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PubMed2000

cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

We have isolated a mouse cDNA clone encoding 3'-phosphoadenylylsulfate-galactosylceramide 3'-sulfotransferase (cerebroside sulfotransferase; CST; EC 2.8.2.11) from a kidney cDNA library, using a human CST cDNA clone [Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A. & Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868] as a probe. A recombinant protein of the cloned cDNA showed CST activity. The deduced protein is composed of the same 423 amino acids as human CST and its sequence exhibits 84% identity with that of the human counterpart. Northern-blot analysis and subquantitative reverse transcription-PCR (RT-PCR) analysis showed that the CST gene is preferentially transcribed in stomach, small intestine, brain, kidney, lung, and testis, in that order. To examine differences in transcripts in various tissues, we isolated CST cDNA clones from stomach, small intestine, brain, kidney, and testis by 5'-RACE analysis. We found seven different nucleotide sequences in the 5'-UTR, while the DNA sequences of all the isolated cDNA clones were identical in the coding region. In addition, we isolated CST genomic DNA clones from a mouse genomic library. The clones covered all the 5'-UTR sequences and coding exons including 3'-UTR. RT-PCR analyses of CST mRNAs from various tissues confirmed that CST transcripts are tissue-specifically spliced by alternative use of multiple exons 1. These observations suggest that the tissue-specific expression of the CST gene is explained by alternative usage of multiple 5'-UTR exons flanked with tissue-specific promoters.

Amino Acid Sequence↗
PubMed2003

Cerebroside sulfotransferase deficiency ameliorates L-selectin-dependent monocyte infiltration in the kidney after ureteral obstruction.

Mononuclear cells infiltrating the interstitium are involved in renal tubulointerstitial injury. The unilateral ureteral obstruction (UUO) is an established experimental model of renal interstitial inflammation. In our previous study, we postulated that L-selectin on monocytes is involved in their infiltration into the interstitium by UUO and that a sulfated glycolipid, sulfatide, is the physiological L-selectin ligand in the kidney. Here we tested the above hypothesis using sulfatide- and L-selectin-deficient mice. Sulfatide-deficient mice were generated by gene targeting of the cerebroside sulfotransferase (Cst) gene. Although the L-selectin-IgG chimera protein specifically bound to sulfatide fraction in acidic lipids from wild-type kidney, it did not show such binding in fractions of Cst(-/-) mice kidney, indicating that sulfatide is the major L-selectin-binding glycolipid in the kidney. The distribution of L-selectin ligand in wild-type mice changed after UUO; sulfatide was relocated from the distal tubules to the peritubular capillaries where monocytes infiltrate, suggesting that sulfatide relocated to the endothelium after UUO interacted with L-selectin on monocytes. In contrast, L-selectin ligand was not detected in Cst(-/-) mice irrespective of UUO treatment. Compared with wild-type mice, Cst(-/-) mice showed a considerable reduction in the number of monocytes/macrophages that infiltrated the interstitium after UUO. The number of monocytes/macrophages was also reduced to a similar extent in L-selectin(-/-) mice. Our results suggest that sulfatide is a major L-selectin-binding molecule in the kidney and that the interaction between L-selectin and sulfatide plays a critical role in monocyte infiltration into the kidney interstitium.

Animals↗
PubMed1983

Topography of cerebroside sulfotransferase in Golgi-enriched vesicles from rat brain.

Cerebroside sulfotransferase (CST) catalyzes the final step in the synthesis of sulfatide (sulfogalactocerebroside) by transferring the sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to galactocerebroside. Orientation of CST was studied in vesicles enriched in this enzyme obtained from 21-d-old rat brain. Several lines of evidence indicate that CST is located on the luminal side of these vesicles. (a) Sulfation of endogenous galactocerebroside occurred in vesicles only in the presence of a detergent to render the membranes permeable to exogenous PAPS. (b) There is a pool of latent enzyme within the vesicle, which is released by Triton X-100. (c) CST is not destroyed by trypsin unless the vesicle membranes are first made permeable by Triton X-100. (d) Glycolipid substrate, when covalently attached to agarose beads, was not sulfated unless the enzyme was solubilized. These results are similar to those obtained with thiamine pyrophosphatase, which is known to be located within the lumen of the vesicles. This study establishes that an enzyme synthesizing a complex glycolipid is localized within Golgi-enriched vesicles. Since the product of the CST reaction must also be localized to the luminal side of the vesicles, it is most likely that sulfatide is located at the intraperiod line (outer layer) of myelin. The orientation of CST within the vesicle provides a mechanism for the asymmetrical assembly of glycolipids in bilayers.

Animals↗
PubMed2000

Methionine oxidation within the cerebroside-sulfate activator protein (CSAct or Saposin B).

The cerebroside-sulfate activator protein (CSAct or Saposin B) is a small water-soluble glycoprotein that plays an essential role in the metabolism of certain glycosphingolipids, especially sulfatide. Deficiency of CSAct in humans leads to sulfatide accumulation and neurodegenerative disease. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A. CSAct has seven methionine residues and a mass of 8,845 Da when deglycosylated. Mildly oxidized, deglycosylated CSAct (+16 Da), separated from nonoxidized CSAct by reversed-phase high-performance liquid chromatography (RP-HPLC), showed significant modulation of the in vitro activity. Because oxidation partially protected against CNBr cleavage and could largely be reversed by treatment with dithiothreitol, it was concluded that the major modification was conversion of a single methionine to its sulfoxide. High-resolution RP-HPLC separated mildly oxidized CSAct into seven or more different components with shorter retention times than nonoxidized CSAct. Mass spectrometry showed these components to have identical mass (+16 Da). The shorter retention times are consistent with increased polarity accompanying oxidation of surface-exposed methionyl side chains, in general accordance with the existing molecular model. A mass-spectrometric CNBr mapping protocol allowed identification of five of the seven possible methionine-sulfoxide CSAct oxoforms. The most dramatic suppression of activity occurred upon oxidation of Met61 (26% of control) with other residues in the Q60MMMHMQ66 motif falling in the 30-50% activity range. Under conditions of oxidative stress, accumulation of minimally oxidized CSAct protein in vivo could perturb metabolism of sulfatide and other glycosphingolipids. This, in turn, could contribute to the onset and progression of neurodegenerative disease, especially in situations where the catabolism of these materials is marginal.

Amino Acid Sequence↗
PubMed1990

Mixtures of semisynthetic species of cerebroside sulfate with dipalmitoyl phosphatidylcholine. Thermotropic phase behavior and permeability.

The phase behavior of mixtures of dipalmitoyl phosphatidylcholine (DPPC) with semisynthetic species of cerebroside sulfate (CBS) containing palmitic acid (C16:0-CBS) or lignoceric acid (C24:0-CBS) in 0.1 M KCl was studied using differential scanning calorimetry. DPPC and C16:0-CBS were miscible in all proportions in the gel phase above 10 mol% CBS and in the liquid-crystalline phase. However, C24:0-CBS was less miscible with DPPC over a wide concentration range in the gel phase. At high CBS concentrations it was probably also not entirely miscible with DPPC in the liquid-crystalline phase. Small amounts of both species of CBS lowered the transition temperature and enthalpy of DPPC, suggesting that they are more soluble in the liquid-crystalline phase of DPPC than the gel phase. The transition temperature at higher CBS concentrations was also less than expected, especially after cycling through the phase transition in the case of C24:0-CBS, suggesting that mixing with DPPC inhibited the intermolecular hydrogen bonding interactions and dehydration of CBS. In C24:0-CBS-DPPC mixtures several populations were present over a wide compositional range, including two solid-solid solutions of fixed composition. At high C24:0-CBS concentrations some C24:0-CBS also phase separated out of the mixture. Structural considerations suggested that the C24:0-CBS which is mixed with DPPC must be interdigitated into the DPPC bilayer. Other populations that are present may have a different structural organization. A fatty acid spin label in these mixtures was a little less ordered than in either lipid by itself. The permeability of these lipids, as well as the two asymmetric species 1-stearoyl-2-caproyl phosphatidylcholine and 1-stearoyl-2-myristoyl phosphatidylcholine (18:10PC and 18:14PC), to a water-soluble spin label tempocholine chloride was also measured. The studies with 18:10PC and 18:14PC indicated that both triple-chain mixed interdigitated bilayers and double-chain partially interdigitated bilayers can trap water-soluble substances and have low permeability. Both species of CBS could also entrap the spin label and had low permeability at 4 degrees C. However, they rapidly lost the entrapped compound when they transformed into their stable dehydrated phases or into the liquid-crystalline phase. Mixing with DPPC prevented both of these losses. These studies supported the conclusion that a significant amount of the CBS was mixed with the DPPC and that this mixing prevented the dehydration changes which CBS undergoes by itself.(ABSTRACT TRUNCATED AT 400 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1984

[Use of fluorescence-labelled cerebroside as a substrate for galactosylceramidase of human skin fibroblasts].

Possible use of the fluorescently labelled cerebroside, 1-O-(beta-D-galactosyl)-2-N-[6-(2-antroyl)hexanoyl]-4-sphingeni n (Gal-A-sphingenin) as a substrate for galactosylceramidase (GC) from human skin fibroblasts was investigated. Studies involving TLC and fluorimetric methods revealed enzymatic splitting of Gal-A-sphingenin whose degree correlated with the amount of the enzyme and incubation time. Some kinetic parameters of GC were determined, using Gal-A-sphingenin as substrate. It was shown that the values of specific activity of GC (1.6 nmol/mg protein/hr) and Km (0.025 mM) for Gal-A-sphingenin agree well with the corresponding values obtained with the use of the galactocerebroside as a natural substrate. In experiments with mixtures of Gal-A-sphingening and galactocerebroside used as substrates in different molar ratios, it was shown that the enzyme splits each of the substrates with equal velocity. The results of experiments with the enzyme samples from skin fibroblasts of healthy individuals and of patients with GM1-gangliosidosis (GM1-beta-D-galactosidase deficiency) suggest that Gal-A-sphingenin is a specific substrate for GC.

Enzyme Activation↗