DISULFIDE BONDS ASSIGNMENT IN THE INTER-ALPHA-INHIBITOR HEAVY-CHAINS - STRUCTURAL AND FUNCTIONAL IMPLICATIONS

Human inter-alpha-inhibitor (I alpha I) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked b y a glycosaminoglycan: a light one named bikunin, carrying the antipro teinase activity and two heavy chains H1 and H2. The amino acid sequen ces of these heavy chains are highly similar; however when I alpha I i s 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 el astase (HLE) preferentially cleaves the heavy chain H2 of inter-alpha- trypsin inhibitor (ITI), Biol. Chem. Hoppe-Seyler 374, 895-901]. We ma pped the disulphide topology of the I alpha I heavy chains in order to investigate whether or not disulphide bonds might be responsible for their differential susceptibility to proteolysis. Using amino acid seq uencing and mass spectrometry analysis, we demonstrate that the H1 hea vy chain contains one free thiol group and two disulphide bridges of w hich 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 immuno print analysis, we show that, when I alpha I is subjected to a limited digestion by Staphylococcus aureus V-8 proteinase, the two polypeptid e chains are similarly susceptible to proteolysis. This enzyme prefere ntially cleaves the I alpha I heavy chains from their N-terminal extre mity. These results are consistent with the circular dichroism (CD) an alysis, suggesting that the conformation of the polypeptide backbone o f H1 is not very different from that of H2, with calculated alpha-heli cities 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 I alpha I molecule, the heavy chains are linke d to the glycosaminoglycan chain via their C-terminal aspartic acid re sidue. Thus we suggest that the affinity of cationic neutrophil protei nases for the anionic glycosaminoglycan is responsible for the cleavag e of the heavy chains (mainly H2) near their C-terminal end and the hi gh susceptibility of Icd to these proteinases.