Accumulation Of Glycolipids Research Articles

Molecular Basis and Clinical Management of Gaucher Disease

Gaucher disease (GD) type I is an autosomal recessive disease caused by a genetic deficiency of lysosomal β-glucocerebrosidase that leads to accumulation of undergraded substrate glucocerebroside and other glycolipids, thus causing damage in different organs. GBA is the only gene in which mutations are known to cause GD. Nearly 300 mutations have been identified in GD patients, including frame-shift mutations, point mutations, deletions, insertions, splice site mutations and recombinants. The variety of phenotypes associated to GD shows imperfect correlation with mutations. GD encompasses a spectrum of clinical findings from a perinatal lethal form to an asymptomatic form. However the classification of GD by clinical subtype is still useful in describing the wide range of clinical findings and broad variability in presentation. Three major clinical types are delineated: type I (chronic nonneuropathic), type II (acute neuropathic), and type III (chronic neuropathic). Patients with type I GD present with visceromegaly, hematological complications, and bone disease. Cardiac and pulmonary complications are rare. Type I GD adult patients have elevated risk of malignancies, Parkinson’s disease or Parkinsonism. Neuropathic forms of GD are rare and clinically ranging from lethal perinatal form to very mild form limited to abnormalities of horizontal ocular saccades. Diagnosis of acid β-glucosylceramidase relies on enzyme activity in peripheral blood leukocytes or skin fibroblasts and/or identification of GBA mutations. Enzyme replacement therapy is an effective treatment for non-neuropathic GD. Substrate inhibitor is the alternative therapy for some patients with GD is miglustat, iminosugar inhibitor of glucocerebroside synthase.

Synchrotron infrared imaging of advanced glycation endproducts (AGEs) in cardiac tissue from mice fed high glycemic diets.

Recent research findings correlate an increased risk for dieases such as diabetes, macular degeneration and cardiovascular disease (CVD) with diets that rapidly raise the blood sugar levels; these diets are known as high glycemic index (GI) diets which include white breads, sodas and sweet deserts. Lower glycemia diets are usually rich in fruits, non-starchy vegetables and whole grain products. The goal of our study was to compare and contrast the effects of a low vs. high glycemic diet using the biochemical composition and microstructure of the heart. The improved spatial resolution and signal-to-noise for SR-FTIR obtained through the coupling of the bright synchrotron infrared photon source to an infrared spectral microscope enabled the molecular-level observation of diet-related changes within unfixed fresh frozen histologic sections of mouse cardiac tissue. High and low glycemic index (GI) diets were started at the age of five-months and continued for one year, with the diets only differing in their starch distribution (high GI diet = 100% amylopectin versus low GI diet = 30% amylopectin/70% amylose). Serial cryosections of cardiac tissue for SR-FTIR imaging alternated with adjacent hematoxylin and eosin (H&E) stained sections allowed not only fine-scale chemical analyses of glycogen and glycolipid accumulation along a vein as well as protein glycation hotspots co-localizing with collagen cold spots but also the tracking of morphological differences occurring in tandem with these chemical changes. As a result of the bright synchrotron infrared photon source coupling, we were able to provide significant molecular evidence for a positive correlation between protein glycation and collagen degradation in our mouse model. Our results bring a new insight not only to the effects of long-term GI dietary practices of the public but also to the molecular and chemical foundation behind the cardiovascular disease pathogenesis commonly seen in diabetic patients.

Fabry nephropathy: indications for screening and guidance for diagnosis and treatment by the European Renal Best Practice

Fabry disease (FD) is an X-linked disorder of glycosphingolipid catabolism resulting in the accumulation of glycolipids including globotriaosylceramide in cells of various tissues resulting in end-organ manifestations. Initially, FD is typically characterized by angiokeratoma and recurrent episodes of neuropathic pain in the extremities occurring during childhood or adolescence. Most affected patients also exhibit a decreased ability to sweat. Later in life, FD results in left ventricular hypertrophy, proteinuria, renal failure and stroke. These later disease manifestations are non-specific and also common in diabetes, hypertension and atheromatosis and thus for most practitioners do not point into the direction of FD. As a consequence, FD is under-diagnosed and screening of high-risk groups is important for case finding, as is a thorough pedigree analysis of affected patients. In the nephrology clinic, we suggest to screen patients for FD when there is unexplained chronic kidney disease in males younger than 50 years and females of any age. In men, this can be performed by measuring α-galactosidase A activity in plasma, white blood cells or dried blood spots. In women, mutation analysis is necessary, as enzyme measurement alone could miss over one-third of female Fabry patients. A multidisciplinary team should closely monitor all known Fabry patients, with the nephrologist screening kidney impairment (glomerular filtration rate and proteinuria) on a regular basis. Transplanted Fabry patients have a higher mortality than the regular transplant population, but have acceptable outcomes, compared with Fabry patients remaining on dialysis. It is unclear whether enzyme replacement therapy (ERT) prevents deterioration of kidney function. In view of the lack of compelling evidence for ERT, and the low likelihood that a sufficiently powered randomized controlled trial on this topic will be performed, data of all patients with FD should be collected in a central registry.

Is it or is it not a pathogenic mutation? Is it or is it not the podocyte?

Is it or is it not a pathogenic mutation? Is it or is it not the podocyte?

Fabry disease: the many faces of a single disorder.

In 1898, two dermatologists, William Anderson in England and Johannes Fabry in Germany, separately described a disease characterized by skin lesions, known as angiokeratomas. This issue contains two reports of Fabry disease (FD) patients presenting with proteinuric chronic kidney disease (CKD) but lacking angiokeratomas [1, 2]. These cases illustrate the phenotypic heterogeneity of the disease in females [1] and FD variants [2], the role of genetic diagnosis [1] and renal biopsy [2] and recent advances in pathophysiology [1]. FD is caused by deficient activity of alpha-galactosidase A (α-GalA) due to mutations in the X-chromosome GLA gene, leading to accumulation of neutral glycolipids and eventual tissue injury and organ dysfunction [3]. In classical FD males, α-GalA activity is absent or nearly absent and there is an early onset of acroparesthesias, angiokeratoma and hypohydrosis followed by life-threatening cardiac, central nervous system and kidney disease leading to end-stage renal disease (ESRD) at a mean age of 40 years [3, 4].

Accumulation of glycolipids and other non-phosphorous lipids in Agrobacterium tumefaciens grown under phosphate deprivation

Phosphate deficiency is characteristic for many natural habitats, resulting in different physiological responses in plants and bacteria including the replacement of phospholipids by glycolipids and other phosphorous-free lipids. The plant pathogenic bacterium Agrobacterium tumefaciens, which is free of glycolipids under full nutrition, harbors an open reading frame (ORF) coding for a processive glycosyltransferase (named as Pgt). This glycosyltransferase was previously shown to synthesize glucosylgalactosyldiacylglycerol (GGD) and digalactosyldiacylglycerol (DGD) after heterologous expression. The native function of this enzyme and the conditions for its activation remained unknown. We show here that Pgt is active under phosphate deprivation synthesizing GGD and DGD in Agrobacterium. A corresponding deletion mutant (Δpgt) is free of these two glycolipids. Glycolipid accumulation is mainly regulated by substrate (diacylglycerol) availability. Diacylglycerol and the total fatty acid pool are characterized by an altered acyl composition in dependence of the phosphate status with a strong decrease of 18:1 and concomitant increase of 19:0 cyclo during phosphate deprivation. Furthermore, Agrobacterium accumulates two additional unknown glycolipids and diacylglycerol trimethylhomoserine (DGTS) during phosphate deprivation. Accumulation of all these lipids is accompanied by a reduction in phospholipids from 75 to 45% in the wild type. A further non-phosphorous lipid, ornithine lipid, was not increased but its degree of hydroxylation was elevated under phosphate deprivation. The lack of GGD and DGD in the Δpgt mutant has no effect on growth and virulence of Agrobacterium, suggesting that these two lipids are functionally replaced by DGTS and the two unknown glycolipids under phosphate deprivation.

P05—Peripheral Vasculature Involvement in Fabry Patients: Is There any Difference Between Genders?

Controversial results exist concerning the changes in vascular structure and function in Fabry disease (FD), and the mechanism by which glycolipid accumulation causes Fabry vasculopathy is not completely understood. Boutouyrie and colleagues1 showed a significant increase in the intima-medial thickness of both radial and carotid arteries in Fabry patients, but not in a control healthy group, while Moore et al2 did not find any significant differences. Kalliokoski and colleagues3 had shown that Fabry patients have significant endothelial function impairment, compared to healthy controls, measured as brachial artery flow–mediated vasodilatation (FMVD).

Glycolipid Content of the Nuclear Membrane in Hypoxic Rat Brain

ABSTRACTIn this study, we report glycolipid changes in the nuclear subcellular fraction from hypoxic rat brains. Male Wistar rats at the age of three months were subjected to sodium nitrite-induced hypoxia. Te nuclear fraction was isolated and lipids were extracted. The glycolipid content was measured by spectrophotometry and thin-layer chromatography.In controls, gangliosides and cerebrosides were the major glycolipid classes. In the nuclear membrane of hypoxic brains, we found increased levels of total glycolipids (5.2-fold), gangliosides (4.1-fold), and cerebrosides (5.9-fold). These changes reflect the disturbances in the glycolipid turnover. Most probably they are associated with impaired energy metabolism. The accumulation of glycolipids may be interpreted as a physiological adaptive response to hypoxia.

Contribution of Decreased Migration of Monocytes to Impaired Inflammation in Gaucher Disease

Abstract 2162 Background:Gaucher disease (GD) is an autosomal recessively inherited lysosomal storage disorder caused by an inborn glucocerebrosidase (GBA) deficiency. This leads to the accumulation of glycolipids in macrophages, mainly in the bone marrow, spleen and liver, resulting in organ damage. However, recent studies suggest that clinical presentation of GD is primarily attributed to the impaired inflammatory process induced by “loaded macrophages”, rather than to their physical presence. This impairment could be explained by dysfunction of circulating monocytes, normally capable of migrating into sites of vascular injury and inflammation. The current in vitro study investigated potential changes in migratory capacity of GD-derived monocytes that would contribute to the inflammatory response. Methods: CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of untreated GD patients and healthy volunteers, using immunomagnetic bead separation (Miltenyi Biotec). Monocyte migration capacity was assessed with 5μm Polycarbonate Membrane Transwell inserts which contained either stromal cell-derived factor 1 (SDF1; CXCL12) (500 and 1000 ng/ml) or serum (0.25%) at the bottom chamber. Monocytes were allowed to migrate from the upper chamber through the membrane, over 4 hours. Migrating monocytes were collected from the bottom chamber and quantified by FACS. Blockage of the CXCR4 receptor was examined using AMD3100 (10μg/ml) added to the migration assay. Serum SDF-1 levels were evaluated by ELISA (Quntikine® human CXCL12/SDF-1α R&D systems). Results: Upon exposure to SDF-1, migration capacity of GD monocytes (n=7) was significantly lower than that observed in monocytes obtained from healthy volunteers (n=6), approaching 5.7% vs. 14%, p=0.02, for SDF1of 500 ng/ml, and 8.7% vs 21.8%, p=0.013, for SDF1of 1000 ng/ml). Similar results were observed in the presence of autologous serum: migration capacity of GD monocytes (n=7) was lower than demonstrated by healthy monocytes (n=5), (18.6% vs. 45%, p=0.05). Notably, substitution of “GD serum” (n=7) with “healthy serum” (n=7) resulted in significant improvement in migration capacity of GD-derived monocytes (from 18.6% to 33.6%, p=0.038), while a reciprocal substitution of the serum caused a marked reduction in migration of monocytes derived from healthy volunteers (34% instead of 45%). Blockage of the CXCR4 receptor led to a decreased migration capacity of healthy monocytes cultured in autologous serum (from 45.2% to 39.6%) (n=4). Remarkably, this blockage had no impact on migration of both healthy and GD-derived monocytes cultured with GD serum (n=6). However, GD monocytes treated with AMD3100 in the presence of healthy serum, exhibited a reduced migration rate compared to that observed in untreated cells (22.6% vs. 33.6%; p=0.06, n=7).CXCR4 expression on GD-derived monocytes appeared to be lower than that measured on cells derived from healthy donors [64.5% (n=6) vs. 83.6% (n=3)]. Of note, serum levels of SDF-1 were significantly increased in GD subjects compared to healthy ones (2603pg/ml vs 2039 pg/ml; p=0.004, n=10). Conclusions: GD patients exhibit an impaired SDF1-dependent migration of monocytes, despite expressing a significantly increased SDF-1serum level. The impaired migration capacity is partly explained by low levels of CXCR4 on monocyte surface. Additionally, the reduced migration appears to be dependent on as yet undetermined serum-derived factors, as demonstrated by the reversibility of this impairment with substitution of the GD serum for “healthy serum”. The observed reduction in migratory capacity may significantly contribute to the abnormal inflammation accounting for GD-associated vascular and bone complications. Further studies exploring the precise mechanisms involved in this process and their direct impact on GD-related organ damage are warranted. Disclosures:No relevant conflicts of interest to declare.

Helper-dependent canine adenovirus vector-mediated transgene expression in a neurodegenerative lysosomal storage disorder

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a severe neurodegenerative lysosomal storage disorder caused by a deficiency of N-sulfoglucosamine sulfohydrolase (SGSH) activity with subsequent accumulation of partially-degraded heparan sulfate and other glycolipids. In this study, we have evaluated a gene therapy approach using a helper-dependent canine adenovirus vector that expresses human SGSH as a means of delivering sustained transgene expression to the brain. Initial testing in a mixed neural cell culture model demonstrated that the vector could significantly increase SGSH activity in transduced cells, resulting in near-normalization of heparan sulfate-derived fragments. While administration of vector by direct injection into the brain of adult MPS-IIIA mice enabled transgene expression for at least 8.5 months post-treatment, it was only in discrete areas of brain. Heparan sulfate storage was reduced in some regions following treatment, however there was no improvement in secondary neuropathological changes. These data demonstrate that helper-dependent canine adenovirus vectors are capable of neural transduction and mediate long-term transgene expression, but increased SGSH expression throughout the brain is likely to be required in order to effectively treat all aspects of the MPS-IIIA phenotype.

Requirement of Npc1 and availability of cholesterol for early embryonic cell movements in zebrafish

Niemann-Pick disease, type C (NP-C), often associated with Niemann-Pick disease, type C1 (NPC1) mutations, is a cholesterol-storage disorder characterized by cellular lipid accumulation, neurodegeneration, and reduced steroid production. To study NPC1 function in vivo, we cloned zebrafish npc1 and analyzed its gene expression and activity by reducing Npc1 protein with morpholino (MO)-oligonucleotides. Filipin staining in npc1-morphant cells was punctate, suggesting abnormal accumulation of cholesterol. Developmentally, reducing Npc1 did not disrupt early cell fate or survival; however, early morphogenetic movements were delayed, and the actin cytoskeleton network was abnormal. MO-induced defects were rescued with ectopic expression of mouse NPC1, demonstrating functional gene conservation, and by treatments with steroids pregnenolone or dexamethasone, suggesting that reduced steroidogenesis contributed to abnormal cell movements. Cell death was found in anterior tissues of npc1 morphants at later stages, consistent with findings in mammals. Collectively, these studies show that npc1 is required early for proper cell movement and cholesterol localization and later for cell survival.

Phytoplankton driven distribution of dissolved and particulate lipids in a semi-enclosed temperate sea (Mediterranean): Spring to summer situation

The relationship between changes in lipid classes and phytoplankton composition and abundance in the northern Adriatic was studied during spring and summer 2008 at two stations with different nutrient levels, i.e. at the western mesotrophic and eastern oligotrophic areas. Changes in the phytoplankton community depended on temporal surface nutrient depletion and bottom accumulation; that is, microphytoplankton, mainly diatoms Pseudo-nitzschia sp., developed at nutrient richer surface layers of the mesotrophic area in spring and at deeper layers of the oligotrophic site in late summer. In other periods nanophytoplankton dominated. Dissolved organic carbon (DOC) and lipid content were comparable for the two stations, while particulate organic carbon (POC) was richer at the mesotrophic side. Total lipid concentrations varied in the range from 8.0 to 92.2 μg l −1 and from 16.9 to 76.9 μg l −1 in the dissolved and particulate fractions, respectively. DOC and POC contents were in the ranges from 0.77 to 1.58 mg l −1 and from 0.06 to 0.56 mg l −1, respectively. Lipid and organic carbon distribution did not follow phytoplankton progression, indicating decoupling between organic matter production and decomposition throughout the investigation period. The main sources of lipids were marine phytoplankton and bacteria. Low nutrient conditions caused increased biosynthesis of lipids. Also, increasing oligotrophy led to an increasing number of phytoplankton taxa. The synthesis and accumulation of glycolipids by the developed taxa were enhanced during nutrient exhaustion, contributing in late summer, on average, 20.2 and 22.0% at the mesotrophic and oligotrophic stations, respectively, in the particulate fraction. The distribution of bacterial lipids and lipid breakdown products implies that bacterial lipid degradation was significant in spring, while very probably lipid abiotic degradation took place during summer.

Minimum requirement of donor cells to reduce the glycolipid storage following bone marrow transplantation in a murine model of Fabry disease

Fabry disease (FD) is a lysosomal storage disorders characterized by a deficiency of the lysosomal enzyme, α-galactosidase A. This results in the accumulation of glycolipids, mainly globotriaosylceramide (GL-3), in the lysosomes of various organs. Although bone marrow transplantation and hematopoietic stem cell-based gene therapy can offer the potential of a curative therapeutic outcome for FD, the minimum requirement of donor cells or gene-corrected cells to reduce GL-3 levels is not known. Lethally-irradiated FD mice were transplanted intravenously with normal bone marrow cells (Ly5.1 positive) mixed with those of FD mice (Ly5.2 positive) at various ratios to investigate the level of engraftment and enzyme activity necessary to effect a reduction in GL-3 storage. Chimerism of whole white blood cells of recipients' peripheral blood remained stable at 8 weeks after transplantation, and chimerism of granulocytes, monocytes, B cells and T cells was equal to that of white blood cells. GL-3 levels were significantly reduced in the lung and heart of animals with a 30% and 50% chimera, respectively. The extent of reduction in these mice was almost identical to that with 100% chimera. In FD mice, reconstitution with 100% donor cells is not required to obtain a therapeutic effect following bone marrow transplantation. These results suggest that a 30% gene correction might be sufficient to reverse disease manifestations in FD.

Tartronate Semialdehyde Reductase Defines a Novel Rate-Limiting Step in Assimilation and Bioconversion of Glycerol in Ustilago maydis

BackgroundGlycerol is a by-product of biodiesel production. Currently, it has limited applications with low bioconversion efficiency to most metabolites reported. This is partly attributed to the poor knowledge on the glycerol metabolic pathway in bacteria and fungi.Methodology/Principal FindingsWe have established a fast screening method for identification of genes that improve glycerol utilization in Ustilago maydis. This was done by comparing the growth rates of T-DNA tagged mutant colonies on solid medium using glycerol as the sole carbon source. We present a detailed characterization of one of the mutants, GUM1, which contains a T-DNA element inserted into the promoter region of UM02592 locus (MIPS Ustilago maydis database, MUMDB), leading to enhanced and constitutive expression of its mRNA. We have demonstrated that um02592 encodes a functional tartronate semialdehyde reductase (Tsr1), which showed dual specificity to cofactors NAD+ and NADP+ and strong substrate specificity and enantioselectivity for D-glycerate. Improved glycerol assimilation in GUM1 was associated with elevated expression of tsr1 mRNA and this could be phenocopied by over-expression of the gene. Glycolipid accumulation was reduced by 45.2% in the knockout mutant whereas introduction of an extra copy of tsr1 driven by the glyceraldehyde phosphate dehydrogenase promoter increased it by 40.4%.Conclusions/SignificanceOur results demonstrate that tartronate semialdehyde reductase (TSR) plays an important role in glycerol assimilation in U. maydis and defines a novel target in genetic engineering for improved conversion of glycerol to higher value products. Our results add significant depth to the understanding of the glycerol metabolic pathway in fungi. We have demonstrated, for the first time, a biological role of a eukaryotic TSR.

Crystal Structure of β-Hexosaminidase B in Complex with Pyrimethamine, a Potential Pharmacological Chaperone

β-Hexosaminidases (β-hex) are a group of glycosyl hydrolase isozymes that break down neutral and sialylated glycosphingolipids in the lysosomes, thereby preventing their buildup in neuronal cells. Some mutants of β-hex have decreased folding stability that results in adult-onset forms of lysosomal storage diseases. However, prevention of the harmful accumulation of glycolipids only requires 10% of wild-type activity. Pyrimethamine (PYR) is a potential pharmacological chaperone that works by stabilizing these mutant enzymes sufficiently to allow more β-hex to arrive in the lysosome, where it can carry out its function. An X-ray structure of the complex between human β-hexosaminidase B (HexB) and PYR has been determined to 2.8 Å. PYR binds to the active site of HexB where several favorable van der Waals contacts and hydrogen bonds are introduced. Small adjustments of the enzyme structure are required to accommodate the ligand, and details of the inhibition and stabilization properties of PYR are discussed.

The pathophysiology of GD – current understanding and rationale for existing and emerging therapeutic approaches

Gaucher disease is a genetic disorder of sphingolipid metabolism resulting from dysfunction of the lysosomal membrane-associated glycoprotein glucocerebrosidase (GBA) and resulting in intracellular accumulation of glucosylceramide and other glycolipids. Although the gene defect and relevant biochemical pathways have been defined, the mechanisms by which substrate accumulation causes disease manifestations are not well understood. The direct effects of a build up of substrate laden cells may account for some aspects of disease but the overall pathology is likely to be more complex with effects of stored material on a variety of intra and extra cellular functions. In this article we review the GBA gene and its protein product, with associated defects, lipid metabolism and storage, enzyme misfolding and endoplasmic reticulum stress, calcium homeostasis, oxidative stress and autophagy and at each point examine how therapies that are currently available, in clinical development or at earlier stages of basic research might address the pathological mechanisms.

Lésions rénales dans la maladie de Fabry

Kidney involvement is one of the main manifestations of Fabry's disease. In the absence of enzyme replacement therapy, hemizygous males and some heterozygous females progress to end stage renal failure. In hemizygous males, diffuse glycolipid accumulation is observed in all glomerular and vascular cells whereas distal tubular cells are focally involved. In heterozygous females, the glycolipid storage is irregular in glomeruli and vessels, some cells being massively involved, others being normal. In both sexes, degenerative changes occur, linked to the necrosis of overloaded mesangial and vascular smooth muscle cells. Their progression leads to unspecific arteriopathy and glomerulosclerosis not prone to reverse under enzymotherapy. Kidney biopsy is useful for confirming the diagnosis if clinical presentation of Fabry's disease is atypical. Moreover, histological analysis of renal tissue allows to assess the severity of degenerative changes and to evaluate the beneficial impact of enzyme replacement therapy.

9 Identifying Np-C Early in Paediatric Patients

Niemann-Pick disease, type C (NP-C) is an autosomal recessive, progressive and ultimately fatal, neurovisceral lysosomal storage disorder involving the accumulation of unesterified cholesterol and glycolipids. Two genes (NPC1 and NPC2) are involved in its pathogenesis. The estimated incidence of NP-C is 1:150,000 live births; however, the true incidence is probably significantly higher, as there is ample evidence that many patients remain undiagnosed. Clinical presentation in NP-C is extremely heterogenous, often resulting in misdiagnosis. The first signs and symptoms may occur from the prenatal period; through to childhood and late adulthood. The absence of a simple diagnostic test for NP-C precludes the inclusion of the disease in general metabolic screening panels. The following signs and symptoms should lead to inclusion of NP-C in a differential diagnosis in paediatric patients: prolonged unexplained neonatal jaundice; hepatosplenomegaly or isolated splenomegaly; and neurological signs including vertical supranuclear gaze palsy, ataxia and/or dystonia, seizures and/or cataplexia, and early and progressive dementia. At present, definitive diagnosis depends on demonstrating impaired cholesterol transport and homeostasis in cultured skin fibroblasts followed by genetic testing for mutations in NPC1 and NPC2. A simple diagnostic tool based on a scoring system for various visceral, neurological and psychiatric signs and symptoms is currently being developed to facilitate decision-making regarding the need for diagnostic biochemical or genetic studies for NP-C. With the recent approval of miglustat (Zavesca®) as disease-modifying therapy for NP-C, treatment can now be aimed toward stabilising neurological disease. Early diagnosis of NPC has, thus, become even more important.

Tissue and plasma globotriaosylsphingosine could be a biomarker for assessing enzyme replacement therapy for Fabry disease

Fabry disease is a genetic disease caused by a deficiency of α-galactosidase A (GLA), which leads to systemic accumulation of glycolipids, predominantly globotriaosylceramide (Gb3). With the introduction and spread of enzyme replacement therapy (ERT) with recombinant GLAs for this disease, a useful biomarker for assessing the response to ERT is strongly required. We measured the tissue level of lyso-globotriaosylsphingosine (lyso-Gb3) in Fabry mice by means of high performance liquid chromatography, and compared it with the Gb3 level. The results revealed a marked increase in the lyso-Gb3 level in most tissues of Fabry mice, and which decreased after the administration of a recombinant GLA as in the case of Gb3, which is usually used as a biomarker of Fabry disease. The response was more impressive for lyso-Gb3 compared with for Gb3, especially in kidney tissues, in which a defect significantly influences the morbidity and mortality in patients with this disease. The plasma level of lyso-Gb3 also decreased after the injection of the enzyme, and it was well related to the degradation of tissue lyso-Gb3. Thus, lyso-Gb3 is expected to be a useful new biomarker for assessing the response to ERT for Fabry disease.

Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases

The human lysosomal enzymes alpha-galactosidase (alpha-GAL, EC 3.2.1.22) and alpha-N-acetylgalactosaminidase (alpha-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of alpha- GAL and alpha-NAGAL. The engineered alpha-GAL (which we call alpha-GAL(SA)) retains the antigenicity of alpha-GAL but has acquired the enzymatic specificity of alpha-NAGAL. Conversely, the engineered alpha-NAGAL (which we call alpha-NAGAL(EL)) retains the antigenicity of alpha-NAGAL but has acquired the enzymatic specificity of the alpha-GAL enzyme. Comparison of the crystal structures of the designed enzyme alpha-GAL(SA) to the wild-type enzymes shows that active sites of alpha-GAL(SA) and alpha-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.