X-Linkage of Human α-Galactosidase

A deficiency in α-galactosidase (α-Gal) activity–as measured aspecifically with the use of artificial substrates–is a regular feature in leucocytes and fibroblasts of patients affected by angiokeratoma corporis diffusum or Fabry's disease, a well-known X-linked trait in man^1–4. Fibroblast clones derived from mothers of affected males exhibit either normal or deficient activity of α-galactosidase. This demonstrates that the deficiency of α-galactosidase is caused by an X-linked mutation, but does not necessarily prove that the structural locus for this enzyme is itself located on the X-chromosome.     

Residual activity of α-galactosidase A in Fabry's disease

The α-galactosidase A activity from fibroblasts of five Fabry patients and five controls has been separated from α-galactosidase B through small DEAE-cellulose columns and in some experiments by treatment of the fibroblast extracts with Sepharose coupled to anti-α-galactosidase B antibodies. By these independent methods, it has been shown that there is a residual α-galactosidase A in Fabry's disease, which is immunologically similar to the α-galactosidase A from the controls. The α-galactosidase A from all of the patients and controls has the same apparent K_m value for the synthetic substrate 4-methylumbelliferyl-α-galactoside. Four out of five patients have a thermostable α-galactosidase A, while the fifth has a thermolabile enzyme like that from the controls. The amount of immunologically active α-galactosidase A seems to be decreased in the patients tested.     

Molecular pathology of Fabry's disease physical and kinetic properties of α-galactosidase a in cultured human endothelial cells

Fabry's disease (α-galactosidase A deficiency) is characterized by the progressive lysosomal accumulation of trihexosyl ceramide primarily in the vascular endothelium. The pathophysiologic mechanisms responsible for this preferential site of deposition were investigated in primary endothelial cell cultures established from veins of Fabry hemizygotes and umbilical veins from normal newborns. Morphologically, cultured endothelial cells from Fabry hemizygotes were strikingly different from normal cultured cells; they contained numerous, large, birefringent, cytoplasmic inclusions, presumably substrate-engorged lysosomes, observable by phase-contrast microscopy. The physical and kinetic properties of α-galactosidase A activity in normal cultured endothelial cells were determined, including (1) thermal inactivation at 50°C, $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 15}\text{min}$; (2) elution profile on DEAE-cellulose; (3) pH optimum, 4.7; (4) specific activity, 349 nmol/h per mg protein; and (5) kinetics, apparent K_m = 2.6 mM with the 4-methylumbelliferyl substrate. These results were similar to the α-galactosidase A properties reported for other enzyme sources. In addition, preliminary glycosphingolipid analysis demonstrated relatively low levels of trihexosyl ceramide in normal cultured endothelial cells. Based on these results, a mechanism for the molecular pathology of Fabry's disease was postulated consistent with previously reported findings. It is suggested that in Fabry hemizygotes the accumulated, circulating trihexosyl ceramide may gain access to the lysosomal apparatus of the vascular endothelium by receptor-mediated lipoprotein uptake, where it is accumulated due to the defective α-galactosidase A activity.     

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.     

Tissue distribution of glycosphingolipids in a case of Fabry's disease

A survey was made of the glycolipid composition of various tissues, including liver, spleen, kidney (cortex and medulla), lymph node, pancreas, prostate gland, heart muscle, thenar muscle, gastrointestinal smooth muscle, frontal cerebral cortex, anterior thalamus, brain stem, a peripheral autonomic ganglion, and renal arterial intima and media, from a patient who died with Fabry's disease. The tissues had been fixed in formalin for 3 yr. Analytical data on trihexosyl ceramide from heart muscle and pancreas indicate a structure identical to trihexosyl ceramide from kidney: galactosylgalactosylglucosyl ceramide. Fatty acid compositions of trihexosyl ceramide and dihexosyl ceramide revealed a wide range of fatty acids, with 16:0, 18:0, 20:0, 22:0, 24:0, and 24:1 predominating. These glycolipids comprised 10-41% of the total lipid in the formalin-fixed organs studied. Trihexosyl ceramide predominated in all tissues and was the only glycolipid found in muscle tissues, lymph node, and arterial tissues. Dihexosyl ceramide was found in kidney, pancreas, liver, spleen, and cerebral tissues. The accumulation of trihexosyl ceramide in cardiac muscle and arterial tissues may account in part for the cardiovascular complications so prominent in Fabry's disease.     

Neurochemical abnormality in I-cell disease: chemical analysis and a possible importance of beta-galactosidase deficiency.

Sphingolipid composition in both gray and white matter of a patient with I-cell disease was normal except for the higher proportion.of G_MI-ganglioside in gray and white matter. In the patient's liver and kidney there was a significant accumulation of ceramide dihexoside and ceramide trihexoside and of sulphatide in kidney. Non-lipid hexosamine and sialic acid concentration in brain was increased 1.2-1.5 times above normal. Recovery of myelin from I-cell's white matter was 80-100%, suggesting that demyelination, if present, is minimal. Myelin lipid and myelin specific glycoprotein patterns were normal. Except for β-galactosidase activity the activity of other brain lysosomal enzymes were within the normal range. This finding was similar to that of Hurler's syndrome. Only β-galactosidase activity was reduced to less than 10% of normal in the patient's brain. To examine the possible metabolic significance of β-galactosidase deficiency in I-cell disease the physical characteristics of this enzyme, isolated from tissues from I-cell, Hurler and control patients, were compared using isoelectric focusing, Con A-Sepharose and Sephadex G-150 chromatography. The isoelectric point and the binding affinity of I-cell β-galactosidase with Con A-Sepharose was comparable to normal. However, the isoenzyme patterns of brain and liver I-cell β-galactosidase with Sephadex G-150 gel filtration revealed decreased acid β-galactosidase. Effects of the addition of sodium chloride on each fraction of β-galactosidase isoenzymes isolated from I-cell tissues were markedly different from controls, whereas the pH optimum of these enzymes were similar to normal. These enzyme characteristics in I-cell tissues were different from normal and Hurler's syndrome. These findings suggest that β-galactosidase deficiency in I-cell disease is a more specific phenomenon rather than secondary inhibition as found in the mucopolysaccharidoses and thus may have an important role for the pathogenesis of brain damage and disease occurrence.     

Binding of soybean agglutinin to glycolipid components of porcine lymphocyte plasma membranes

¹²⁵I-Labeled soybean agglutinin binds primarily to glycolipids contained in pig lymphocyte plasma membranes as measured by in situ “staining” of membranes subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Separation of these glycolipids by differential extraction, silicic acid chromatography, and high-performance thin-layer chromatography showed that three different species of plasma membrane glycolipid bind this lectin; trihexosyl ceramide, globoside, and ganglioside G_M2 in order of increasing affinity (over a range of 10- to 20-fold). Trihexosyl ceramide and globoside, major neutral membrane glycolipids, are the major binders; while G_M2, a minor acidic glycolipid, is a quantitatively smaller lectin-binding component.     

Enzymic hydrolysis of sphingolipids: Hydrolysis of ceramide lactoside by an enzyme from rat brain

Ceramide lactoside [1-O-(galactosido-4-β-glucosido)-2-N-acyl-sphingosine] was hydrolysed to ceramide glucoside and galactose by β-galactosidase of rat brain. The reaction was not reversible, required cholate or taurocholate, had optimum pH5·0 and K_m 2·2×10⁻⁵m. It was inhibited by γ-galactonolactone and galactose as well as by ceramide, sphingosine and fatty acid. Ceramide lactoside could be degraded to ceramide, galactose and glucose by mixtures of rat-brain β-galactosidase and ox-brain β-glucosidase.     

The differentiation of α- and β -glucosidase and α- and β-galactosidase isoenzymes from maize and broad bean root tips using disc electrophoresis in polyacrylamide gels

For the separation of α- and β-glucosidase and α- and β-galactosidase isoenzymes fromZea mays L. andVicia fabaL. root tips the system of disc electrophoresis in polyacrylamide gel developed for basic protein separation proved most suitable. The detection was carried out by a simultaneous azocoupling reaction. In maize α-glucosidase was not detected, β-glucosidase gave 3, α-galactosidase 4, and β-galactosidase 3 zones. In broad bean a- and β-glucosidases were absent, α-galactosidase gave 2 and β-galactosidase 3 zones, α- and β-galactosidase activity zones correspond principially to each other in their position. In maize one zone gives a positive reaction for both β-glucosidase and α- and β-galactosidaso.     

Decreased caveolae in AGPAT2 lacking adipocytes is independent of changes in cholesterol or sphingolipid levels: A whole cell and plasma membrane lipidomic analysis of adipogenesis

BackgroundAdipocytes from lipodystrophic Agpat2^?/? mice have impaired adipogenesis and fewer caveolae. Herein, we examined whether these defects are associated with changes in lipid composition or abnormal levels of caveolae-associated proteins. Lipidome changes were quantified in differentiated Agpat2^?/? adipocytes to identify lipids with potential adipogenic roles.MethodsAgpat2^?/? and wild type brown preadipocytes were differentiated in vitro. Plasma membrane was purified by ultracentrifugation. Number of caveolae and caveolae-associated proteins, as well as sterol, sphingolipid, and phospholipid lipidome were determined across differentiation.ResultsDifferentiated Agpat2^?/? adipocytes had decreased caveolae number but conserved insulin signaling. Caveolin-1 and cavin-1 levels were equivalent between Agpat2^?/? and wild type adipocytes. No differences in PM cholesterol and sphingolipids abundance were detected between genotypes. Levels of phosphatidylserine at day 10 of differentiation were increased in Agpat2^?/? adipocytes. Wild type adipocytes had increased whole cell triglyceride, diacylglycerol, phosphatidylglycerol, phosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, and trihexosyl ceramide, and decreased 24,25-dihydrolanosterol and sitosterol, as a result of adipogenic differentiation. By contrast, adipogenesis did not modify whole cell neutral lipids but increased lysophosphatidylcholine, sphingomyelin, and trihexosyl ceramide levels in Agpat2^?/? adipocytes. Unexpectedly, adipogenesis decreased PM levels of main phospholipids in both genotypes.ConclusionIn Agpat2^?/? adipocytes, decreased caveolae is not associated with changes in PM cholesterol nor sphingolipid levels; however, increased PM phosphatidylserine content may be implicated. Abnormal lipid composition is associated with the adipogenic abnormalities of Agpat2 ?/? adipocytes but does not prevent insulin signaling.     

Purification an α-galactosidase from Coriolus versicolor with acid-resistant and good degradation ability on raffinose family oligosaccharides

An acid-tolerant α-galactosidase (CVGI) was isolated from the fruiting bodies of Coriolus versicolor with a 229-fold of purification and a specific activity of 398.6 units mg^?1. It was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The purified enzyme gave a single band corresponding to a molecular mass of 40 kDa in SDS-PAGE and gel filtration. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The optimum temperature and pH of the enzyme were determined as 60 °C and 3.0, respectively. The enzyme was very stable at a temperature range of 4–50 °C and at a pH range of 2–5. Among the metal ions tested, Cu²⁺, Cd²⁺ and Hg²⁺ ions have been shown to partially inhibit the activity of α-galactosidase, while the activity of CVGI was completely inactivated by Ag⁺ ions. N-bromosuccinamide inhibited enzyme activity by 100 %, indicating the importance of tryptophan residue(s) at or near the active site. CVGI had wide substrate specificity (p-nitrophenyl galactoside, melidiose, raffinose and stachyose). After treatment with CVGI, raffinose family oligosaccharide was hydrolyzed effectively to yield galactose and sucrose. The results showed that the general properties of the enzyme offer potential for use of this α-galactosidase in several production processes.     

Characterization of lysosomal hydrolases that are elevated in Gaucher's disease.

Although Gaucher's disease occurs in three distinct forms with greatly varying degrees of severity, there is no correlation between the clinical course of the disease and levels of residual glucocerebrosidase, the fundamental enzymatic deficiency. In an effort to study secondary changes which might contribute to the pathology of Gaucher's disease, homogenates of spleen, liver, and brain tissue, as well as serum from patients with Gaucher's disease were analyzed for their content of a number of lysosomal enzymes. Extracts of 8 Gaucher spleens contained 3- to 4-fold increases in acid phosphatase activity as well as 5-to 10-fold increases in galactocerebrosidase⁵ activity. The marked elevation in galactocerebrosidase activity in Gaucher spleen was documented using various [³H]galactose labeled galactocerebrosides as substrates and with [³H]galactose labeled lactocerebroside under the “lactosylceramidase I”⁵ assay conditions established by Suzuki (Tanaka, H., and Suzuki, K., 1975, J. Biol. Chem., 250, 2324–2332) that measure galactocerebrosidase activity specifically in the presence of G_mi-ganglioside β-galactosidase. Acid phosphatase determinations using extracts of liver from a case of infantile, neuropathic Gaucher's disease revealed a 2-fold elevation in this activity, whereas brain acid phosphatase activity in this case was similar to that of control tissue. Separation of hexosaminidase A and B activities on DEAE-Sephadex columns indicated increases in both forms of the enzyme in Gaucher tissue with the major increase occurring in the hexosaminidase B component. Glucuronidase and nonspecific esterase were observed to be elevated approximately 2-fold. However, not all lysosomal enzyme activities were increased. Levels of splenic arylsulfatase A and B, α-arabinosidase, sphingomyelinase, α-mannosidase, and G_mi-ganglioside β-galactosidase activities in Gaucher spleen were unremarkable. G_mi-ganglioside β-galactosidase was measured using 4-methylumbelliferyl-β-d-galactopyranoside and [³H]galactose labeled lactocerebroside under the specific assay conditions described by Suzuki for the determination of “lactosylceramidase II” activity. Although levels of arylsulfatase A and B in Gaucher spleen were similar to those of control tissue, arylsulfatase A activity was markedly reduced (20% of control) in homogenates of brain from the case of infantile (type 2) Gaucher's disease. The metabolic and pathologic consequences of these changes in lysosomal enzymes in Gaucher's disease are discussed.     

Enzymatic synthesis of novel oligosaccharides from N-acetylsucrosamine and melibiose using Aspergillus niger α-galactosidase,and properties of the products

Two kinds of oligosaccharides, N-acetylraffinosamine (RafNAc) and N-acetylplanteosamine (PlaNAc), were synthesized from N-acetylsucrosamine and melibiose using the transgalactosylation activity of Aspergillus niger α-galactosidase. RafNAc and PlaNAc are novel trisaccharides in which d-glucopyranose residues in raffinose (Raf) and planteose are replaced with N-acetyl-d-glucosamine. These trisaccharides were more stable in acidic solution than Raf. RafNAc was hydrolyzed more rapidly than Raf by α-galactosidase of green coffee bean. In contrast, RafNAc was not hydrolyzed by Saccharomyces cerevisiae invertase, although Raf was hydrolyzed well by this enzyme. These results indicate that the physicochemical properties and steric structure of RafNAc differ considerably from those of Raf.     

The α-galactosidase from Escherichia coli K12

Growth of Escherichia coli on melibiose requires the induced synthesis of α-galactoside permease and α-galactosidase. Hydrolysis of the chromogenic substrate p-nitrophenyl-σ-galactoside by whole bacteria is dependent on intact oxidative metabolism. The α-galactosidase from E. coli was isolated for the first time as a soluble enzyme. In cell-free extracts p-nitrophenyl-α-galactoside hydrolisis was observed only at high protein concentrations and the activity decreased exponentially with the square of the dilution. The reason for this behaviour was shown to be that, unlike other known α-galactosidases, the enzyme of E. coli requires NAD. For optimal activity the enzyme also requires Mn²⁺, a high concentration of 2-mercaptoethanol, and a pH of 8.1. The approximate molecular weight of the active from of α-galactosidase as determined by sedimentation in a sucrose gradient is 200 000. Due to the instability of the enzyme, its purification has not been achieved.     

Ca modulating α-synuclein membrane transient interactions revealed by solution NMR spectroscopy

α-Synuclein is involved in Parkinson's disease and its interaction with cell membrane is crucial to its pathological and physiological functions. Membrane properties, such as curvature and lipid composition, have been shown to affect the interactions by various techniques, but ion effects on α-synuclein membrane interactions remain elusive. Ca^2 + dynamic fluctuation in neurons plays important roles in the onset of Parkinson's disease and its influx is considered as one of the reasons to cause cell death. Using solution Nuclear Magnetic Resonance (NMR) spectroscopy, here we show that Ca^2 + can modulate α-synuclein membrane interactions through competitive binding to anionic lipids, resulting in dissociation of α-synuclein from membranes. These results suggest a negative modulatory effect of Ca^2 + on membrane mediated normal function of α-synuclein, which may provide a clue, to their dysfunction in neurodegenerative disease.     

Mass spectrometric analysis of permethylated glycosphingolipids II. Comparative studies on different blood-group active and related erythrocyte membrane glycosphingolipids

Three isomeric ceramide tetrasaccharides — P blood-group active globoside, lacto-N-neotetraosyl ceramide as ABH blood-group precursor, both isolated from human erythrocytes and “asiologanglioside” from human brain as reference standard — and two ceramide pentasaccharides — H blood-group active glycosphingolipid, obtained from blood-group B active ceramide hexasaccharide of human B erythrocytes after α-galactosidase treatment and ceramide pentasaccharide from rabbit erythrocytes with B-like blood-group activity — were investigated by mass spectrometry after permethylation. The carbohydrate moiety exhibits differences not only concerning the sugar sequence but also with regard to the position of some glycosidie linkages: Oligosaccharides containing N-acetylhexosamine substituted at position 4 produce spectra that are distinctly different from those containing C-3 substituted N-acetylhexosamines, thus allowing the differentiation between type 1 and type 2 carbohydrate chains. Moreover, oligosaccharide ions with a hexose at the cleavage site exhibit a fragmentation pattern different from those with a N-acetylhexosamine at the “reducing terminal”. The intensity ratio between parent ion and parent ion — 32 mass units is Q ? 3 in the first case, whereas in the latter case Q is <1. The Q-values are given for 14 oligosaccharide ions. Differences in the composition of the ceramide residues can also be deduced from the mass spectra.     

Calcium accelerates SNARE-mediated lipid mixing through modulating α-synuclein membrane interaction

α-Synuclein is involved in Parkinson's disease, and its interaction with cell membrane is vital to its pathological and physiological functions. We have shown that Ca²⁺ can regulate α-synuclein membrane interaction, but the physiological role of Ca²⁺ in modulating α-synuclein membrane interaction is still unexplored. Based on the previous findings that α-synuclein inhibits membrane fusion and its inhibitory effect is highly related to its membrane binding, here we employed solution state Nuclear Magnetic Resonance (NMR) spectroscopy and the ensemble fluorescence fusion assay to show that Ca²⁺ can modulate the inhibitory effect of α-synuclein on SNARE-mediated membrane fusion through disrupting α-synuclein membrane interaction, resulting in acceleration of SNARE-mediated membrane fusion. These results suggest a modulatory effect of Ca²⁺ on membrane mediated normal function of α-synuclein, which of importance for the study of the Parkinson's disease.     

Regulation of α-galactosidase activity and the hydrolysis of galactomannan in the endosperm of the fenugreek (Trigonella foenum-graecum L.) seed

When endosperms were isolated from fenugreek seeds 5 h after sowing and incubated in a small volume of water, the development of α-galactosidase activity and the breakdown of the galactomannan storage polysaccharide were both inhibited relative to control endosperms incubated in larger volumes. The inhibition could be relieved by pre-washing the endosperms, and reimposed by the wash-liquors. If the endosperms were isolated 24 h after sowing, no inhibition was observed. Removal of the embryonic axis from germinating fenugreek seeds and from germinated seedlings also inhibited the development of α-galactosidase activity and galactomannan breakdown in the endosperms; the inhibition was more pronounced the earlier the axis was removed. Axis excision 5 h after sowing caused a delay in the onset of galactomannan breakdown and of the appearance of α-galactosidase activity in the endosperms. It also led to a decrease in the rates of galactomannan breakdown and α-galactosidase production. Axis excision 24 h after sowing caused only a slowing of the rates of galactomannan breakdown and α-galactosidase increase. The inhibition caused by axis removal at 5 h could be relieved partially by gibberellin (10^-4 M), benzyladenine (10^-5 M), mixtures of these and by the herbicide SAN 9789 [4-chloro-5-(methylamine)-2-(α,α,α-trifluoro-m-tolyl)-3-(2H)-pyridazinone]. These substances had no effect on the inhibition caused by axis-removal at 24 h. Excision of the cotyledons at 5 h-leaving the separated axis and the endosperm-also caused inhibition of galactomannan breakdown and α-galactosidase development. The results are consistent with the presence in the fenugreek seed endosperm of diffusible inhibitors of galactomannan mobilisation which are removed or inactivated during normal germination and early seedling development. They are also consistent with a role for the seedling axis in the control of galactomannan breakdown in the endosperm. Initially the axis appears to have a regulatory function (via gibberellins and/or cytokinins?) in determining the onset of α-galactosidase production in the endosperm. Thereafter its continued presence is necessary to ensure maximal rates of α-galactosidase production and galactomannan hydrolysis. The role of the axis may be initially to counteract the endogenous inhibitors in the endosperm and then to act as a sink for the galactomannan breakdown products released in the endosperm and taken up by the cotyledons.     

The properties of alpha-galactosidase remaining in kidney and liver of patients with Fabry's disease

α-Galactosidase activity is diminished in the kidney and liver of patients with Fabry's disease. Less than 2% of the normal activity was found in their kidney, while more than 20% of the normal activity was retained in their liver. The residual enzyme in these two organs showed a single component of pI 4.5 with activity toward 4-methylumbelliferyl α-galactoside on isoelectric focusing. This component seemed to correspond to Fr. II of normal liver or kidney. Ceramide trihexosidase activity was observed as a single component in the same fractions as the α-galactosidase activity for the synthetic substrate.In normal liver, 4-methylumbelliferyl α-galactoside hydrolase was separated into four components with pI's of 4.9, 4.5, 4.2 and 3.9 by isoelectric focusing. Fr. II with pI 4.5 differed from Fr. I in its heat stability and inhibition by myoinositol. In spite of some dissimilarities in their properties, the ratios of enzyme activities for ceramide trihexoside and 4-methylumbelliferyl α-galactoside were similar in all the components of both normal liver and kidney.