Michiko Sekine

Abnormality of Long-Chain Fatty Acids in Erythrocyte Membrane Sphingomyelin from Patients with Adrenoleukodystrophy

Abstract: We have devised an analytical method for the determination of fatty acid composition of erythrocyte membrane sphingomyelin by chemical ionization mass spectrometry combined with capillary column gas‐liquid chromatography. Fatty acid composition of erythrocyte membrane sphingomyelin from 8 patients with adrenoleukodystrophy (ALD) and 16 healthy controls were examined by this method. The ratio of hexacosanoic acid (C26.0) to docosanoic acid (C22:0) in erythrocyte membrane sphingomyelin from ALD patients was 2.6‐fold higher than that of the controls. This result suggests that biochemical diagnosis of ALD is possible by the analysis of fatty acid composition of erythrocyte membrane sphingomyelin. Furthermore, it demonstrates that biochemical abnormality in ALD is the generalized abnormal metabolism of very long‐chain saturated fatty acids.

The cis-Regulatory Element Gsl5 Is Indispensable for Proximal Straight Tubule Cell-specific Transcription of Core 2 β-1,6-N-Acetylglucosaminyltransferase in the Mouse Kidney

Gsl5 regulates the expression of a glycolipid and glycoproteins that contain the LeX epitope in the mouse kidney through tissue-specific transcriptional regulation of the core 2 β-1,6-N-acetylglucosaminyltransferase (core 2 GnT) gene. The core 2 GnT gene has six exons and produces three alternatively spliced transcripts. Gsl5 regulates only the expression of the kidney-type mRNA, which is transcribed from the most 5′-upstream exon. By introducing a 159-kb bacterial artificial chromosome (BAC) clone that carries the mouse core 2 GnT gene and its 5′-upstream region into DBA/2 mice that carry a defective Gsl5 allele, we were able to rescue the deficient phenotype. The BAC clone was subsequently engineered to replace the core 2 GnT gene with the sequence of enhanced green fluorescent protein (EGFP) as a reporter by an inducible homologous recombination system in Escherichia coli. The transgenic mice derived from the modified BAC clone expressed EGFP in the kidney, which suggests that the candidate Gsl5 is in the 5′-upstream region of the core 2 GnT gene. Sequence analysis of the 5′-upstream regions of the BAC clone and DBA/2 genomic DNA revealed a candidate sequence for Gsl5 at about 5.5 kb upstream of exon 1. This sequence consisted of eight repeats of two GT-rich units in the wild-type mice, whereas it consisted of only one pair of GT-rich units with a minor modification in the DBA/2 mice. Transgenic mice produced with the EGFP reporter gene construct that included this candidate sequence expressed EGFP exclusively in the proximal straight tubular cells of the kidney. These results indicated that this unique repeat is indeed the Gsl5, and it is a cis-regulatory element responsible for proximal straight tubule cell-specific transcriptional regulation.

Monoclonal antibodies that bind to galactosylgloboside (SSEA-3 antigen).

The 3-fucosyllactosamine (3-FL) antigenic determinant, Gal(beta 1-4)[Fuc(alpha 1-3)]-GlcNAc-R, is very immunogenic in BALB/c mice. A panel of monoclonal antibodies was raised against a novel glycosphingolipid that contains a terminal 3-FL structure (structure 1): (formula: see text) Surprisingly, most of these antibodies reacted with the internal galactosylgloboside structure rather than with the terminal 3-FL epitope. The specificities of two of these antibodies, 5A3 and 8A7, were analyzed by enzyme-linked immunosorbent assay and immunostaining procedures, and compared with MC631 (anti-SSEA-3), the only other monoclonal antibody known to bind to galactosylgloboside. 5A3 and 8A7 bound equally well to structure 1 and to galactosylgloboside. These antibodies also bound to structure 1 from which the terminal galactose, or the galactose and fucose, had been removed, but not to globoside or sialylated galactosylgloboside. In contrast, MC631 did not bind to structure 1 but, as described previously, did bind to globoside and sialylated galactosylgloboside. Galactosylgloboside is a developmentally regulated antigen of mouse embryos, human teratocarcinomas, and mouse embryonic brain. Antibodies 5A3 and 8A7 will complement MC631 in the analysis of the distribution and regulation of galactosylgloboside.

Generation of mouse models for type 1 diabetes by selective depletion of pancreatic beta cells using toxin receptor-mediated cell knockout.

By using the toxin receptor-mediated cell knockout (TRECK) method, we have generated two transgenic (Tg) murine lines that model type 1 (insulin-dependent) diabetes. The first strain, C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg, carries the diphtheria toxin receptor (hDTR) driven by the human insulin gene promoter, while the other strain, C57BL/6-ins2(BAC)-TRECK-Tg, expresses hDTR cDNA under the control of the mouse insulin II gene promoter. With regard to the C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg strain, only one of three Tg strains exhibited proper expression of hDTR in pancreatic β cells. By contrast, hDTR was expressed in the pancreatic β cells of all four of the generated C57BL/6-ins2(BAC)-TRECK-Tg strains. Hyperglycemia, severe ablation of pancreatic β cells and depletion of serum insulin were observed within 3days after the administration of diphtheria toxin (DT) in these Tg mice. Subcutaneous injection of a suitable dosage of insulin was sufficient for recovery from hyperglycemia in all of the examined strains. Using the C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg model, we tried to perform regenerative therapeutic approaches: allogeneic transplantation of pancreatic islet cells from C57BL/6 and xenogeneic transplantation of CD34(+) human umbilical cord blood cells. Both approaches successfully rescued C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg mice from hyperglycemia caused by DT administration. The high specificity with which DT causes depletion in pancreatic β cells of these Tg mice is highly useful for diabetogenic research.

Adrenal Medulla Gangliosides

The gangliosides were examined in adrenal glands of mouse, rat, guinea pig, rabbit, monkey, pig, ox and chicken. GM3 ganglioside was predominant in all examined animals except pig. In pig GD3 ganglioside was the major one. GM4 ganglioside was found in guinea pig and chicken. The distribution of sialic acid varied in each species. NeuNGly containing gangliosides were not detected in rat, guinea pig, rabbit and chicken. The other animals have both NeuNAc and NeuNGly containing gangliosides. Chromaffin granules from bovine adrenal medulla contain gangliosides at concentrations 3 and 7 times as great as microsomal and mitochondrial fractions, respectively. These gangliosides were NeuNAc-containing GM3 and NeuNGly containing GM3 in the same amount.

Isolation and characterization of a novel disialoganglioside from bovine adrenal medulla

A novel GDlb ganglioside which contains only N-glycolylneuraminic acid was isolated from bovine adrenal medulla by DEAE-Sephadex A-25 and Iatrobeads column chromatography. The concentration of this ganglioside was 0.9 nmol lipid-bound sialic acid per gram fresh tissue, which accounted for 2.7% of the disialoganglioside fraction. The structure was elucidated by sugar analysis, neuraminidase digestion, and permethylation studies. The complete structure of this ganglioside was identified as GDlb (NeuGc)2 or II3 (NeuGc)2-GgOse4Cer.

Core 2 GlcNAc transferase and kidney tubular cell-specific expression.

The expression of glycan chains is precisely regulated in a time- and space-dependent manner. We summarize here our recent work on the kidney tubular cell-specific regulation of core 2 β-1,6-GlcNAc transferase. Gsl5 gene was first identified by genetic analysis on the basis of polymorphic expression of kidney glycolipids among inbred strains of mice and turned out to be a regulatory gene controlling the level of mRNA of kidney-specific core 2 β-1,6-GlcNAc transferase. This kidney-specific core 2 GlcNAc transferase takes glycolipids having Galβ1-3GalNAc at their termini, Galβ1-3GalNAcα1- and β1-oligosaccharide derivatives, and glycoproteins having core 1 structure, as substrates. Immunohistochemistry with anti-core 2-Lex monoclonal antibody demonstrated that vesicles located just below the microvillous membrane of proximal tubule cells were clearly stained in a Gsl5-wild type mouse. Western blotting with the monoclonal antibody detected a major glycoprotein with a molecular mass of 500 kDa in the microsomal fraction of the wild type mouse kidney. In situ hybridization with anti-sense cDNA of kidney-specific core 2 GlcNAc transferase confirmed that Gsl5 gene controls the expression of the core 2 β-1,6-GlcNAc transferase mRNA in a proximal tubular cell-specific manner. The 5′ upstream sequences of the kidney-specific core 2 GlcNAc transferase gene in inbred and wild-derived strains of mice were analyzed, and the phylogenetic analysis of these sequences suggests that functional Gsl5 gene might be produced by the time of subspeciation of M. musculus, about one million years ago. Published in 2004.

Tissue specific control of glyco-chains

The expression of glyco-chains is precisely regulated in a time-and-space dependent manner. We summarize two types of regulation: kidney tubular cell-specific regulation of core 2 β1-6GlcNAc transferase by the Gsl5 gene, and the suppression of N-glycolylneuraminic acid (NeuGc) by the regulation of CMP-NeuAc hydroxylase activity. The Gsl5 gene identified by genetic analysis on the basis of the polymorphic expression of kidney glycolipids among inbred strains of mice regulates β1,6-GlcNAc transferase activity. The GlcNAc transferase transfers GlcNAc on Galβ1-3GalNAcβ-and also synthesizes core 2 structure, GlcNAcβ1-6(Galβ1-3)GalNAcα-. Immunohistochemistry with anti-core 2-Lex monoclonal antibody demonstrated that the lysosome-like vesicles of the proximal tubule cells were clearly stained in a Gsl5 wild-type mouse. Western blotting confirmed that the positive staining was due to the microsomal glycoproteins. These results, together with those of in situ hybridization, confirmed that the Gsl5 gene controls the expression of β-1,6-GlcNAc transferase mRNA in a proximal tubular cell-specific manner. The expression of NeuGc is controlled by the activity of CMP-NeuAc hydroxylation, which requires three enzyme proteins, cytochrome b5, cytochrome b5 reductase, and a terminal hydroxylase, in the presence of NADH. We cloned mouse and human hydroxylase cDNAs. Mouse brain contains a very high amount of NeuAc, but the hydroxylase mRNA is not detectable by Northern blotting. Surprisingly, the same phenotype is conserved in humans, even though humans have lost the ability to produce intact hydroxylase enzyme, due to the deletion of 92 bp in the genome.