The Compact and Biologically Relevant Structure of Inter-α-inhibitor Is Maintained by the Chondroitin Sulfate Chain and Divalent Cations

J Biol Chem. 2016 Feb 26;291(9):4658-70. doi: 10.1074/jbc.M115.678748. Epub 2016 Jan 4.


Inter-α-inhibitor is a proteoglycan of unique structure. The protein consists of three subunits, heavy chain 1, heavy chain 2, and bikunin covalently joined by a chondroitin sulfate chain originating at Ser-10 of bikunin. Inter-α-inhibitor interacts with an inflammation-associated protein, tumor necrosis factor-inducible gene 6 protein, in the extracellular matrix. This interaction leads to transfer of the heavy chains from the chondroitin sulfate of inter-α-inhibitor to hyaluronan and consequently to matrix stabilization. Divalent cations and heavy chain 2 are essential co-factors in this transfer reaction. In the present study, we have investigated how divalent cations in concert with the chondroitin sulfate chain influence the structure and stability of inter-α-inhibitor. The results showed that Mg(2+) or Mn(2+), but not Ca(2+), induced a conformational change in inter-α-inhibitor as evidenced by a decrease in the Stokes radius and a bikunin chondroitin sulfate-dependent increase of the thermodynamic stability. This structure was shown to be essential for the ability of inter-α-inhibitor to participate in extracellular matrix stabilization. In addition, the data revealed that bikunin was positioned adjacent to both heavy chains and that the two heavy chains also were in close proximity. The chondroitin sulfate chain interacted with all protein components and inter-α-inhibitor dissociated when it was degraded. Conventional purification protocols result in the removal of the Mg(2+) found in plasma and because divalent cations influence the conformation and affect function it is important to consider this when characterizing the biological activity of inter-α-inhibitor.

Keywords: chondroitin sulfate; glycosaminoglycan; metal ion-protein interaction; protease inhibitor; protein cross-linking; proteoglycan structure.

MeSH terms

  • Alpha-Globulins / chemistry*
  • Alpha-Globulins / isolation & purification
  • Alpha-Globulins / metabolism
  • Binding Sites
  • Chondroitin Sulfates / chemistry*
  • Chondroitin Sulfates / metabolism
  • Cross-Linking Reagents / chemistry
  • Hot Temperature / adverse effects
  • Humans
  • Ligands
  • Magnesium / chemistry*
  • Magnesium / metabolism
  • Manganese / chemistry*
  • Manganese / metabolism
  • Models, Molecular*
  • Molecular Conformation
  • Protein Conformation
  • Protein Footprinting
  • Protein Interaction Domains and Motifs
  • Protein Multimerization
  • Protein Stability
  • Protein Subunits / chemistry
  • Protein Subunits / isolation & purification
  • Protein Subunits / metabolism
  • Protein Unfolding
  • Proteoglycans / chemistry*
  • Proteoglycans / metabolism


  • Alpha-Globulins
  • Cross-Linking Reagents
  • Ligands
  • Protein Subunits
  • Proteoglycans
  • alpha-1-microglobulin
  • inter-alpha-inhibitor
  • Manganese
  • Chondroitin Sulfates
  • Magnesium