REGULATION OF LACTOSYLCERAMIDE SYNTHESIS IN RENAL CELLS

我们已经发现，在正常人肾近端小管（PT）细胞中， 血浆低密度脂蛋白（LDL）发挥浓度依赖性 乳糖神经酰胺（LacCer）和酶水平降低 参与LacCer生物合成，即，UDP-半乳糖：葡萄糖神经酰胺 B1->4半乳糖基转移酶（GalT-2）。 然而，进入体内的甲基低密度脂蛋白 细胞通过LDL受体非依赖性途径刺激活性 在正常PT细胞中，GalT-2的表达约为正常PT细胞的三倍。 而反观 来自患有家族性高胆固醇血症（FH）的患者的PT细胞，以及来自人类的PT细胞。 肾肿瘤PT细胞没有LDL受体，LDL增加了PT细胞的LDL受体。 GalT-2的活性约为3倍。 这导致了15倍 FH PT细胞中LacCer细胞水平的增加和 更复杂的鞘糖脂。 我们净化了 并部分表征了来自人肾脏的GalT-2。 产生的抗体 针对这种酶特异性免疫沉淀GalT-2， 正常PT细胞。 我们的总体目标是：1）追求分子 GalT-2的表征/表达并研究LDL的作用，以及 M-LDL对PT细胞mRNA水平和周转的影响; 2）测定 GalT-2在正常PT、FH-PT和肿瘤PT细胞中的细胞定位; 3）研究LDL和修饰的LDL对大鼠心肌细胞的作用及其机制 低密度脂蛋白（M-LDL）对PT细胞中GalT-2水平的影响。 假说. 合成（生产过剩）和储存 FH-PT细胞中的乳糖神经酰胺是由于缺乏GalT的调节， 2、LDL。 研究建议，将阐明生物化学 LDL和M-LDL对GalT-2的作用机制及其在 分子水平。 这些研究提供了关于 LacCer合成在正常PT细胞中是如何调节的， FH-PT中LDL介导的GalT-2调节缺乏的病理生理学， 肿瘤PT细胞。

We have found that in normal human kidney proximal tubular (PT) cells, plasma low density lipoproteins (LDL) exerts a concentration dependent decrease in the level of lactosylceramide (LacCer) and the enzyme involved in LacCer biosynthesis, i.e., UDP-galactose: gluco-sylceramide B1->4 galactosyltransferase (GalT-2). However, methyl-LDL which enters cells through a LDL receptor independent pathway stimulated the activity of GalT-2 approximately three fold in normal PT cells. In contrast, in PT cells from patients with familial hypercholesterolemia (FH), and human kidney tumor PT cells which do not have LDL receptors, LDL increased the activity of GalT-2 approximately three fold. This resulted in a 15 fold increase in the cellular level of LacCer in FH PT cells and the synthesis of more complex glycosphingolipids in tumor PT cells. We have purified and partially characterized GalT-2 from human kidney. Antibodies raised against this enzyme specifically immunoprecipitate GalT-2 from cultured normal PT cells. Our overall objects are: 1) to pursue molecular characterization/expression of GalT-2 and to study the effect of LDL, and M-LDL on the levels and the turnover of mRNA in PT cells; 2) to determine the cytolocalization of GalT-2 in normal PT, FH-PT and tumor PT cells; and 3) to study the effects and mechanisms of action of LDL and modified LDL (M-LDL) on the level of GalT-2 in PT cells. Hypothesis. The synthesis (overproduction) and storage of lactosylceramide in FH-PT cells is due to the lack of regulation of GalT- 2 by LDL. Studies are proposed that will elucidate biochemical mechanisms of action of LDL and M-LDL on GalT-2 and its regulation at the molecular level. such studies provide fundamental knowledge regarding how LacCer synthesis is regulated in normal PT cells and the pathophysiology of LDL mediated lack of regulation of GalT-2 in FH-PT and tumor PT cells in man.