N-butyldeoxygalactonojirimycin reduces neonatal brain ganglioside content in a mouse model of GM1 gangliosidosis

GM1 gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by a genetic deficiency of acid beta-galactosidase (beta-gal), the enzyme that catabolyzes GM1 within lysosomes. Accumulation of GM1 and its asialo form (GA1) occurs primarily in the brain, leading to progressive neurodegeneration and brain dysfunction. Substrate reduction therapy aims to decrease the rate of GSL biosynthesis to counterbalance the impaired rate of catabolism. The imino sugar N-butyldeoxygalactonojirimycin (NB-DGJ) is a competitive inhibitor of the ceramide-specific glucosyltransferase that catalyzes the first step in GSL biosynthesis. Neonatal C57BL/6J (B6) and beta-gal knockout (-/-) mice were injected daily from post-natal day 2 (p-2) to p-5 with either vehicle or NB-DGJ at 600 mg or 1200 mg/kg body weight. These drug concentrations significantly reduced total brain ganglioside and GM1 content in the B6 and the beta-gal (-/-) mice. Drug treatment had no significant effect on viability, body weight, brain weight, or brain water content in the B6 and beta-gal (-/-) mice. Significant elevations in neutral lipids (GA1, ceramide, and sphingomyelin) were observed in the NB-DGJ-treated beta-gal (-/-) mice, but were not associated with adverse effects. Also, NB-DGJ treatment of B6 and beta-gal (-/-) mice from p-2 to p-5 had no subsequent effect on brain ganglioside content at p-21. Our results show that NB-DGJ is effective in reducing total brain ganglioside and GM1 content at early neonatal ages. These findings suggest that substrate reduction therapy using NB-DGJ may be an effective early intervention for GM1 gangliosidosis and possibly other GSL lysosomal storage diseases.     

Substrate reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum in GM1 gangliosidosis mice

II3NeuAc-GgOse4Cer (GM1) gangliosidosis is an incurable lysosomal storage disease caused by a deficiency in acid beta-galactosidase (beta-gal), resulting in the accumulation of ganglioside GM1 and its asialo derivative GgOse4Cer (GA1) in the central nervous system, primarily in the brain. In this study, we investigated the effects of N-butyldeoxygalacto-nojirimycin (N B-DGJ), an imino sugar that inhibits ganglioside biosynthesis, in normal C57BL/6J mice and in beta-gal knockout (beta-gal-/-) mice from postnatal day 9 (p-9) to p-15. This is a period of active cerebellar development and central nervous system (CNS) myelinogenesis in the mouse and would be comparable to late-stage embryonic and early neonatal development in humans. N B-DGJ significantly reduced total ganglioside and GM1 content in cerebrum-brainstem (C-BS) and in cerebellum of normal and beta-gal-/- mice. N B-DGJ had no adverse effects on body weight or C-BS/cerebellar weight, water content, or thickness of the external cerebellar granule cell layer. Sphingomyelin was increased in C-BS and cerebellum, but no changes were found for cerebroside (a myelin-enriched glycosphingolipid), neutral phospholipids, or GA1 in the treated mice. Our findings indicate that the effects of N B-DGJ in the postnatal CNS are largely specific to gangliosides and suggest that N B-DGJ may be an effective early intervention therapy for GM1 gangliosidosis and other ganglioside storage disorders.     

Neurochemical, morphological, and neurophysiological abnormalities in retinas of Sandhoff and GM1 gangliosidosis mice

Retinal abnormalities are well documented in patients with ganglioside storage diseases. The total content and distribution of retinal glycosphingolipids was studied for the first time in control mice and in Sandhoff disease (SD) and GM1 gangliosidosis mice. Light and electron microscopy of the SD and the GM1 retinas revealed storage in ganglion cells. Similar to previous findings in rat retina, GD3 was the major ganglioside in mouse retina, while GM2 and GM1 were minor species. Total ganglioside content was 44% and 40% higher in the SD and the GM1 retinas, respectively, than in the control retinas. Furthermore, GM2 and GM1 content were 11-fold and 51-fold higher in the SD and the GM1 retinas than in the control retinas, respectively. High concentrations of asialo-GM2 and asialo-GM1 were found in the SD and the GM1 retinas, respectively, but were undetectable in the control retinas. The GSL abnormalities in the SD and the GM1 retinas reflect significant reductions in beta-hexosaminidase and beta-galactosidase enzyme activities, respectively. Although electroretinograms appeared normal in the SD and the GM1 mice, visual evoked potentials were subnormal in both mutants, indicating visual impairments. Our findings present a model system for assessing retinal pathobiology and therapies for the gangliosidoses.     

Hepatic storage of oligosaccharides and glycolipids in a cat affected with GM1 gangliosidosis.

1. Glycopeptides and glycolipids were isolated from normal cat liver and liver from a cat affected with GM1 gangliosidosis. 2. Bio-Gel P-6 chromatography of the crude glycopeptide fractions demonstrated three major peaks of hexose-containing compounds that were greatly increased in the mutant liver sample; these peaks contained oligosaccharides that comprised over 2% of the liver wet weight. 3. Two of the major pathological oligosaccharides, GP5 and GP6, were purified by chromatography on charcoal/Celite and Sephadex G-25. Oligosaccharides GP5 and GP6 had apparent mol.wts. of 1800 +/- 200 and 1350+/-200 respectively, and contained galactose, mannose and N-acetylglucosamine in molar proportions of 2.0:3.1:4.1 (GP5) and 1.0:2.2:2.7 (GP6). Periodate oxidation studies demonstrated the presence of galactose in a non-reducing terminal position. 4. The neutral glycolipid fraction from the mutant cat liver has a 1.3-fold increase in hexose content accompanied by an increased concentration of asialo-(ganglioside GM1). 5. There was a 2-fold increase of gangliosides in the mutant cat liver compared with normal cats. Ganglioside GM1 and a compound tentatively identified as N-glycolloyl-(ganglioside GM1) were the major glycolipids accumulated.     

Ganglioside GM1 forces the redistribution of cholesterol in a biomimetic membrane

Neutron reflectivity has been applied to investigate different mixed asymmetric lipid systems, in the form of single "supported+floating" bilayers, made of phospholipids, cholesterol and GM1 ganglioside (Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer)) in bio-similar mole ratios. Bilayer preparation was carried out layer-by-layer with the Langmuir-Blodgett Langmuir-Schaefer techniques, allowing for compositional asymmetry in the system buildup. It is the first time that such a complex model membrane system is reported. Two important conclusions are drawn. First, it is experimentally shown that the presence of GM1 enforces an asymmetry in cholesterol distribution, opposite to what happens for a GM1-free membrane that, submitted to a similar procedure, results in a full symmetrization of cholesterol distribution. We underline that natural cholesterol has been used. Second, and most interesting, our results suggest that a preferential asymmetric distribution of GM1 and cholesterol is attained in a model membrane with biomimetic composition, revealing that a true coupling between the two molecular species occurs.     

Fluorescence Polarization Analysis, Lipid Composition, and Na+, K+-ATPase Kinetics of Synaptosomal Membranes in Feline GM1 and GM2 Gangliosidosis

Neurochemical studies were performed on synaptosomal membranes from cats with GM1 or GM2 gangliosidosis to examine possible mechanisms of neuronal dysfunction in these disorders. The basic hypothesis tested was that deficient ganglioside catabolism causes increased ganglioside content of synaptosomal plasma membrane which in turn disrupts normal function. Fluidity characteristics of synaptosomal membranes were examined using fluorescence polarization. Results showed markedly reduced membrane fluidity in both GM1 and GM2 gangliosidosis. These results were supported by a second study which revealed that isolated synaptosomal membranes of GM1 gangliosidosis cats had a 24-fold increase in total ganglioside content caused predominantly by excess GM1, a 2.3-fold increased cholesterol content, and a 1.4-fold increased phospholipid content. Finally, kinetic analysis of synaptosomal plasma membrane Na+,K+-ATPase from cats with GM1 gangliosidosis showed negligible differences in kinetic parameters compared with controls. Thus, the enzyme appeared protected from the global membrane changes in fluidity and composition. These observations provide evidence for a pathogenetic mechanism of neuronal dysfunction in the gangliosidoses while demonstrating protection of certain vital functional components, such as Na+,K+-ATPase.     

Quantitation of the enzymically deficient cross reacting material in GM1 gangliosidoses.

Normal quantities of GM1 beta-galactosidase cross reacting material (CRM) (0.31-0.47 microgram/mg protein) were detected by a sensitive radial immunodiffusion assay in skin fibroblasts from patients with GM1 gangliosidosis type 1 and adult variants, whereas elevated levels were found in GM1 gangliosidosis type 2 (0.41-0.72 microgram/mg protein). The specific activity of the immunologically CRM towards GM1 ganglioside of normal fibroblasts was about 500 times that of type 1, 100 times that of type 2, and 30 times that of the adult variants.     

Biosynthesis of the major brain gangliosides GD1a and GT1b

Gangliosides-sialylated glycosphingolipids-are the major glycoconjugates of nerve cells. The same four structures-GM1, GD1a, GD1b and GT1b-comprise the great majority of gangliosides in mammalian brains. They share a common tetrasaccharide core (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1'Cer) with one or two sialic acids on the internal galactose and zero (GM1 and GD1b) or one (GD1a and GT1b) α2-3-linked sialic acid on the terminal galactose. Whereas the genes responsible for the sialylation of the internal galactose are known, those responsible for terminal sialylation have not been established in vivo. We report that St3gal2 and St3gal3 are responsible for nearly all the terminal sialylation of brain gangliosides in the mouse. When brain ganglioside expression was analyzed in adult St3gal1-, St3gal2-, St3gal3- and St3gal4-null mice, only St3gal2-null mice differed significantly from wild type, expressing half the normal amount of GD1a and GT1b. St3gal1/2-double-null mice were no different than St3gal2-single-null mice; however, St3gal2/3-double-null mice were >95% depleted in gangliosides GD1a and GT1b. Total ganglioside expression (lipid-bound sialic acid) in the brains of St3gal2/3-double-null mice was equivalent to that in wild-type mice, whereas total protein sialylation was reduced by half. St3gal2/3-double-null mice were small, weak and short lived. They were half the weight of wild-type mice at weaning and displayed early hindlimb dysreflexia. We conclude that the St3gal2 and St3gal3 gene products (ST3Gal-II and ST3Gal-III sialyltransferases) are largely responsible for ganglioside terminal α2-3 sialylation in the brain, synthesizing the major brain gangliosides GD1a and GT1b.     

Membrane cholesterol content plays a key role in the neurotoxicity of β-amyloid: implications for Alzheimer's disease

Beta amyloid (βA) plays a central role in the pathogenesis of the most common and devastating neurodegenerative disorder, Alzheimer's disease (AD). The mechanisms of βA neurotoxicity remain controversial, but include dysregulation of calcium homeostasis and oxidative stress. A large body of data suggest that cholesterol plays a significant role in AD. In mixed cultures containing hippocampal neurons and astrocytes, we have shown that neurotoxic βA peptides (1-42 and 25-35) cause sporadic cytosolic calcium ([Ca(2+) ](c) ) signals in astrocytes but not in neurons, initiating a cascade that ends in neuronal death. We now show, using the cholesterol-sensitive fluorescent probe, Filipin, that membrane cholesterol is significantly higher in astrocytes than in neurons and mediates the selective response of astrocytes to βA. Thus, lowering [cholesterol] using mevastatin, methyl-β-cyclodextrin or filipin prevented the βA-induced [Ca(2+) ](c) signals, while increased membrane [cholesterol] increased βA-induced [Ca(2+) ](c) signals in both neurons and astrocytes. Addition of βA to lipid bilayers caused the appearance of a conductance that was significantly higher in membranes containing cholesterol. Increasing membrane [cholesterol] significantly increased βA-induced neuronal and astrocytic death. We conclude that a high membrane [cholesterol] promotes βA incorporation into membranes and increased [Ca(2+) ](c) leading to cell death.     

Incorporation and degradation of GM1 ganglioside and asialoGM1 ganglioside in cultured fibroblasts from normal individuals and patients with beta-galactosidase deficiency

The uptake and degradation of GM1 ganglioside (GM1) and asialoGM1 ganglioside (GA1) were studied in cultured fibroblasts from normal individuals and patients with beta-galactosidase deficiency, using the lipid-loading test. The glycolipids were incorporated from the media into the fibroblasts and the terminal galactose was hydrolyzed in normal cells. The hydrolysis rates of GA1 were 80-86% of normal on the 3rd day after loading, while GM1 was hydrolyzed slowly; 35-54% on the 14th day. In infantile GM1 gangliosidosis and I-cell disease, little GM1 and GA1 was hydrolyzed on any day of culture, while fibroblasts from patients with adult GM1 gangliosidosis, Morquio disease type B and galactosialidosis hydrolyzed the lipids at nearly normal rates. The intracellular accumulation of the glycolipids, on the basis of protein content, was abnormally high in the case of infantile GM1 gangliosidosis and I-cell disease, but normal in the other disorders examined. These observations indicate that the in situ metabolism of GM1 and GA1 is probably normal in fibroblasts from patients with adult GM1 gangliosidosis, Morquio disease type B and galactosialidosis, although in vitro beta-galactosidase activities in these disorders are very low. The results are compatible with findings that GM1 and GA1 do not accumulate in the somatic organs of patients with adult GM1 gangliosidosis and galactosialidosis. In I-cell disease, however, the results of the loading test did not agree with the finding that there is little accumulation of glycolipids in postmortem tissues.     

Increase in membrane cholesterol: A possible trigger for degradation of HMG CoA reductase and crystalloid endoplasmic reticulum in UT-1 cells

The crystalloid endoplasmic reticulum (ER) houses large amounts of HMG CoA reductase, the rate-controlling enzyme in cholesterol synthesis. The crystalloid ER appears in UT-1 cells, a line of Chinese hamster ovary cells that has been chronically starved of cholesterol as a result of growth in the presence of compactin, an inhibitor of reductase. When cholesterol was provided to UT-1 cells in the form of low density lipoprotein (LDL), the reductase and crystalloid ER were destroyed. This destruction was preceded by an increase in the cholesterol content of crystalloid ER membranes, as judged by a 4- to 8-fold increase in their ability to form complexes with filipin, a cholesterol-binding compound that can be visualized in freeze-fracture electron micrographs. Filipin binding to other membranes was unchanged. Thus insertion of cholesterol into the crystalloid ER membrane may trigger the degradation of reductase and the membrane itself.     

Physiologically-relevant levels of sphingomyelin,but not GM1, induces a β-sheet-rich structure in the amyloid-β(1-42) monomer

To resolve the contribution of ceramide-containing lipids to the aggregation of the amyloid-β protein into β-sheet rich toxic oligomers, we employed molecular dynamics simulations to study the effect of cholesterol-containing bilayers comprised of POPC (70% POPC, and 30% cholesterol) and physiologically relevant concentrations of sphingomyelin (SM) (30% SM, 40% POPC, and 30% cholesterol), and the GM1 ganglioside (5% GM1, 70% POPC, and 25% cholesterol). The increased bilayer rigidity provided by SM (and to a lesser degree, GM1) reduced the interactions between the SM-enriched bilayer and the N-terminus of Aβ42 (and also residues Ser26, Asn27, and Lys28), which facilitated the formation of a β-sheet in the normally disordered N-terminal region. Aβ42 remained anchored to the SM-enriched bilayer through hydrogen bonds with the side chain of Arg5. With β-sheets in the at the N and C termini, the structure of Aβ42 in the sphingomyelin-enriched bilayer most resembles β-sheet-rich structures found in higher-ordered Aβ fibrils. Conversely, when bound to a bilayer comprised of 5% GM1, the conformation remained similar to that observed in the absence of GM1, with Aβ42 only making contact with one or two GM1 molecules. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

Regulatory role of β-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of β-arrestin-2 (βarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that βarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged βarr2 (βarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The βarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This βarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting βarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/βarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for βarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of βarr2 expression on Niemann-Pick type C-1 (NPC1)-containing organelle movement is proposed as the mechanism of βarr2-mediated alterations on cholesterol processing. It is concluded that βarr2 expression contributes to altered cholesterol trafficking observed in CF cells.     

A mouse embryonic stem cell line showing pluripotency of differentiation in early embryos and ubiquitous β-galactosidase expression

For analysis of chimeric mice made by injecting embryonic stem (ES) cells into host blastocysts, it is very desirable if the ES cells have a good cell marker that can distinguish them from host cells. It is ideal if the marker can be easily visualized in every type of cell and tissue throughout the embryogenesis. We tried to produce such ES cell lines by introducing an E. coli β-galactosidase (β-gal) gene construct by electroporation. One of the transformant lines (MS1-EL4) showed β-gal activity in every undifferentiated stem cell. After being induced to differentiate in vitro, cells with various morphologies showed β-gal activity. We also detected β-gal activity in a wide variety of tissue elements in solid tumors made by injecting the MS1-EL4 cells into syngeneic mice. Then we produced chimeric embryos by injecting the MS1-EL4 cells into blastocysts and recovering the embryos at various developmental stages. We found that the MS1-EL4 cells contributed to various tissues and expressed β-gal activity, including not only descendants of the inner cell mass but also the trophectoderm-derived extraembryonic ectoderm.     

GM1 gangliosidosis and Morquio B disease: An update on genetic alterations and clinical findings

GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000-1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation. Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses. Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes showed that all the amino acid replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease.     

Genotypic and phenotypic characterization of Brazilian patients with GM1 gangliosidosis

GM1 gangliosidosis is a lysosomal disorder caused by β-galactosidase deficiency due to mutations in the GLB1 gene. It is a rare neurodegenerative disorder with an incidence of about 1:100,000–1:200,000 live births worldwide. Here we review GLB1 mutations and clinical features from 65 Brazilian GM1 gangliosidosis patients. Molecular analysis showed 17 different mutations and c.1622–1627insG was the most frequent, accounting for 50% of the alleles. Cognitive impairment was the main clinical sign, observed in 82% of patients, followed by hepatosplenomegaly observed in 56% of patients. It was possible to establish a significant correlation between age at onset of symptoms preceding the first year of life and the presence of the mutation c.1622–1627insG (p = 0.03). Overall our findings differ from literature and represent the exclusive genotypic profile found in Brazilian GM1 gangliosidosis patients.     

Accumulation of Lysosphingolipids in Tissues from Patients with GM1 and GM2 Gangliosidoses

By using a sensitive method, we assayed lysocompounds of gangliosides and asialogangliosides in tissues from four patients with GM2 gangliosidosis (one with Sandhoff disease and three with Tay-Sachs disease) and from three patients with GM1 gangliosidosis [one with infantile type (fetus), one with late-infantile, and one with adult type]. In the brain and spinal cord of all the patients except for an adult GM1 gangliosidosis patient, abnormal accumulation of the lipids was observed, though the concentration in the fetal tissue was low. In GM2 gangliosidosis, the amounts of lyso GM2 ganglioside accumulated in the brain were similar among the patient with Sandhoff disease and the patients with Tay-Sachs disease, whereas the concentration of asialo lyso GM2 ganglioside in the brain was higher in the former patient than in the latter patients. By comparing the sphingoid bases of neutral sphingolipids, gangliosides, and lysosphingolipids, it was suggested that lysosphingolipids in the diseased tissue are synthesized by sequential glycosylation from free sphingoid bases, but not by deacylation of the sphingolipids. Because lysosphingolipids are known to be cytotoxic, the abnormally accumulated lysophingolipids may well be the pathogenetic agent for the neuronal degeneration in gangliosidoses.     

AAV-mediated gene delivery in adult GM1-gangliosidosis mice corrects lysosomal storage in CNS and improves survival

Background

GM1-gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by a genetic deficiency of acid β-galactosidase (βgal), which results in the accumulation of GM1-ganglioside and its asialo-form (GA1) primarily in the CNS. Age of onset ranges from infancy to adulthood, and excessive ganglioside accumulation produces progressive neurodegeneration and psychomotor retardation in humans. Currently, there are no effective therapies for the treatment of GM1-gangliosidosis.

Methodology/Principal Findings

In this study we examined the effect of thalamic infusion of AAV2/1-βgal vector in adult GM1 mice on enzyme distribution, activity, and GSL content in the CNS, motor behavior, and survival. Six to eight week-old GM1 mice received bilateral injections of AAV vector in the thalamus, or thalamus and deep cerebellar nuclei (DCN) with pre-determined endpoints at 1 and 4 months post-injection, and the humane endpoint, or 52 weeks of age. Enzyme activity was elevated throughout the CNS of AAV-treated GM1 mice and GSL storage nearly normalized in most structures analyzed, except in the spinal cord which showed ∼50% reduction compared to age-matched untreated GM1 mice spinal cord. Survival was significantly longer in AAV-treated GM1 mice (52 wks) than in untreated mice. However the motor performance of AAV-treated GM1 mice declined over time at a rate similar to that observed in untreated GM1 mice.

Conclusions/Significance

Our studies show that the AAV-modified thalamus can be used as a ‘built-in’ central node network for widespread distribution of lysosomal enzymes in the mouse cerebrum. In addition, this study indicates that thalamic delivery of AAV vectors should be combined with additional targets to supply the cerebellum and spinal cord with therapeutic levels of enzyme necessary to achieve complete correction of the neurological phenotype in GM1 mice.     

人β-半乳糖苷酶R299L突变体的获得与活性研究*

目的: GM1神经节苷脂贮积症是一种由半乳糖苷酶beta 1(galactosidase beta 1, GLB1)基因突变引起的β-半乳糖苷酶(β-galactosidase,β-gal)活性降低导致的严重的溶酶体贮积病。该病以进行性、致命性神经退行性病变为特征,目前尚无有效的治疗手段,AAV载体介导的基因治疗被认为是最有希望的治疗方法。通过基因定点突变获得具有较高β-gal活性的GLB1突变体,以期用于后续AAV介导的基因治疗。方法: 对人类和其他6种脊椎动物GLB1基因进行多序列比对分析,筛选出部分氨基酸位点进行定点突变,采用携带突变位点的重组质粒和AAV9载体转染或感染HEK-293细胞,比较突变体与未突变体的活性差异。对GM1模型鼠注射携带coGLB1-R299L的rAAV9病毒,探究该突变体的体内活性表达。结果: 从15个突变体中筛选出coGLB1-R299L突变体,经质粒转染导入细胞后,其β-gal活性比具有野生型氨基酸序列的coGLB1增加了30%~40%。AAV体外感染实验中,rAAV9-coGLB1-R299L组的β-gal活性较未感染的细胞对照组提升了约2.2倍。体内结果显示,rAAV9-coGLB1-R299L在模型鼠体内广泛表达,心脏、肝脏、脾脏、肺、脑组织中β-gal活性显著提升。结论: 获得了具有更高β-gal活性的突变体coGLB1-R299L,初步探究了rAAV9-coGLB1-R299L的体外表达效果和模型鼠体内β-半乳糖苷酶的表达与分布,为该突变体应用于AAV介导的GM1神经节苷脂病治疗奠定基础。     

Proteomic of lipid rafts in the exocrine pancreas from diet-induced obese rats

In the present work, we induced obesity in rats with high-energy-starch diet and studied exocrine pancreas response. The zymogen granule (ZG) or purified plasma membrane (PM) from the exocrine pancreas was used for the isolation of the detergent-resistant membranes (DRMs). Based on high content of cholesterol, GM1, the bile salt dependent lipase (BSDL), and GP2 enrichment, the low-density fractions were defined as lipid rafts. Additionally, the rafts vesicles were determined by immunogold labeling with anti BSDL. By combining MALDI-TOF/MS and nano-LC ESI Q-TOF MS/MS proteomic identification we have selected 33 proteins from the lipid rafts which were classified into at least four functional families. Our data suggest that the acinar PM from the diet-induced obesity rats may be organized into lipid rafts, and characterization of rafts proteome can contribute to improve our understanding of food digestion under obesity.