a conformational interaction that weakens the hyaluronan‐binding of the full‐length protein compared to the Link domain alone.Support or Funding InformationSupported by internal funding of Touro University Nevada