Analysis of glycolipids metabolism in peroxisome-defective cells and its relation to pathogenesis of Zellweger syndrome

我们观察到ZS和IRD患者成纤维细胞中a系列神经节苷脂、GM 2、GM 1和GD 1a的量增加。神经节苷脂GM 1和GD 1a在正常受试者中未检测到。α-系列神经节苷脂合成的一个关键步骤是将GalNAc转化为神经节苷脂GM 3，因此我们采用免疫组织化学方法测定了神经节苷脂GM 3的水平。在成纤维细胞胞浆中发现抗神经节苷脂GM 3抗体阳性的颗粒状结构。在对照细胞中，细胞膜对抗GM 3抗体呈轻微阳性。为了阐明糖脂含量增加的代谢基础，我们测定了Z65和CHO-K1细胞中神经酰胺葡萄糖基转移酶和β-葡萄糖苷酶的活性，发现前者负责葡萄糖脑苷脂在Z65细胞中的积累。然后，我们从CHO-K1细胞中克隆了编码神经酰胺葡萄糖基转移酶的cDNA，其与人基因产物的序列同源性为98.7%。北方印迹分析表明，与CHO-K1细胞相比，神经酰胺葡萄糖基转移酶在Z65细胞中的表达增加，可能导致GM 3的同时增加。用免疫组化方法，GM 3在Z65细胞的细胞膜上的表达比在CHO-K1细胞的细胞膜上的表达更强。这些结果可能有助于阐明PBD的发病机制与鞘糖脂在细胞分化，增殖和凋亡中的功能作用。

We observed an increased amount of a-series gangliosides, GM2, GM1 and GD1a in the fibroblasts of patients with ZS and IRD. Gangliosides GM1 and GD1a were not contained in a detectable amount in normal subjects. A key step for the synthesis of a-series gangliosides is a transfer of GalNAc to ganglioside GM3, so we determined the level of ganglioside GM3 by immunohistochemical method. We found granular structure, which was positive toward antiganglioside GM3 antibody in the cytoplasm of the patients' fibroblasts. In the control cells, cell membrane was slightly positive toward anti-GM3 antibody. To clarify the metabolic bases of incresed amounts of glycolipids, we measured the ceramide glucosyltransferase and β-glucosidase activities in Z65 and CHO-K1 cells, and found that the former enzyme was responsible for the accumulation of glucocerebroside in Z65 cells. Then, we cloned the cDNA encoding ceramide glucosyltransferase from CHO-K1 cells, which exhibited sequence homology with the human gene product (98.7%). Northern blot analysis of ceramide glucosyltransferase revealed increased expression of it in Z65 cells compared with in CHO-K1 cells, probably causing the simultaneous increase in GM3. With an immunohistochemical procedure, GM3 was more strongly expressed in the cell membrane of Z65 cells than in that of CHO-K1 cells. These results may help to clarify the pathogenesis of PBD with respect to the functional roles of glycosphingolipids in cell differentiation, proliferation and apoptosis.