Flahaut C, Mizon C, Aumercier-Maes P, Colson P, Bailly C, Sautiere P, Mizon J
Laboratoire de Biochimie, Faculté de Pharmacie, Lille, France.
Eur J Biochem. 1998 Jul 1;255(1):107-15. doi: 10.1046/j.1432-1327.1998.2550107.x.
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.
人α-间抑制因子（IαI）是一种血浆丝氨酸蛋白酶抑制剂。它由三条通过糖胺聚糖共价连接的多肽链组成：一条轻链名为比基尼，具有抗蛋白酶活性，以及两条重链H1和H2。这些重链的氨基酸序列高度相似；然而，当IαI被中性粒细胞蛋白酶消化时，它们的蛋白水解敏感性差异很大[Balduyck, M., Piva, F., Mizon, C., Maes, P., Malki, N., Gressier, B., Michalski, C. & Mizon, J. (1993) 人白细胞弹性蛋白酶（HLE）优先切割α-胰蛋白酶抑制剂（ITI）的重链H2，《生物化学与霍普-赛勒学报》374, 895 - 901]。我们绘制了IαI重链的二硫键拓扑结构，以研究二硫键是否可能是它们对蛋白水解敏感性差异的原因。通过氨基酸测序和质谱分析，我们证明H1重链含有一个游离巯基和两个二硫键，其中一个连接两个相距很远的半胱氨酸残基（Cys239和Cys511）。因此，H1明显不同于H2，H2在紧密相邻的半胱氨酸残基之间含有两个二硫键。然而，通过免疫印迹分析，我们发现，当IαI用金黄色葡萄球菌V - 8蛋白酶进行有限消化时，两条多肽链对蛋白水解的敏感性相似。这种酶优先从其N末端切割IαI重链。这些结果与圆二色性（CD）分析一致，表明H1多肽主链的构象与H2的构象差异不大，计算出的α-螺旋度分别为24%和28%。CD测量结果表明，H1和H2的芳香族氨基酸处于不同的不对称环境中。在IαI分子内部，重链通过其C末端天冬氨酸残基与糖胺聚糖链相连。因此，我们认为阳离子中性粒细胞蛋白酶对阴离子糖胺聚糖链的亲和力是导致重链（主要是H2）在其C末端附近被切割以及IαI对这些蛋白酶高度敏感的原因。
