Leukocyte and Dried Blood Spot Arylsulfatase A Assay by Tandem Mass Spectrometry.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spots (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed a clear deficit in the enzymatic activity and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patients and for monitoring the efficacy of therapeutic treatments. ARSA activity was measured in DBS for the first time without an antibody. This new ARSA DBS assay can serve as a second-tier test following the sulfatide measurement in DBS for newborn screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay.

A new analytical bench assay for the determination of arylsulfatase a activity toward galactosyl-3-sulfate ceramide: implication for metachromatic leukodystrophy diagnosis.

Here, we present the design and validation of a new assay for the diagnosis of metachromatic leukodystrophy. The method is highly specific, simple, reproducible, and straightforward. In our spectrophotometric method, the determination of arylsulfatase A (ARSA) activity toward the natural substrate, galactosyl-3-sulfate ceramide (or sulfatide), is performed using neat sulfatide without chemical modification. This confers to the assay high analytical specificity. The hydrolyzed sulfatide is monitored upon inclusion of the colorimetric reagent Azure A. The nonhydrolyzed sulfatide-Azure A is recovered and measured at a wavelength of λ = 650 nm. Thus, ARSA activity toward the sulfatide is obtained by subtracting the nonhydrolyzed sulfatide from the total sulfatide used in the enzyme reaction (sulfatide-Azure A present in a parallel assay performed in the absence of ARSA). Within a clinical context, our method definitely discriminated between healthy subject samples and metachromatic leukodystrophy patient samples, and, therefore, it is suitable for diagnostic applications and for monitoring the efficacy of therapeutic treatments in patients or animal models.

Studies of pH-dependent self-association of a recombinant form of arylsulfatase A with electrospray ionization mass spectrometry and size-exclusion chromatography.

Arylsulfatase A is an endogenous enzyme that is responsible for the catabolism and control of sulfatides in humans. Its deficiency results in the accumulation of sulfatides in the cells of the central and peripheral nervous system leading to the destruction of the myelin sheath and resulting in metachromatic leukodystrophy (MLD), a neurodegenerative lysosomal storage disease. A recombinant human form of this glycoprotein (rhASA) is currently under development as an enzyme replacement therapy. At neutral and alkaline pH, this protein exists as a homodimer but converts to an octameric state in the mildly acidic environment of the lysosome, and a failure to form an octamer results in suboptimal catalytic activity (most likely due to a diminished protection from lysosomal proteases). Despite the obvious importance of the rhASA oligomerization process, its mechanistic details remain poorly understood. In this work, we use size exclusion chromatography (SEC) and electrospray ionization mass spectrometry (ESI MS) to monitor the dimer-to-octamer transition as a function of both solution pH and protein concentration. While SEC clearly shows different profiles (i.e., retention time differences) for rhASA when the chromatography is performed at neutral and lysosomal pH, consistent with changing oligomerization states, no resolved peaks could be observed for either octamer or dimer when analyzed at intermediate pH (5.5-6.5). This could be interpreted either as the result of a rapid dimer-to-octamer interconversion on the chromatographic time scale or as a consequence of the presence of previously unidentified intermediate species (e.g., tetramer and/or hexamer). In contrast, ESI MS provides strong evidence of the dimer-to-octamer transition state that occurs when the analysis is performed within a narrow pH range (6.0-7.0). Octamer assembly was shown to be a highly cooperative process with no intermediate states that are populated to detectable levels. A tetrameric state of rhASA exists at equilibrium with a dimer at neutral pH but does not appear to be involved in the octamer assembly process.

Cell-surface arylsulfatase A and B on sinusoidal endothelial cells, hepatocytes, and Kupffer cells in mammalian livers.

Arylsulfatase A (ARSA) and B (ARSB) have been regarded as lysosomal enzymes because of their hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of their enzymatic activity. Using sea urchin embryos, we previously demonstrated that the bulk of ARS is located on the cell surface of the epithelium, colocalizing with sulfated polysaccharides, and that it does not exhibit enzymatic activity. To examine whether ARSA and ARSB exist on the cell surface in mammalian tissues, we raised antibodies against ARSA and ARSB and examined immunohistochemically their localization in the liver using light and electron microscopy. Here we show that mammalian ARSA and ARSB exist on the cell surface of sinusoidal endothelial cells, hepatocytes, and sinusoidal macrophages (Kupffer cells), as well as in the lysosome. They are also colocalized with heparan sulfate proteoglycan. These results suggest that ARSA and ARSB also may function in the cell surface of mammals. This is the first report to show cell-surface localization of ARS in mammalian somatic cells. The extracellular localization of ARS will provide new insight for human ARS deficiency disorders, such as metachromatic leukodystrophy and mucopolysaccharidosis VI.

Safety of arylsulfatase A overexpression for gene therapy of metachromatic leukodystrophy.

Successful gene therapy approaches for metachromatic leukodystrophy (MLD), based either on hematopoietic stem/progenitor cells (HSPCs) or direct central nervous system (CNS) gene transfer, highlighted a requirement for high levels of arylsulfatase A (ARSA) expression to achieve correction of disease manifestations in the mouse model. Full assessment of the safety of ARSA expression above physiological levels thus represents a prerequisite for clinical translation of these approaches. Here, using lentiviral vectors (LVs), we generated two relevant models for the stringent evaluation of the consequences of ARSA overexpression in transduced cells. We first demonstrated that ARSA overexpression in human HSPCs does not affect their clonogenic and multilineage differentiation capacities in clonogenic assays and in a neonatal hematochimeric mouse model. Further, we studied ARSA overexpression in all body tissues by generating transgenic mice overexpressing the ARSA enzyme by LV up to 15-fold above the normal range and carrying multiple copies of LV in their genome. Characterization of these mice demonstrated the safety of ARSA overexpression in two main gene therapy targets, HSPCs and neurons, with maintenance of the complex functions of the hematopoietic and nervous system in the presence of supraphysiological enzyme levels. The activity of other sulfatases dependent on the same common activator, sulfatase-modifying factor-1 (SUMF1), was tested in ARSA-overexpressing HSPCs and in transgenic mice, excluding the occurrence of saturation phenomena. Overall, these data indicate that from the perspective of clinical translation, therapeutic levels of ARSA overexpression can be safely achieved. Further, they demonstrate an experimental platform for the preclinical assessment of the safety of new gene therapy approaches.

In vitro analysis of multipotent mesenchymal stromal cells as potential cellular therapeutics in neurometabolic diseases in pediatric patients.

Multipotent mesenchymal stromal cells (MSCs) play an important role in stromal support for hematopoietic stem cells, immune modulation, and tissue regeneration. We investigated their potential as cellular therapeutic tools in neurometabolic diseases as a growing number of affected children undergo to bone marrow transplantation. MSCs were isolated from bone marrow aspirates and expanded ex vivo under various culture conditions. MSCs under optimal good medical practice (GMP)-conform culture conditions showed the typical morphology, immunophenotype, and plasticity. Biochemically, the activities of beta-hexosaminidase A, total beta-hexosaminidase, arylsulfatase A (ASA), and beta-galactosidase measured in MSCs were comparable to those in fibroblasts of healthy donors. These four enzymes were interesting for their expression in MSCs, as each of them is defective, respectively, in well-known neurometabolic diseases. We found that MSCs released significant amounts of ASA into the media. In coculture experiments, fibroblasts from patients with metachromatic leukodystrophy, who are deficient for ASA, took up a substantial amount of ASA that was released into the media from MSCs. Mannose-6-phosphate (M6P) inhibited this uptake, which was in accordance with the M6P receptor-mediated uptake of lysosomal enzymes. Taken together, we show that MSCs produce appreciable amounts of lysosomal enzyme activities, making these cells first-choice candidates for providing metabolic correction when given to enzyme-deficient patients. With the example of ASA, it was also shown that an enzyme secreted from MSCs is taken up by enzyme-deficient patient fibroblasts. Given the plasticity of MSCs, these cells represent an interesting add-on option for cellular therapy in children undergoing bone marrow transplantation for lysosomal storage diseases and other neurometabolic diseases.

Embryonic stem cell-based reduction of central nervous system sulfatide storage in an animal model of metachromatic leukodystrophy.

Pluripotency, virtually unlimited self-renewal and amenability to genetic modification make embryonic stem (ES) cells an attractive donor source for cell-mediated gene therapy. In this proof of concept study, we explore whether glial precursors derived from murine ES cells (ESGPs) and engineered to overexpress human arylsulfatase A (hASA) can cross-correct the metabolic defect in an animal model of metachromatic leukodystrophy (MLD). Transfected ES cells showed an up to 30-fold increase in ASA activity. Following in vitro differentiation, high expression of ASA was found in all stages of neural and glial differentiation. hASA-overexpressing ESGPs maintained their ability to differentiate into astrocytes and oligodendrocytes in vitro and in vivo. After transplantation into the brain of neonatal ASA-deficient mice, hASA-overexpressing ESGPs were found to incorporate into a variety of host brain regions. Four weeks after engraftment, immunofluorescence analyses with an antibody to sulfatide revealed a 46.7+/-4.0% reduction of immunoreactive sulfatide deposits in the vicinity of the hASA-positive engrafted cells, thereby significantly extending the rate of sulfatide reduction achieved by the endogenous ASA activity of non-hASA-transfected control cells (21.1+/-5.8%). These findings provide first in vivo evidence that ES cells may serve as a potential donor source for cell-mediated enzyme delivery in storage disorders such as MLD.

Somatic intragenic recombination of the arylsulfatase A gene in a metachromatic leukodystrophy patient.

A metachromatic leukodystrophy (MLD) patient was found to carry two additional arylsulfatase A (ARSA) alleles besides the two inherited. The additional alleles arose from an event of mitotic intragenic recombination between the inherited alleles, thus leading to a case of somatic mosaicism. As suggested by in vitro expression, the recombination was ineffective in generating a significantly advantaged ARSA allele compared to the inherited alleles. Although the phenotype in this patient was not modified by the recombination, similar events could potentially yield significant clinical benefits.

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.

Expression and purification of a human, soluble Arylsulfatase A for Metachromatic Leukodystrophy enzyme replacement therapy.

The production of active Arylsulfatase A is a key step in the development of enzyme replacement therapy for Metachromatic Leukodystrophy. To obtain large amounts of purified Arylsulfatase A for therapeutic use, we combined a retroviral expression system with a versatile and rapid purification protocol that can easily and reliably be adapted to high-throughput applications. The purification method consists of an initial ion-exchange DEAE-cellulose chromatography step followed by immuno-affinity purification using a polyclonal antibody against a 29-mer peptide of the Arylsulfatase A sequence. Immuno-adsorbed protein was eluted with a combination of acidic pH and an optimal concentration of the 29-mer peptide. This protocol reproducibly yielded approximately 100 microg of >99% pure human Arylsulfatase A, corresponding to 152 mU of enzyme activity, per liter of culture medium with properties similar to those of human non-recombinant protein.

Sphingolipidoses in Turkey.

During the last 5 years 2057 children under the age of 5 with various neurologic symptoms with the suspected diagnosis of lysosomal storage diseases were referred to our hospital from different universities and state hospitals. We were able to separate sphingolipidoses by lysosomal enzyme screening. A total of 300 patients (15%) with sphingolipidoses were diagnosed; there were deficiencies of arylsulfatase A [metachromatic leukodystrophy (MLD)] in 93 (31%), hexosaminidase [Sandhoff disease (SHD)] in 62 (20.7%), hexosaminidase A [Tay-Sachs disease (TSD)] in 15 (5%), beta-galactosidase (GM1 gangliosidosis) in 35 (11.7%), alpha-galactosidase (Fabry disease) in one (0.3%) cerebroside beta-galactosidase (Krabbe disease) in 65 (21.7%) and glucosylceramidase (Gaucher disease) in 29 (9.6%). SHD (20.7%), MLD (31%) and Krabbe disease (21.7%) were common. Prenatal enzymatic diagnosis was made in 70 at risk pregnancies, 64 for TSD and SHD, three for MLD and three for GM1 gangliosidosis by using chorionic villus biopsy in 54, cord blood samples in 12 and cultured amniotic fluid cells in four. Seventeen fetuses were found to be affected. We have calculated the relative frequency and minimum incidence of sphingolipidoses in Turkey. The combined incidence of sphingolipidoses is 4.615 per 100,000 live births. The calculated incidences are 1.43, 0.95, 1, 0.23, 0.54, 0.45, 0.015 per 100,000 live births for MLD, SHD, Krabbe, Gaucher, TSD, GM1 gangliosidosis and Fabry diseases, respectively. The real incidence, which covers all subtypes of this group of diseases, should be greater than this number. The results suggested that, as a group, sphingolipidoses are relatively common and represent an important health problem in Turkey and some rare autosomal recessive diseases of Turkey are due to 'founder effect' created by consanguineous marriages.

Analysis of arylsulfatases A and B, acid phosphatase, lactate dehydrogenase, and aspartate transaminase in chronic periapical lesions of endodontic origin.

Attempts were made to detect and measure the activities of arylsulfatases. A&B acid phosphatase, lactate dehydrogenase, and glutamate oxaloacetate transaminase (aspartate transaminase) enzymes in human chronic lesions of endodontic origin. Thirteen periapical lesions of endodontic origin and 11 noninflamed control periapical tissues were obtained. The specimens were carried to the laboratory on liquid nitrogen and kept at -70 degrees C. Samples were thawed, homogenized, and then assayed for enzyme activities. The specific activities of arylsulfatase A (nmol/hr/mg protein) were 55.0+/-10.7 (chronic lesions) vs. 3.4+/-2.2 (controls) (p < 0.01). Arylsulfatase B specific activities (nmol/hr/mg protein) were 50.3+/-6.4 (chronic lesions) vs 91.8+/-18.4 (controls). Total acid phosphatase activities (mU/mg protein) were 45.8+/-6.6 (chronic lesions) vs. 26.8+/-3.1 (controls). Lactate dehydrogenase activities (Berger-Broida units/mg protein) of the chronic periapical lesions were significantly higher than the control group (362+/-63.2) vs. (140+/-46.0) (p < 0.05). There was no significant difference between the specific activities of aspartate transaminase in chronic lesions and the control group (68.0+/-14.5) vs. (53.0+/-10.4) mU/mg protein).

Leukocyte arylsulfatase A activity in patients with alcohol-related cirrhosis.

Arylsulfatase (ASA) enzyme deficiency is associated with metachromatic leukodystrophy (MLD), which is a hereditary myelin metabolic disease. It has been proposed that in alcoholic subjects with abnormal ASA, the accumulation of sulfatides may lead to demyelinization and generalized cerebral atrophy. ASA may be diminished in subjects with alcoholic cirrhosis having encephalopathic manifestations. This idea has not been previously proposed. Leukocyte arylsulfatase A (ASA) activity was measured in 30 healthy male volunteers and 28 patients with alcohol-related cirrhosis. The patients were divided into two groups: patients with alcohol-related cirrhosis with hepatic encephalopathy history and patients with alcoholic cirrhosis without history of hepatic encephalopathy. Alcoholic cirrhotic patients with history of encephalopathy showed 58.21% (40.95 nmol/mg protein/h) less enzymatic activity than a control group (98.00 nmol/mg protein/h), whereas the group without history of encephalopathy showed an ASA value which was 38.2% (60.55 nmol/mg protein/h) less than the control group. The results suggest that the low ASA activity is a factor associated to the appearance of encephalopathy in patients with alcohol-related cirrhosis.

Evaluation of leukocyte arylsulfatase-A activity in patients with breast cancer and benign breast disease.

This study was planned to evaluate the feasibility of using the assay of leukocyte arylsulfatase-A (AS-A) activity as a non-invasive diagnostic tool in patients with benign and malignant breast disease. The leukocyte AS-A activity of a total of 81 women was analyzed, including 28 healthy women, 29 women with benign breast disease (BBD) and 24 patients with primary breast cancer (BC). The mean leukocyte AS-A activity in patients with BBD was slightly higher (14.3%) that observed in the healthy subjects, but the difference was not statistically significant. In patients with BC the enzyme activity was significantly higher than in the healthy subjects (60.3%, P<0.05) and in the benign group (40.2%, P<0.05). In addition, since no significant differences have been observed between premenopausal patients and their controls, it is suggested that the measurement of leukocyte AS-A activity may not be a reliable test for differential diagnosis of benign and malignant proliferation in mammary glands due to the possible interfering effect of gonadal hormones on AS-A activity. In contrast, since peri- and postmenopausal BC patients have negligible or no gonadal activity function, the elevation in the activity of leukocyte AS-A in these age groups of patients may only be expected to originate from malignant proliferation. Based on our results, it is concluded that in patients in whom high leukocyte AS-A activities were observed the possibility of the presence of malignancy might also be high. Therefore, this test might be valuable as a non-invasive biochemical technique in combination with other established markers for the identification of masses in the breast.

Leucodistrofia metacromática / Metachromatic leukodystrophy

Introducción. La leucodistrofia metacromática (LMC) es una enfermedad hereditaria autosómica recesiva, caracterizada por una anormalidad en el metabolismo de la mielina, cuyos datos neurpatológicos más importantes son: la desmielinización en el sistema nervioso central (SNC) y periférico (SNP). Caso clínico. Pacientes con LMC iniciada después del año de edad con involucramiento progresivo de las funciones neurológicas de tipo motoras, trastornos del lenguaje, atrofia óptica, y signos extrapiramidales y cerebelosos. El diagnóstico se realizó tanto por imagen de tomografía axial computada y resonancia magnética nuclear de cráneo, así como en la disminución de la arilsulfatasa A en suero y aumento en la excreción de sulfátidos en orina. El estudio histopatológico confirmó la desmielinización de la sustancia blanca y el acúmulo de material metacromático en el SNC, SNP y otros órganos. Conclusión. La LMC debe sospecharse en la infancia temprana o tardía, cuando los pacientes tienen regresión del desarrollo psicomotor asociado a deterioro progresivo de las funciones cerebrales superiores, datos piramidales, extrapiramidales, cerebrales y neuropatía periférica

Metachromatic leukodystrophy: identification of the first deletion in exon 1 and of nine novel point mutations in the arylsulfatase A gene.

Metachromatic leukodystrophy (MLD), a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA), is inherited as an autosomal recessive trait, and its frequency is estimated to be 1 in 40,000 live births. Genomic DNA from 21 MLD patients (14 late-infantile and 7 juvenile cases) was amplified in four overlapping PCR fragments and tested by allele-specific oligonucleotide (ASO) for the two common mutations 459 + 1G-->A and P426L. These mutations were found in only 28.6% of the alleles studied. The remaining alleles were analyzed by chemical mismatch cleavage (CMC) and automatic sequencing. In addition to five previously reported mutations (459 + 1G-->A, A212V, R244C, R390W, P426L), 10 novel mutations were identified: 9 missense mutations (S95N, G119R, D152Y, R244H, S250Y, A314T, R384C, R496H, K367N) and one 8 bp deletion in exon 1, the first mutation reported in this exon. These methods allowed us to identify 76% of the alleles tested. Genotype-phenotype correlations could be established for some of these mutations. These results confirm the heterogeneity of mutations causing MLD and suggest that CMC is a reliable and informative screening method for point mutation detection in the arylsulfatase A gene.

Arylsulfatase A pseudodeficiency: altered kinetic and heat-inactivation properties.

Arylsulfatase A (ASA) pseudodeficiency (PD) was described in clinically healthy individuals with ASA-deficient activity. To confirm that the PD individual in the present study is homozygous for the PD allele without any other mutations, direct solid-phase sequencing was done and the two A-to-G transitions--one at the third N-glycosylation site (N350S) and the other at the first polyadenylation signal (ATTAAC to AGTAAC)--were identified. No other mutations were detected in the entire coding region nor in the intron-exon boundary region of the ASA gene in the PD cells. Kinetic studies to compare the partially purified ASA from controls to that from a homozygote (PD allele) were carried out using p-nitrocatechol sulfate (p-NCS) as a substrate. The apparent Km for the control ASA was 0.6 mM and for the PD enzyme 2.0 mM (p < 0.01). The heat inactivation at 60 degrees C revealed 50% inactivation within 90 min for control ASA and 28 min for PD ASA. At 65 degrees C, the 50% inactivation was reached at 18 min for the control and at 8.5 min for the PD. These results document the decreased affinity of ASA toward p-NCS and increased heat inactivation from a PD individual. Western blot analysis following SDS-PAGE and isoelectric focusing revealed differences in both the molecular weight and the isoelectric point between the control ASA and that of the PD allele. To the best of our knowledge, this is the first report showing the altered properties of ASA from a PD homozygote.

Neuraminidase assay in cultured human fibroblasts: in situ versus in vitro procedures.

Further investigations have been carried out to characterize a published procedure of neuraminidase assay, in which the activity is measured directly on the cell culture layer. The pH optimum was 4.0. A Vmax value of 130 nmol/mg/h and a K(m) of 0.3 mmol/l were found. During incubation in the acid buffer, arylsulphatase activity was released into the medium, whereas neuraminidase activity remained attached to the cells. The in situ method allowed an unequivocal diagnosis of primary and secondary neuraminidase deficiencies. Because of its simplicity and reliability, the method appears useful as a routine method in clinical laboratories.