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RISS 인기검색어
- 검색키워드
- 저자 : Akemi Suzuki (검색결과 3 건)
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Tissue Specific Regulation of Glycolipids and Related Functions
Akemi Suzuki 한국당과학회 2008 한국당과학회 학술대회 Vol.2008 No.1
The expression of glycan chains and hydrophobic structures of glycolipids is precisely regulated in a time- and space-dependent manner. We focus our research on two genes, Gsl5 (glycosphingolipid regulatory gene 5) and Des2 (degenerative spermatocyte 2). Gsl5 regulates the expression of GlcNAc 1-6(Gal 1-3)GalNAc containing glycolipids and glycoproteins through controlling the activity of 6GlcNAc transferase in mouse kidney. Des2 encodes a hydroxylase which hydroxylates dihydroceramide:sphinganine at C4 position and is responsible for the expression of glycolipids containing C4 hydroxysphinganine (phytosphingosine) in the small intestine and kidney of mice. We found that Gsl5 locates at about 5.5 kb upstream of exon 1 of 6GlcNAc T1, consists of eight pairs of two GT-repeats, and controls 6GlcNAc T1 mRNA expression in kidney proximal tubular cellspecific manner. 6GlcNAc transferase transfers GlcNAc on the GalNAc of Gal 1-3GalNAc and of glycolipids and glycoproteins, respectively. One of glycoproteins modified by the GlcNAc transferase in the proximal tubular epithelial cells is megalin, which binds and reabsorbs small molecular weight proteins from urine produced by the blood filtration at glomeruli. We purified two types of megalin, one carries Gal 1-4(Fuc 1-3)GlcNAc 1-6(Gal 1- 3)GalNAc 1-Ser/Thr (Lewis X-core 2 structure) and the other the structure without the Gal 1-4(Fuc 1-3)GlcNAc 1-6 branch from mouse kidneys. Ligand binding capacity of the megalin with Lewis Xcore 2 structure was much higher than that without the branch structure. Des2 hydroxylase requires NADH, NADH-dependent cytochrome b5 reductase, and cytochrome b5 for producing the activity. mRNA expression of Des2 is regulated in a tissue-specific manner, highest in the small intestine and high in the kidney. We are in the process of establishing Des2 knock out mice and are going to analyze phenotypes. We consider that C4-hydroxylated sphinganine containing glycolipids play a significant role for maintaining functions of nutrient absorption.
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Matrices for the Analysis of Glycosphingolipids by LC-MS
Akemi Suzuki,Hideshi Fujiwaki,Yoshikatsu Umemura 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
Recent advances in mass spectrometry make our view of glycosphingolipids (GSLs) com- pletely different from the previous one. LC-MS is applicable to GSL mixture in a smallamount, and provides structural information of carbohydrate chains and ceramides andpossibly quantitative results. However, several subjects remain to be carefully clarified.Critical subjects are matrices used for enhancing ionization of GSLs and the prepara- tion of standard or isotope-labeled GSLs for quantitation. We have tested several matrices for neutral GSLs and acidic GSLs (gangliosides) in the analysis of negative ion mode. Negative ion mode provides more fragment ions derived from carbohydrate chains and ceramides. Matrices used for LC analysis with a C30 reversed phase column of neutral GSLs, such as ammonium formate, formic acid, ammonium acetate, and acetic acid, give adduct ions and the ratios of molecularions [M - H]- and adduct ions are different among molecular species of GlcCer as the simplest neutral GSL. Ammonium bicarbonate containing solvents give lesser amount of adducts and can be used for comparing changes of molecular species of neutral GSLs produced by the different conditions of cultured cells or comparing neutral GSLs of different subsets of cells isolated from living organisms. In the analysis of gangliosides, we detected GM1(NeuAc) as a single charged ion [M – H]-,and GM1(NeuGc) as a double charged ion [M - 2H]-2. Other ganglisoides such as GD1, GT1, and GQ1 molecules were detected as double charged ions, therefore, GM1(NeuAc) gives the highest m/z values among these gangliosides, thus detection effi- ciency becomes very low. We have successfully applied LC-MS analysis to gangliosides of mouse thymocytes and CD4+ and CD8+ T cells (Nagafuku et al. 2012). Fortunately, these gangliosides contain N-glycolylneuraminic acid and give double charged ions, even in the case of GM1. When they are activated, the down regulation of CMP- NeuAc hydroxylase which is the rate liming step for the expression of NeuGc containing ganglisodies (Naito et al. 2007), is possibly triggered. Then, the sensitive detection of GM1(NeuAc) must be assured. This is also required for the analysis of microdomains of neuronal cell mem-brane of neural degenerative diseases such as Alzheimer disease and Parkinson disease.
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