脂多糖

糖生物学分析手册,第2版

脂多糖(LPS)是革兰氏阴性细菌外膜的主要成分。脂多糖位于胞膜的外层,在非包封菌株中暴露在细胞表面上。


 脂多糖的结构

结构体完整的细菌脂多糖是分子量为10-20 kDa的大分子,由三个结构组分组成(参见图1):

  • 疏水脂质部分,脂质A,造成分子毒性,
  • 亲水核心多糖链,
  • 重复的亲水性O-抗原性寡糖侧链,对细菌血清型具有特异性1
  • 细菌脂多糖的一般结构

图1. 细菌脂多糖的一般结构。有关每个部分的详细说明,请参阅文本。缩写:KDO:3脱氧-α-D-甘露辛酮糖酸;Hep:庚糖(庚酮糖);NGa:半乳糖胺;NGc:氨基葡萄糖。

 

脂质A核心由β-葡糖胺-(1→6)-葡糖胺-1-磷酸碱基构成,其中脂肪酸酯附着在两种碳水化合物上。酰基链长度和酰基数可以随细菌种类的不同而不同,但在同一物种内变化相对较小。内多糖核心通常含有1至4个与二糖核心连接的KDO(3脱氧-α-D-甘露辛酮糖酸)分子。KDO与脂多糖特异性结合,并且具有生物活性的脂质A过去被认为需要至少一个KDO残基才能使细菌存活。2 然而,缺乏KDO的大肠杆菌K-12抑制剂菌株证明,是否有KDO对于细菌的存活并不是绝对的。3  
 
含有KDO的内核也用庚糖(庚酮糖)单糖修饰,其中最常见的是L-丙三基-α-D-甘露-吡喃庚糖。内核聚糖残基通常用含磷酸基团磷酸化或修饰,例如焦磷酸盐或2-氨基乙基磷酸盐。脂多糖的磷酸基团增加细胞膜的总负电荷,并有助于稳定结构。 
 
脂多糖的外核含有更常见的己糖,包括葡萄糖、半乳糖和N-乙酰葡糖胺,并且在结构上比内核更多样化。 
 
O-抗原是重复的寡糖单元,通常由2-6个糖组成。O-抗原是脂多糖的主要结构成分,基于它细菌被区分开来。已经使用独特的O-抗原结构来鉴定和分类大肠杆菌、肠沙门氏菌和霍乱弧菌的血清群。4 来自大肠杆菌的粗糙突变菌株的脂多糖缺乏该结构的O-抗原部分。 
 
脂多糖的核心部分和脂质A部分可能具有一些结构可变性,而O-抗原具有高度的结构可变性以及重复单元数量的可变性。这些差异导致LPS制剂的显著异质。由于LPS是异质的,并且倾向于形成不同大小的聚集体,有报道称这些聚集体的“分子量”范围为1-4百万道尔顿或更高。当用十二烷基硫酸钠(SDS)和加热处理LPS时,分子量为~50-100 kDa。5 


 脂多糖的功能和应用

在革兰氏阴性菌中,胞膜脂多糖可保护细菌免受胆汁盐和亲脂性抗生素的作用6

脂多糖是热稳定的内毒素,并且长期以来被认为是人类感染性休克(败血症)的关键因素1,7 更一般而言,它是在正常哺乳动物细胞中诱导强烈免疫应答的关键因素。已经鉴定脂质A基团对脂多糖的内毒素活性是关键的。通过在合成和天然来源的大肠杆菌脂质A制剂之间发现相同的生物活性结果(包括内毒素活性),Galanos等证明了点。8 脂多糖的活性受体已被鉴定为CD14 / TLR4 / MD2受体复合物,它促进促炎细胞因子的分泌,包括肿瘤坏死因子-α和白细胞介素-1。9 虽然脂质A成分主要负责免疫反应激活,但肠道沙门氏菌LPS的多糖成分也是NF-κB活化所必需的。10

脂多糖制剂已被用于研究LPS结构、11 代谢、12 免疫学、13 生理学、14 毒性、15 和生物合成。16 它们还被用于诱导白细胞介素等生长促进因子的合成和分泌。17,18由于脂多糖与败血症的关系,已经对脂多糖进行了研究,以确定抗体和LPS生物合成抑制剂的可能靶标。19,20

 


 脂多糖的提取和纯化

脂多糖可以通过从TCA、21苯酚、22,23或苯酚-氯仿-石油醚(对粗糙菌株)提取来制备。24 TCA提取的脂多糖在结构上类似于苯酚提取的脂多糖,具有相似的电泳图案和内毒性。主要差异在于提取后残留的核酸和蛋白质污染物的量。TCA提取物含有~2%的RNA和~10%的变性蛋白质,而苯酚提取物含有高达60%的RNA和<1%的蛋白质。随后通过凝胶过滤色谱法纯化除去苯酚提取的LPS中存在的大量蛋白质,但产生含有10-20%核酸的制剂。使用离子交换色谱进一步纯化产生含有<1%蛋白质和<1% RNA的脂多糖产物。


 材料

     


 参考文献

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