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.

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National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Medical Biochemistry Study Section (MEDB)
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Johns Hopkins University
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Chatterjee, Subroto; Alsaeedi, Nezar (2012) Lactosylceramide synthase as a therapeutic target to mitigate multiple human diseases in animal models. Adv Exp Med Biol 749:153-69
Chatterjee, Subroto; Pandey, Ambarish (2008) The Yin and Yang of lactosylceramide metabolism: implications in cell function. Biochim Biophys Acta 1780:370-82
Chatterjee, Subroto; Neill, Roger; Shupp, Jeffrey W et al. (2007) Identification of staphylococcal enterotoxin B domains involved in binding to cultured human kidney proximal tubular cells: imparting proliferation and death. Exp Biol Med (Maywood) 232:1142-51
Martin, Sergio F; Williams, Niesha; Chatterjee, Subroto (2006) Lactosylceramide is required in apoptosis induced by N-Smase. Glycoconj J 23:147-57
Chatterjee, Subroto; Berliner, Judith A; Subbanagounder, Ganesamoorthy G et al. (2004) Identification of a biologically active component in minimally oxidized low density lipoprotein (MM-LDL) responsible for aortic smooth muscle cell proliferation. Glycoconj J 20:331-8
Yeh, L H; Kinsey, A M; Chatterjee, S et al. (2001) Lactosylceramide mediates shear-induced endothelial superoxide production and intercellular adhesion molecule-1 expression. J Vasc Res 38:551-9
Chatterjee, S (2000) Assay of lactosylceramide synthase and comments on its potential role in signal transduction. Methods Enzymol 311:73-81
Jan, J T; Chatterjee, S; Griffin, D E (2000) Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide. J Virol 74:6425-32
Chatterjee, S (1999) Neutral sphingomyelinase: past, present and future. Chem Phys Lipids 102:79-96
Chatterjee, S; Han, H; Rollins, S et al. (1999) Molecular cloning, characterization, and expression of a novel human neutral sphingomyelinase. J Biol Chem 274:37407-12

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