Cerebroside Sulfate Research Articles

Synthesis and structure-activity relationships of cerebroside analogues as substrates of cerebroside sulphotransferase and discovery of a competitive inhibitor

Metachromatic leukodystrophy (MLD) is a rare genetic disease characterised by a dysfunction of the enzyme arylsulphatase A leading to the lysosomal accumulation of cerebroside sulphate (sulphatide) causing subsequent demyelination in patients. The enzyme galactosylceramide (cerebroside) sulphotransferase (CST) catalyses the transfer of a sulphate group from 3′-phosphoadenosine-5'-phosphosulphate (PAPS) to cerebrosides producing sulphatides. Substrate reduction therapy for arylsulphatase A by inhibition of CST was proposed as a promising therapeutic approach. To identify competitive CST inhibitors, we synthesised and investigated analogues of the substrate galactosylceramide with variations at the anomeric position, the acyl substituent and the carbohydrate moiety, and investigated their structure–activity relationships. While most of the compounds behaved as substrates, α-galactosylceramide 16 was identified as the first competitive CST inhibitor. Compound 16 can serve as a new lead structure for the development of drugs for the treatment of this devastating disease, MLD, for which small molecule therapeutics are currently not available.

Metachromatic leukodystrophy associated with choledochal cysts and gallbladder papillomatosis

Abstract Metachromatic Leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by the deficiency of the enzyme arylsulfatase A which is responsible for the desulfation of cerebroside sulfate, a myelin glycolipid. Accumulation of these sulfatides in the macrophages of various tissues causes a wide spectrum of presentations including central and peripheral nervous system dysfunction and gallbladder abnormalities. We report a case of a 6-year old girl with infantile Metachromatic Leukodystrophy who was found to have elevated liver enzymes, biliary markers and total and direct bilirubin during work-up for unexplained high-grade fever. Imaging showed dilated intra and extra hepatic biliary tree, markedly expanded CBD by dense content and multiple variable sized filling defects with narrowing in between. The dense content was confirmed to be mucosal papillomatosis with hyperplastic epithelium. To the authors' knowledge, obstructive extra-hepatic biliary tree polypoid masses and cystic dilation with metachromatic leukodystrophy have not previously been reported.

Cerebral Spinal Fluid levels of Cytokines are elevated in Patients with Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease resulting from a deficiency of arylsulfatase A causing an accumulation of cerebroside sulfate, a lipid normally abundant in myelin. Sulfatide accumulation is associated with progressive demyelination and a clinical presentation in severe disease forms that is dominated by motor manifestations. Cerebral inflammation may contribute to the pathophysiology of MLD. To date, cytokine levels in the cerebral spinal fluid of MLD patients have not previously been reported. The objective of this study was to evaluate the concentration of inflammatory cytokines in the CSF of patients with MLD and to compare these levels to unaffected controls. Of 22 cytokines evaluated, we documented significant elevations of MCP-1, IL-1Ra, IL-8, MIP-1b and VEGF in the MLD patients compared to unaffected controls. The elevated cytokines identified in this study may play a significant role in the pathophysiology of MLD. Better understanding of the inflammatory and neurodegenerative process of MLD may lead to improved targeted therapies.

Determination of arylsulfatase A pseudodeficiency allele and haplotype frequency in the Tunisian population.

Arylsulfatase A (ASA) is a lysosomal enzyme involved in the catabolism of cerebroside sulfate. ASA deficiency is associated with metachromatic leukodystrophy (MLD). Low ASA activities have also been reported in a more common condition with no apparent clinical consequences termed ASA pseudo-deficiency (ASA-PD) which is associated with two linked mutations in the ASA gene (c.1049A>G and c.*96A>G). This study aimed to investigate the frequency of the two ASA-PD variants and their linkage disequilibrium (LD) among Tunisians. ASA-PD variants were detected in 129 healthy Tunisians and their frequencies were compared to those described worldwide. The frequency of the PD allele was estimated at 17.4% for the overall sample, with c.1049A>G and c.*96A>G frequencies of 25.6 and 17.4%, respectively. This study also revealed a high LD between the two ASA-PD variants (r(2) = 0.61). Inter-population analysis revealed similarities in the ASA-PD genetic structure between Tunisians and populations from Middle East with c.*96A>G frequencies being the highest in the world. A significant North vs. South genetic differentiation in the ASA-PD frequency was also observed in Tunisian population who seems genetically intermediate between Africans, Middle-Easterners and Europeans. This is the first report on the allele frequency of the ASA-PD in North Africa, revealing a relatively high frequency of the PD allele among Tunisians. This study gives also evidence on the importance of discriminating ASA-PD allele from pathological mutations causing MLD and supporting enzymatic activity testing with both sulfatiduria determination and genetic testing in the differential diagnosis of MLD in the Tunisian population.

A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase

Metachromatic leukodystrophy (MLD) is a rare and severe genetic disease. Inhibition of cerebroside sulfotransferase (CST) has been proposed as a promising new therapeutic strategy for the treatment of MLD. CST catalyzes the transfer of a sulfate group from the coenzyme 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to cerebroside yielding cerebroside sulfate and adenosine-3′,5′-diphosphate (PAP). So far only a few weak CST inhibitors have been described. The goal of the present study was to establish a suitable assay for identifying and characterizing novel CST inhibitors. To this end, we developed and optimized a capillary electrophoresis (CE) based assay for monitoring the catalytic activity of CST by measuring the formation of PAP. A sample matrix consisting of 5mM phosphate buffer with about 0.0001% polybrene at pH 7.4 and a background electrolyte (BGE) containing 75mM phosphate buffer with 0.002% polybrene at pH 5.6 were utilized to achieve a stacking effect for PAP by dynamic pH junction. This led to a limit of detection for the enzymatic product PAP of 66.6nM. The CE method was sensitive, robust, and suitable for CST inhibitor screening, Ki value determination, and enzyme kinetic studies. Selected reference compounds were tested in order to validate the assay, including the substrates cerebroside and psychosine, and the inhibitor Congo Red. The newly developed CE method will be useful for the identification and development of novel CST inhibitors which are urgently needed for the treatment of MLD.

Multi-organ Abnormalities and mTORC1 Activation in Zebrafish Model of Multiple Acyl-CoA Dehydrogenase Deficiency

Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxavu463) that has an inactivating mutation in the etfa gene. dxavu463 recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxavu463 zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxavu463 zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.

Effects of glycosylation and pH conditions in the dynamics of human arylsulfatase A

Arylsulfatase A (ARSA) is a lysosomal sulfatase that catalyzes the hydrolysis of cerebroside sulfate. Its deficiency results in Metachromatic Leukodystrophy, whereas a minor condition called ARSA pseudodeficiency occurs in healthy individuals, which has been associated with the substitution of the glycosylated Asn350 by a Ser and with the loss of the polyadenylation signal. In this work, we have investigated ARSA dynamics employing molecular dynamics simulations in response to (1) different pH’s, as, beyond its natural lysossomal environment, it has been recently identified in cytoplasmatic medium and (2) glycan occupancies, including its normal glycosylation state, presenting three high mannose-type oligosaccharides. Accordingly, four systems were studied considering ARSA under different conditions: (1) nonglycosylated at pH ∼ 7 (ARSApH7); (2) non-glycosylated at pH ∼ 5 (ARSApH5); (3) triple glycosylated at pH ∼ 5 (ARSAglyc,pH5); and (4) ARSA-N350S mutant at pH ∼ 5 (ARSAN350S,pH5). Lowering pH and increasing glycosylation was found to reduce the flexibility of the enzyme. In addition, at acidic pH, the glycosylated enzyme presented a higher secondary conformational stability when compared to its nonglycosylated counterpart, supporting experimental findings on triple glycosylation as the essential state of ARSA. The N350S mutant exhibited a consistent degree of unfolding, which may be related to its in vitro reduced stability. Finally, the obtained data are discussed in the search for structural evidences able to contribute to the understanding of biological activity of ARSA and molecular etiology of ARSA pseudodeficiency, as determined by ARSA-N350S in the absence of polyadenylation defect.

Enhanced detection of olefins using ambient ionization mass spectrometry: Ag+ adducts of biologically relevant alkenes

Spray solvent doped with silver ions increases the ease of olefin detection by desorption electrospray ionization (DESI). Characteristic silver adducts were generated in up to 50 times greater abundance when compared to conventional DESI spray solvents for the biologically significant olefin, arachidonic acid, in the positive ion mode. In the analysis of 26 lipids, silver adduct formation was highly favorable for fatty acids, fatty acid esters and prostaglandins but not applicable to some other classes (e.g., polar lipids such as ceramide and its derivative cerebroside sulfate). An investigation exploring competitive Ag(+) cationization with a mixture of components demonstrated that polyunsaturated compounds form Ag(+) adducts most readily. Silver cationization allowed the distinction between three sets of isomers in the course of multiple-stage collision-induced dissociation, so providing insight into the location of the olefin bonds. A silver ion-doped solvent was used in DESI imaging of normal and tumor canine bladder tissue sections. The Ag(+) fatty acid adducts permitted post facto differentiation between the normal and tumor regions. In addition, silver adduct formation in the course of DESI imaging of tissue sections revealed the presence of triacylglycerides, a class of compounds not previously identified through DESI imaging. A simple silver nitrate spray solvent has the potential to further improve DESI analysis of unsaturated biomolecules and other molecules containing π-bonds through selective silver cationization.

Variation of urinary sulfate in relation to renal arylsulfatase A and B in rat models of hypertension

The enzyme arylsulfatase B (ARSB; N‐acetylgalactosamine‐4‐sulfate) removes the sulfate group from the non‐reducing end of the sulfated glycosaminoglycans chondroitin‐4‐sulfate and dermatan sulfate. Recent work in renal epithelial cells in cell culture has demonstrated that ASB activity regulates cellular chondroitin‐4‐sulfate (C4S) and kininogen content and bradykinin release in the spent media. In experiments in salt‐sensitive (SS), salt‐resistant (SR), and spontaneously hypertensive (SHR) rats, measurements of urinary sulfate and bradykinin and cellular arylsulfatase A (ARSA) and arylsulfatase B (ARSB) activity and chondroitin‐4‐sulfate content demonstrate significant relationships. Significant differences in the ARSA and ARSB activity (in nmol/mg protein/hr) were demonstrated between the SS (151 ± 8; 106 ± 10), SR (180 ± 9; 132 ± 10) and SHR (227 ± 10; 161 ± 10) rats, in contrast to no differences in the steroid sulfatase, galactose‐6‐sulfatase, or iduronate‐2‐sulfatase activity. (Iduronate‐2‐sulfatase activity was absent.) Urinary sulfate was highly correlated with the ARSB (r=0.96) and the ARSA (r=0.96) activity. Urinary bradykinin was inversely associated with the C4S (r= −0.88). Since ARSB activity is affected by chloride and phosphate, as well as other anions, and ARSA can remove 3‐sulfate groups from cerebroside sulfate, as well as from sulfated tyrosine residues, the study findings suggest new approaches to mechanisms of blood pressure regulation. VA Merit Review funding

Uptake by Human Astrocytes of Lipid Vesicles Modeling the Lipid Composition of Myelin

The myelin sheath is composed of around 80% lipids including cholesterol, phospholipids, sphingomyelin, cerebroside sulfate and cerebrosides, and in smaller proportions, ceramides and glycerophosphatides. During pathological processes inside the central nervous system (CNS), the damage of this axonal insulation may expose the surrounding glial cells to lipid aggregates that result from this demyelination process. Indeed, microglia, considered to function as the local macrophages, can phagocyte myelin and cell detritus. Human astrocytes are another key example of glial cells where uptake of myelin debris may take place. Astrocytes are key regulators of several neuronal protective mechanisms, but they are also involved in the pathogenesis of certain autoimmune and inflammatory CNS diseases. Aiming to probe the behavior of human astrocytes exposed to different myelin lipids, we monitor the dynamics of lipid vesicle uptake by culture cells, and explore how varying specific myelin lipid components regulate the uptake kinetics and cell viability. A human astrocyte cell line obtained from a glioblastoma is used for all the experiments. Cells are exposed to NBD-PE or calcein labeled 50 nm small unilamellar vesicles (SUVs) of various lipid compositions reflecting various combinations of the myelin lipid components. Vesicle uptake is then monitored through fluorescence spectroscopy at different time points. Significant uptake is observed within 30 minutes, reaching saturation levels around 2 hours. These results are corroborated through flow cytometry, where astrocyte fluorescence stabilizes at around 2 hours. Additionally, we observe the presence of a smaller population of scatter cells thatshowed higher liposome uptake. Finally, by using fluorescence/DIC microscopy, liposomes are found to spread in the astrocyte cytoplasm after 4 hours of incubations. Interestingly, crowding of liposomes around the nucleus is observed after 12 hours of incubation, suggesting a sorting mechanism to be determined.

Autoantibodies to cerebroside sulphate (sulphatide) in leprosy.

Sera from 40 leprosy patients were screened for autoantibodies to cerebroside sulphate (sulphatide). Anti-sulphatide IgM in groups of patients with lepromatous (LL) and borderline (BL + BB + BT), but not with tuberculoid (TT) disease, were significantly elevated above the levels found in endemic control subjects. Eight-six percent (18 out of 21; mean 1.59 OD units) of LL, 33% (four out of 12; mean 1.08 OD units) of borderline and 13% (one out of eight; mean 0.69 OD units) of tuberculoid patients had anti-sulphatide IgM in their sera above a cut-off value of 2 s.d. above the mean value (0.66 OD units) for control sera. Elevated anti-sulphatide IgG was detected in only one patient's serum, an individual with LL disease. The level of anti-sulphatide IgM was strongly correlated to expression of the TH3 idiotype, an idiotype previously defined by a human MoAb that bound Mycobacterium leprae phenolic glycolipid, Klebsiella capsular polysaccharide, polynucleotides and human tissues. The purified, TH3 MoAb was found in this study to bind sulphatide, but not cholesterol-3-sulphate or cerebroside. It is suggested that anti-sulphatide IgM is elevated in leprosy, in relation to the bacterial load. Anti-sulphatide IgM fell at the onset of erythema nodosum leprosum (ENL) reaction, consistent with the deposition of serum antibodies, and thus may play a part in pathology during periods of inflammation, particularly in multibacillary patients.

Signal transduction pathways involved in interaction of galactosylceramide/sulfatide‐containing liposomes with cultured oligodendrocytes and requirement for myelin basic protein and glycosphingolipids

We showed previously that the addition to cultured oligodendrocytes (OLs) of multivalent carbohydrate in the form of liposomes containing the two major glycosphingolipids (GSLs) of myelin, galactosylceramide (GalC) and cerebroside sulfate (Sulf), or galactose conjugated to bovine serum albumin caused clustering of GalC on the extracellular surface and myelin basic protein (MBP) on the cytosolic surface. Multivalent carbohydrate also caused depolymerization of actin microfilaments and microtubules, indicating that interaction of the carbohydrate with the OL surface transmits a transmembrane signal to the cytoskeleton. In the present study we show that inhibition of GSL synthesis with fumonisin B1 prevents clustering of MBP in GalC/Sulf-negative oligodendrocytes, suggesting that GSLs are required for the effect. Because the effects of multivalent carbohydrate resemble those caused by the addition of anti-GalC/Sulf antibodies to OLs and because GalC and Sulf can interact with each other by trans carbohydrate-carbohydrate interactions across apposed membranes, these results support the conclusion that the OL receptor for GalC/Sulf in liposomes is GalC/Sulf in the OL membrane. Inhibition of MBP expression using MBP siRNA inhibited GalC clustering, suggesting that MBP is required for the effect. We also investigate the signal transduction pathways involved using a number of enzyme inhibitors. These indicated that the Akt and p42/p44 MAPK pathways, Rho GTPases, and GSK-3beta are involved, consistent with their known involvement in regulation of the cytoskeleton. These interactions between GalC/Sulf-containing liposomes and the OL membrane may mimic interactions between GalC/Sulf-enriched signaling domains when OL cell membranes or the extracellular surfaces of compact myelin come into contact.

Conformational Dynamics of a Lipid-Interacting Protein: MD Simulations of Saposin B

Saposin B is a water soluble alpha-helical protein which can bind to membranes and extract selected lipids, especially cerebroside sulfates. The X-ray structure of saposin B is homodimeric. There are two conformations of the dimer in the crystal-one with a closed central cavity (the AB dimer) and one (the CD dimer) with a more open cavity. We have conducted a series of short (5 ns) molecular dynamics simulations of saposin B, starting from both the AB and CD conformations and with/without bound lipid and/or water molecules within the central hydrophobic cavity. The more open (CD) dimer showed greater conformational drift than the AB dimer. The conformational drift was also somewhat higher in the absence of bound lipid. Two more extended (30 ns) simulations of AB and CD dimers were performed and analyzed in terms of changes in intersubunit packing within the dimers. The AB dimer remained largely unchanged in conformation over the duration of the extended simulation. In contrast, the CD dimer underwent a substantial conformational change corresponding to a 'scissor' motion of the two monomers so as to compress the central cavity to a more closed conformation than that seen in the AB dimer structure. A H-bond between the Q53 and Y54 side chains of the alpha3 helices of the two opposing monomers seems to hold the dimer in this 'scissor-closed' conformation. We suggest that a cycle of conformational changes, expanding and compressing the central cavity of the saposin B dimer, may play a key role in facilitating lipid extraction from bilayers.

Regulation of the Sodium/Sulfate Co-transporter by Farnesoid X Receptor α

Fxralpha is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxralpha is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxralpha target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxralpha response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxralpha agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxralpha-/- mice, after treatment with the hepatotoxin alpha-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxralpha-/- mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxralpha target gene expressed in the kidney and intestine and that in the absence of Fxralpha, mice waste sulfate into the urine. Thus, Fxralpha is necessary for normal sulfate homeostasis in vivo.

Saposin B binds and transfers phospholipids

Saposin B (Sap B) is a member of a family of four small glycoproteins, Sap A, B, C, and D. Like the other three saposins, Sap B plays a physiological role in the lysosomal degradation of sphingolipids (SLs). Although the interaction of Sap B with SLs has been investigated extensively, that with the main membrane lipid components, namely phospholipids and cholesterol (Chol), is scarcely known. Using large unilamellar vesicles (LUVs) as membrane models, we have now found that Sap B simultaneously extracts from the lipid surface neutral [phosphatidylcholine (PC)] and anionic [phosphatidylinositol (PI)] phospholipids, fewer SLs [ganglioside GM1 (GM1) or cerebroside sulfate (CS)], and no Chol. More PI than SL (GM1 or CS) was solubilized from LUVs containing equal amounts of PI and SLs. An increase in PI level had a poor effect on the Sap B-induced solubilization of GM1 or CS but strongly inhibited that of PC. Sap B was able not only to bind, but also to transfer phospholipids between lipid surfaces. Both the phospholipid binding and transfer activities were optimal at low pH values. These results represent the first biochemical analysis of the Sap B interaction with phospholipids. The capacity of Sap B to bind and transfer phospholipids occurs under conditions mimicking the interior of the late endosomal/lysosomal compartment and thus might have physiological relevance.

A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A

A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concentrations of alkali metal salts. This method has been adapted for use in the assay of arylsulfatase A (ASA) and the cerebroside sulfate activator protein (CSAct or saposin B). Detection of the neutral glycosphingolipid cerebroside product was achieved via enhancement of ionization efficiency in the presence of lithium ions. Assay samples were extracted into the chloroform phase as for the existing assays, dried, and diluted in methanol-chloroform-containing lithium chloride. Samples were analyzed by electrospray ionization mass spectrometry with a triple quadrupole mass spectrometer in the multiple reaction monitoring tandem mass spectrometric mode. The assay has been used to demonstrate several previously unknown or ambiguous aspects of the coupled ASA/CSAct reaction, including an absolute in vitro preference for CSAct over the other saposins (A, C, and D) and a preference for the non-hydroxylated species of the sulfatide substrate over the corresponding hydroxylated species. The modified assay for the coupled ASA/CSAct reaction could find applicability in settings in which the assay could not be performed previously because of the need for radiolabeled substrate, which is now not required.

The synthesis of internal standards for the quantitative determination of sphingolipids by tandem mass spectrometry

Novel internal standards have been synthesised for the quantitative determination by tandem mass spectrometry (MS/MS) of the sphingolipids that accumulate in lysosomal storage diseases. The [d4]C16- and [d47]C24-isoforms of galactosylceramide (CMH), lactosylceramide (CDH), globotriaosylceramide (CTH), cerebroside sulphate, sphingomyelin and G(M1)-, G(M2)- and G(M3)-gangliosides were synthesised by the reaction of their lyso-forms with the acid chlorides of hexadecanoic 5,5,6,6-d4 acid ([d4]-palmitic acid) and tetracosanoic-d47 acid ([d47]-lignoceric acid), respectively. The acid chlorides were formed using oxalyl chloride. The structures of the internal standards were confirmed by MS/MS. The fragmentation pattern of each novel compound was similar to that of the corresponding natural form of the sphingolipid, making it a good internal standard for the quantitative determination of the natural sphingolipid by ESI-MS/MS. Characteristic product ions were identified for each compound.

Co-clustering of galactosylceramide and membrane proteins in oligodendrocyte membranes on interaction with polyvalent carbohydrate and prevention by an intact cytoskeleton.

We have shown previously that addition of liposomes containing the two major glycosphingolipids of myelin, galactosylceramide (GalC) and cerebroside sulfate (CBS), to cultured oligodendrocytes (OLs) caused clustering of GalC on the extracellular surface and myelin basic protein (MBP) on the cytosolic surface to the same membrane domains. It also caused depolymerization of actin microfilaments and microtubules, indicating that interaction of the liposomes with the OL surface induces transmembrane signal transmission. We show that a multivalent form of galactose conjugated to bovine serum albumin has a similar effect as the multivalent GalC/CBS-containing liposomes. Because GalC and CBS can interact with each other across apposed membranes and because anti-GalC and anti-CBS antibodies also cause redistribution of GalC/CBS and depolymerization of microtubules, we believe that the multivalent carbohydrate interacts with GalC and CBS in the OL membrane. Several myelin-specific transmembrane proteins could be involved in this transmembrane signal transmission from GalC/CBS. We looked at co-clustering of several myelin constituents by confocal microscopy to determine if they are located in or redistribute to GalC/MBP-containing domains. Myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), MAPK, and some phosphotyrosine-containing proteins were found to co-cluster with GalC and MBP, but myelin-associated glycoprotein (MAG) and phosphatidylinositol-4,5-bisphosphate (PIP(2)) did not. These results suggest that MOG and PLP, but probably not MAG, are possible candidates for transmembrane transmission of the signal received by GalC/CBS. To determine if depolymerization of actin microfilaments was required for co-clustering, or was secondary to clustering, we stabilized F-actin with jasplakinolide. This also prevented depolymerization of the microtubules and prevented clustering of all constituents, including GalC. The prevention of clustering or redistribution of these glycolipids and proteins by an intact cytoskeleton is consistent with the picket fence model.

Expression, purification, crystallization, and preliminary X-ray analysis of recombinant human saposin B

Saposin B (also known as cerebroside sulfate activator or CSAct) is a small non-enzymatic glycoprotein required for the breakdown of cerebroside sulfates (sulfatides) in lysosomes. Saposin B contains three intramolecular disulfide bridges, exists as a dimer and is remarkably heat, protease, and pH stable. We have expressed the protein in a thioredoxin reductase deficient strain of Escherichia coli and purified the protein by heat treatment, followed by ion-exchange, gel filtration, and hydrophobic interaction chromatographies. The protein is properly folded as judged by the observed disulfide bond topology, the hydrogen–deuterium exchange rate, and the level of stimulation of sulfatide hydrolysis by arylsulfatase A. Crystals of human saposin B were grown by vapor diffusion and diffract to a resolution of 2.2 Å. Despite obtaining only merohedrally twinned P3 1 native crystals, an untwined seleomethionine-substituted crystal belonging to space group P3 121 was also grown. The three-dimensional structure of saposin B protein will provide insights into how this 79 amino acid protein is able to solubilize relatively large membrane-bound lipid ligands.

Crystal structure of saposin B reveals a dimeric shell for lipid binding.

Saposin B is a small, nonenzymatic glycosphingolipid activator protein required for the breakdown of cerebroside sulfates (sulfatides) within the lysosome. The protein can extract target lipids from membranes, forming soluble protein-lipid complexes that are recognized by arylsulfatase A. The crystal structure of human saposin B reveals an unusual shell-like dimer consisting of a monolayer of alpha-helices enclosing a large hydrophobic cavity. Although the secondary structure of saposin B is similar to that of the known monomeric members of the saposin-like superfamily, the helices are repacked into a different tertiary arrangement to form the homodimer. A comparison of the two forms of the saposin B dimer suggests that extraction of target lipids from membranes involves a conformational change that facilitates access to the inner cavity.