Chark Davin, Nutikka Anita, Trusevych Natasha, Kuzmina Julia, Lingwood Clifford
Research Institute, Division of Infection, Immunity, Injury and Repair, The Hospital for Sick Children, Ontario, Canada.
Eur J Biochem. 2004 Jan;271(2):405-17. doi: 10.1046/j.1432-1033.2003.03941.x.
The role of renal expression of the glycosphingolipid verotoxin receptor, globotriaosylceramide, in susceptibility to verotoxin-induced hemolytic uremic syndrome is unclear. We show that a single glycosphingolipid can discriminate multiple specific ligands. Antibody detection of globotriaosylceramide in renal sections does not necessarily predict verotoxin binding. The deoxyglobotriaosylceramide binding profile for verotoxin 1, verotoxin 2 and monoclonal anti-globotriaosylceramide are distinct. Anti-globotriaosylceramide had greater dependency on the intact alpha-galactose and reducing glucose of globotriaosylceramide than verotoxin 1, while verotoxin 2 was intermediate. These ligands differentially stained human kidney sections. Glomerulopathy is the primary verotoxin-associated pathology in hemolytic uremic syndrome. For most samples, verotoxin 1 immunostaining within adult glomeruli was observed (type A). Some samples, however, lacked glomerular binding (type B). Anti-globotriaosylceramide (and less effectively, verotoxin 2) stained all glomeruli. Verotoxin 1/anti-globotriaosylceramide tubular staining was comparable. Type B glomerular/tubular globotriaosylceramide showed minor, but significant, fatty acid compositional differences. Verotoxin 1 type B glomerular binding became evident following pretreatment with cold acetone, or methyl-beta-cyclodextrin, used to deplete cholesterol. Direct visualization, using fluorescein isothiocyanate-verotoxin 1B, showed paediatric, but no adult glomerular staining; this was confirmed by anti-fluorescein isothiocyanate immunostaining. Acetone induced fluorescein isothiocyanate-verotoxin 1B glomerular staining in type A, but poorly in type B samples. Comparison of fluorescein isothiocyanate-verotoxin 1B and native verotoxin 1B deoxyglobotriaosylceramide analogue binding showed an alteration in subspecificity. These studies indicate a marked heterogeneity of globotriaosylceramide expression within renal glomeruli and differential binding of verotoxin 1/verotoxin 2/anti-globotriaosylceramide to the same glycosphingolipid. Verotoxin 1 derivatization can induce subtle changes in globotriaosylceramide binding to significantly affect tissue binding. Heterogeneity in glomerular globotriaosylceramide expression may play a significant (cholesterol-dependent?) role in determining renal pathology following verotoxemia.
糖鞘脂维罗毒素受体——球三糖神经酰胺在肾脏中的表达在维罗毒素诱导的溶血尿毒综合征易感性中的作用尚不清楚。我们发现单一糖鞘脂能够区分多种特异性配体。在肾脏切片中通过抗体检测球三糖神经酰胺不一定能预测维罗毒素的结合情况。维罗毒素1、维罗毒素2和单克隆抗球三糖神经酰胺的脱氧球三糖神经酰胺结合谱各不相同。与维罗毒素1相比，抗球三糖神经酰胺对球三糖神经酰胺完整的α - 半乳糖和还原性葡萄糖依赖性更强，而维罗毒素2则介于两者之间。这些配体对人肾脏切片的染色存在差异。肾小球病变是溶血尿毒综合征中与维罗毒素相关的主要病理表现。对于大多数样本，在成人肾小球内观察到维罗毒素1免疫染色（A型）。然而，一些样本缺乏肾小球结合（B型）。抗球三糖神经酰胺（维罗毒素2的染色效果稍差）能使所有肾小球染色。维罗毒素1/抗球三糖神经酰胺在肾小管的染色情况相当。B型肾小球/肾小管球三糖神经酰胺显示出微小但显著的脂肪酸组成差异。用冷丙酮或甲基 - β - 环糊精预处理以消耗胆固醇后，维罗毒素1在B型肾小球的结合变得明显。使用异硫氰酸荧光素 - 维罗毒素1B直接观察显示，儿科肾小球有染色，但成人肾小球无染色；抗异硫氰酸荧光素免疫染色证实了这一点。丙酮可诱导异硫氰酸荧光素 - 维罗毒素1B在A型肾小球染色，但在B型样本中染色效果较差。比较异硫氰酸荧光素 - 维罗毒素1B和天然维罗毒素1B的脱氧球三糖神经酰胺类似物结合情况，发现亚特异性发生了改变。这些研究表明肾小球内球三糖神经酰胺表达存在显著异质性，且维罗毒素1/维罗毒素2/抗球三糖神经酰胺与同一糖鞘脂的结合存在差异。维罗毒素1的衍生化可诱导球三糖神经酰胺结合发生细微变化，从而显著影响组织结合。肾小球球三糖神经酰胺表达的异质性可能在决定毒血症后的肾脏病理过程中起重要作用（是否依赖胆固醇？）。
