Neutral glycosphingolipids of serum lipoproteins in Fabry's disease.

The neutral glycosphingolipid compositions of lipoprotein fractions of serum from eight healthy male volunteers and three patients with Fabry's disease were determined. Four fractions were studied: very low density lipoprotein (VLDL, d less than 1.006); low density lipoprotein (LDL, d 1.006-1.063); high density lipoprotein (HDL, d 1.063-1.21); and ultracentrifugal residue (Residue, d less than 1.21). All lipoprotein fractions contained the four major neutral glycosphingolipids (glucosylceramide, lactosylceramide, galactosylgalactosylglucosylceramide and N-acetylgalactosaminylgalactosylgalactosylglucosylceramide). The LDL and HDL, however, accounted for most of the total glycosphingolipid (69 and 20%, respectively); only small amounts were demonstrated in the VLDL and Residue. The relative distributions of the glycosphingolipids within the LDL and HDL fractions were similar to the distribution in unfractionated serum. Galactosylgalactosylglucosylceramide levels were 3-5 times normal in all three patients with Fabry's disease, and in two the distribution of the excess lipid among the major lipoprotein fractions was similar to that in the control group. In the third patient, who exhibited the presence of "sinking pre-beta lipoprotein", the amount of glycosphingolipid isolated with the HDL was greater than that in the LDL.     

Novel trinucleotide deletion in Fabry's disease

We describe the molecular characterization of a novel, in-frame deletion that is located in exon 7 of the -galactosidase A gene in a patient with Fabry's disease. The 3-bp deletion we identified, besides the typical severe clinical features, also expresses diffuse facial telangiectasias, which is a new cutaneous marker of Fabry's disease.     

Unexplained left ventricular hypertrophy: consider a diagnosis of Fabry's disease

Lysosomal storage disorders are a group of disorders characterised by the deficiency of a specific lysosomal hydrolase. These diseases are rare, with only a few hundred patients in the Netherlands. Fabry''s disease, an X-linked lysosomal storage disorder, is caused by a deficiency of the lysosomal enzyme α-galactosidase A which results in, among other things, left ventricular hypertrophy, renal failure and cerebrovascular events. Patients with Fabry''s disease, especially males, have a decreased life expectancy. Recent studies have shown that Fabry''s disease may be much more common among patients with left ventricular hypertrophy (LVH) than previously thought. Up to 7% of male patients with left ventricular hypertrophy and up to 12% of female patients with unexplained LVH were found to suffer from Fabry''s disease. Thus, Fabry''s disease should be considered in patients with unexplained LVH. This case report summarises the main features of the disease. In addition recent developments concerning prevalence, diagnosis and the current available treatments are discussed and an algorithm on who and how to screen for Fabry''s disease is presented.     

Endocytosis and sorting of glycosphingolipids in sphingolipid storage disease

Recent studies on the endocytic itinerary of glycosphingolipids (GSLs) in sphingolipid storage disease (SLSD) fibroblasts have yielded new insights into the mechanisms underlying the endocytosis and intracellular sorting of lipids in normal and disease cells. Here we highlight new data on clathrin-independent endocytosis of GSLs, the involvement of sphingolipid–cholesterol interactions in perturbation of endocytic trafficking, and potential roles for rab proteins in regulation of GSL transport in SLSDs.     

Tissue distribution of acetyl-coenzyme a carboxylase in leaves

Acetyl-CoA carboxylase [acetyl-CoA—carbon dioxide ligase (ADP forming), EC 6.4.1.2] is a biotin-containing enzyme catalyzing the formation of malonyl-CoA. The tissue distribution of this enzyme was determined for leaves of C₃- and C₄-plants. The mesophyll tissues of the C₃-plants Pisum sativum and Allium porrum contained 90% of the leaf acetyl-CoA carboxylase activity, with the epidermal tissues containing the remainder. Western blotting of proteins fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, using ¹²⁵I-streptavidin as a probe, revealed biotinyl proteins of molecular weights 62,000, 51,000, and 32,000 in P. sativum and 62,000, 34,000, and 32,000 in A. porrum.

In the C₄-plant sorghum, epidermal protoplasts, mesophyll protoplasts and strands of bundle sheath cells contained 35, 47, and 17%, respectively, of the total leaf acetyl-CoA carboxylase activity. In Zea mays leaves the respective figures were 10% for epidermal protoplasts, 56% for mesophyll protoplasts, and 32% for bundle sheath strands. Biotinyl proteins of molecular weights 62,000 and 51,000 were identified in leaves of sorghum and Z. mays.

The results are discussed with respect to each tissue's requirements for malonyl-CoA for various metabolic pathways.

     

Critical role for glycosphingolipids in Niemann-Pick disease type C

Niemann-Pick type C (NPC) disease is a cholesterol lipidosis caused by mutations in NPC1 and NPC2 gene loci. Most human cases are caused by defects in NPC1, as are the spontaneously occurring NPC diseases in mice and cats. NPC1 protein possesses a sterol-sensing domain and has been localized to vesicles that are believed to facilitate the recycling of unesterified cholesterol from late endosomes/lysosomes to the ER and Golgi. In addition to accumulating cholesterol, NPC1-deficient cells also accumulate gangliosides and other glycosphingolipids (GSLs), and neuropathological abnormalities in NPC disease closely resemble those seen in primary gangliosidoses. These findings led us to hypothesize that NPC1 may also function in GSL homeostasis. To evaluate this possibility, we treated murine and feline NPC models with N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glucosylceramide synthase, a pivotal enzyme in the early GSL synthetic pathway. Treated animals showed delayed onset of neurological dysfunction, increased average life span (in mice), and reduced ganglioside accumulation and accompanying neuropathological changes. These results are consistent with our hypothesis and with GSLs being centrally involved in the pathogenesis of NPC disease, and they suggest that drugs inhibiting GSL synthesis could have a similar ameliorating effect on the human disorder.     

A case of Fabry's disease in a patient with no alpha-galactosidase A activity caused by a single amino acid substitution of Pro-40 by Ser

We analyzed a male patient with Fabry's disease who had no activity of the lysosomal hydrolase alpha-galactosidase A (alpha-GalA) and female members of his family. We cloned a cDNA that encoded the mutant alpha-GalA, determined its nucleotide sequence, and found two nucleotide differences between the mutant and the wild-type cDNAs. Although one difference was silent, the other difference, a C-to-T transition at nucleotide number 118, resulted in an amino acid substitution of Pro-40 by Ser. A transient expression assay demonstrated that this missense mutation was the cause of the deficiency of alpha-GalA activity in the patient. In vitro mutagenesis experiments demonstrated that Pro-40 is critical for the appearance of alpha-GalA activity.     

Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1(-/-) mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.     

Fabry's disease: Heterozygote detection by hair root analysis

Summary Serum concentrations of immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA) were determined in 15 women with a lack of X chromosome material (Turner's syndrome), and compared with the immunoglobulin concentrations in normal men and women. Further, the investigation is supplemented by a comparison of normal women and the Turner group matched according to age.The serum concentrations of IgG and IgA in women with Turner's syndrome were very close to the concentration in serum from normal men, whereas the concentration of IgM was significantly lower. Compared to normal women the concentrations of IgG and IgM were significantly lower, and the concentration of IgA significantly higher in the Turner group.Whether these differences in serum immunoglobulins are determined by hormonal factors or under direct genetic control linked to the X chromosomes, is discussed.     

Total synthesis of ceramide trihexoside accumulating with Fabry's disease.

We report the synthesis of natural ceramide trihexoside, viz. gal (alpha-1 leads to 4) gal (beta-1 leads to 4) gluc (beta-1 leads to 1) ceramide (XIII). It involves the Koenigs-Knorr reaction of the bromide II with the aglucon XIX, and of the chloride VII with the D-enantiomer of the ceramide ester XII. A positional isomer of XIII was obtained as a by-product. A novel technique in the Koenings-Knorr reaction is described.