Interaction between Bacterial Endotoxin and Lysozyme

细菌内毒素（lipopolysaccharide，LPS）是革兰氏阴性菌的一种细胞表面物质，是人类炎症性牙周炎的重要致病因子。溶菌酶通过酶促裂解构成细胞壁的肽聚糖，对革兰氏阳性菌具有杀菌活性。也有报道溶菌酶与LPS结合并抑制免疫药理学活性。在这项研究中，我们表明，LPS纯化从三个假定的牙周病细菌（中间普氏菌，放线菌放线杆菌，牙龈卟啉单胞菌），以及LPS从大肠杆菌促进破骨细胞形成小鼠骨髓细胞培养。用鸡蛋清溶菌酶（HEL）减弱LPS的这种活性。LPS的其他各种生物活性，即鲎变形细胞裂解物的活化。HEL对人白细胞分泌TNF-α的刺激和小鼠B细胞的多克隆活化均有抑制作用，对合成的脂A的生物活性也有抑制作用，提示HEL可与LPS的脂A部分发生反应。在这些HEL衍生物中，S-三甲基铵-丙基化的HEL，设计为具有更高的水溶性和更高的pI的衍生物，最有效地抑制LPS活性。HEL的抗内毒素能力与其作为溶菌酶的已知酶活性无关。这些结果表明，HEL作为一种抗内毒素剂的牙周治疗的可能性，和潜在的蛋白质工程，以产生一种新的药物，有效地中和内毒素。

Bacterial endotoxin (lipopolysaccharide ; LPS) is a cell-surface substance of gram-negative bacteria, and considered to be an important pethogenic factor for human inflammatory periodontitis. Lysozyme has a bactericidal activity for gram-positive bacteria, by enzymatic cleavage of peptidoglycans that constitute the cell wall. It has also been reported that lysozyme binds to LPS and inhibits the immunopharmacological activities. In this study, we showed that LPS purified from three putative periodontopathic bacteria (Prevotella intermedia, Actinbacillus actinomycetemcomitans, Porphyromonas gingivalis) as well as LPS from Escherichia coli promote osteoclast formation in mouse bone marrow cell cultures. This activity of LPS was attenuated by hen egg-white lysozyme (HEL). Other various biological activities of LPS,i.e. activation of Limulus amoebocyte lysate. stimulation of human leukocytes to secrete TNF-alpha, polyclonal activation of mouse B cells, were inhibited by HEL.The bioactivitiy of synthetic lipid A was also inhibited by HEL,suggesting that HEL reacts to the common lipid A portion of LPS.Various chemically nodified HEL were produced and tested for their ability to inhibit the LPS activities. Among those HEL-derivatives, S-trimethylammonium-propylated HEL,the derivative designed to posses higher water solubility and higher pI,most efficiently inhibit LPS activities. The anti-endotoxic capacity of HEL was not related to its known enzymatic activity as a muramidase. These results suggest a possible application of HEL to the periodontal therapy as an anti-endotoxic agent, and the potential of protein engineering to denerate a novel drug that efficiently neutralize endotoxin.