Sanggaard Kristian W, Hansen Lone, Scavenius Carsten, Wisniewski Hans-Georg, Kristensen Torsten, Thøgersen Ida B, Enghild Jan J
Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO) at the Department of Molecular Biology, Aarhus University, 8000 Aarhus C, Denmark.
Biochim Biophys Acta. 2010 Apr;1804(4):1011-9. doi: 10.1016/j.bbapap.2010.01.013. Epub 2010 Jan 25.
The bikunin proteins are composed of heavy chains (HCs) covalently linked to a chondroitin sulfate chain originating from Ser-10 of bikunin. Tumor necrosis factor stimulated gene-6 protein (TSG-6)/heavy chain 2 (HC2) cleaves this unique cross-link and transfers the HCs to hyaluronan and other glycosaminoglycans via a covalent HCTSG-6 intermediate. In the present study, we have investigated if this reaction is evolutionary conserved based on the hypothesis that it is of fundamental importance. The results revealed that plasma/serum samples from mammal, bird, and reptile were able to form TSG-6 complexes suggesting the presence of proteins with the same function as the human bikunin proteins. To substantiate this, the complex forming protein from Gallus gallus (Gg) plasma was purified and identified as a Gg homolog of human HC2bikunin. In addition, Gg pre-alpha-inhibitor and smaller amount of high molecular weight forms composed of bikunin and two HCs were purified. Like the human bikunin proteins, the purified Gg proteins were all stabilized by a protein-glycosaminoglycan-protein cross-link, i.e. the HCs were covalently attached to a chondroitin sulfate originating from bikunin. Furthermore, the complex formed between Gg HC2*bikunin and human TSG-6 appeared to be identical to that of the human proteins. Akin to human, Gg HC2 was further transferred to hyaluronan when present, and when incubated in vitro, Gg pre-alpha-inhibitor and TSG-6, failed to form the intermediate covalent complex, essential for HC transfer. Significantly, Gg HC2, analogous to human HC2, promoted complex formation between human HC3 and human TSG-6, substantiating the evolutionary conservation of these interactions. The present study demonstrates that the unique interactions between bikunin proteins, glycosaminoglycans, and TSG-6 are evolutionary conserved, emphasizing the physiological importance of the TSG-6/HC2-mediated HC-transfer reaction. In addition, the data show that the evolution of HC transfer is likely to predate the role of HC.HA complexes in female fertility and thus has evolved in the context of inflammation rather than fertility.
比库宁蛋白由重链（HCs）组成，这些重链与源自比库宁丝氨酸10的硫酸软骨素链共价连接。肿瘤坏死因子刺激基因6蛋白（TSG - 6）/重链2（HC2）可切割这种独特的交联结构，并通过共价HCTSG - 6中间体将重链转移至透明质酸和其他糖胺聚糖。在本研究中，基于其具有根本重要性这一假设，我们探究了该反应在进化上是否保守。结果显示，来自哺乳动物、鸟类和爬行动物的血浆/血清样本能够形成TSG - 6复合物，这表明存在与人类比库宁蛋白功能相同的蛋白质。为证实这一点，从鸡（Gg）血浆中纯化出形成复合物的蛋白，并鉴定为人类HC2比库宁的Gg同源物。此外，还纯化出了Gg前α抑制剂以及由比库宁和两条重链组成的少量高分子量形式。与人类比库宁蛋白一样，纯化后的Gg蛋白均通过蛋白 - 糖胺聚糖 - 蛋白交联得以稳定，即重链共价连接至源自比库宁的硫酸软骨素。此外，Gg HC2*比库宁与人类TSG - 6之间形成的复合物似乎与人类蛋白形成的复合物相同。与人类相似，当存在透明质酸时，Gg HC2会进一步转移至透明质酸，并且在体外孵育时，Gg前α抑制剂和TSG - 6无法形成重链转移所必需的中间共价复合物。值得注意的是，与人类HC2类似，Gg HC2促进了人类HC3与人类TSG - 6之间的复合物形成，证实了这些相互作用在进化上的保守性。本研究表明，比库宁蛋白、糖胺聚糖和TSG - 6之间独特的相互作用在进化上是保守的，强调了TSG - 6/HC2介导的重链转移反应的生理重要性。此外，数据表明重链转移的进化可能早于HC.HA复合物在女性生育中的作用，因此是在炎症而非生育的背景下进化而来的。
