Disulphide bonds assignment in the inter-alpha-inhibitor heavy chains--structural and functional implications

Human inter-alpha-inhibitor (IalphaI) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan: a light one named bikunin, carrying the antiproteinase activity and two heavy chains H1 and H2. The amino acid sequences of these heavy chains are highly similar; however when IalphaI is digested by neutrophil proteinases, their proteolytic susceptibility strongly differs [Balduyck, M., Piva, F., Mizon, C., Maes, P., Malki, N., Gressier, B., Michalski, C. & Mizon, J. (1993) Human leucocyte elastase (HLE) preferentially cleaves the heavy chain H2 of inter-alpha-trypsin inhibitor (ITI), Biol. Chem. Hoppe-Seyler 374, 895-901]. We mapped the disulphide topology of the IalphaI heavy chains in order to investigate whether or not disulphide bonds might be responsible for their differential susceptibility to proteolysis. Using amino acid sequencing and mass spectrometry analysis, we demonstrate that the H1 heavy chain contains one free thiol group and two disulphide bridges of which one links two largely spaced cysteine residues (Cys239 and Cys511). Thus H1 is clearly different from H2 which contains two disulphide bonds between closely located cysteine residues. However, using immunoprint analysis, we show that, when IalphaI is subjected to a limited digestion by Staphylococcus aureus V-8 proteinase, the two polypeptide chains are similarly susceptible to proteolysis. This enzyme preferentially cleaves the IalphaI heavy chains from their N-terminal extremity. These results are consistent with the circular dichroism (CD) analysis, suggesting that the conformation of the polypeptide backbone of H1 is not very different from that of H2, with calculated alpha-helicities of 24% and 28%, respectively. The CD measurements reveal that the aromatic amino acids of H1 and H2 are in a different asymmetrical environment. Inside the IalphaI molecule, the heavy chains are linked to the glycosaminoglycan chain via their C-terminal aspartic acid residue. Thus we suggest that the affinity of cationic neutrophil proteinases for the anionic glycosaminoglycan is responsible for the cleavage of the heavy chains (mainly H2) near their C-terminal end and the high susceptibility of IalphaI to these proteinases.