Inter-α-inhibitor heavy chain-1 has an integrin-like 3D structure mediating immune regulatory activities and matrix stabilization during ovulation

David C Briggs,Alexander W.W Langford-Smith,Holly L Birchenough,Thomas A Jowitt,Cay M Kielty,Jan J Enghild,Clair Baldock,Caroline M Milner,Anthony J Day

doi:10.1074/jbc.ra119.011916

Abstract

Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous “heavy chains” (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering–based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation.

Highlights

Inter-␣-inhibitor is a proteoglycan essential for mammalian reproduction and plays a less well-characterized role in inflammation
We show that the metal ion-dependent adhesion site (MIDAS) is important in HC1-mediated inhibition of the alternative pathway C3 convertase via its interaction with C3 and demonstrate that HC1 can interact with vitronectin and other novel ligands (e.g. fibronectin and small latent complexes of transforming growth factor ␤ (TGF␤)) in a noncanonical MIDAS-independent manner
Crystal structures were obtained for the with accession codes 6FPY (WT) recombinant HC1, encompassing the entire 638-residue mature protein sequence, and for the corresponding D298A single-site mutant, at 2.34 and 2.20 Å resolution, respectively (Table 1)

Summary

The abbreviations used are

I␣I, inter-␣-inhibitor; AUC, analytical ultracentrifugation; CMG2, capillary morphogenesis protein-2; COC, cumulus–. Irrespective of the mechanism, the formation of HC1⁄7HA in arthritic joints enhances the binding of HA to its major cell surface receptor, CD44, on leukocytes [25] It is unknown whether this, or the altered hydrodynamic properties of the modified HA [26], are part of a protective process or contribute to arthritis pathology [9]. The inhibition of the alternative and classical pathways of complement is thought to be dependent on HCs rather than bikunin [32, 33]; most of the available data were generated using I␣I, and the HC-mediated mechanisms have not been determined. We show that the MIDAS is important in HC1-mediated inhibition of the alternative pathway C3 convertase via its interaction with C3 and demonstrate that HC1 can interact with vitronectin and other novel ligands (e.g. fibronectin and small latent complexes of transforming growth factor ␤ (TGF␤)) in a noncanonical MIDAS-independent manner

Results

Discussion

Experimental procedures