Ernst Conzelmann

A simple chromogenic assay for arylsulfatase A

Arylsulfatase A hydrolyzes the artificial chromogenic substrate 4-nitrocatechol-sulfate at 0 degree C at a rate of 24% of that at 37 degrees C whereas arylsulfatase B is almost inactive at 0 degree C. Based on this observation, a simple assay was developed which permits the accurate determination of low residual arylsulfatase A activities in cultured skin fibroblasts of infantile, juvenile and adult MLD patients and pseudodeficient individuals. In cultured skin fibroblasts, the following residual activities were found with this assay system: late-infantile patients, 0.0%, one juvenile patient, 1.0%, adult patients, 4.4-14% of normal average. healthy pseudodeficient probands ranged between 18% and 32%.

Pseudodeficiency of arylsulfatase A: a common genetic polymorphism with possible disease implications

SummaryAt the locus for arylsulfatase A (ASA) at least four to five alleles exist: besides the normal ASA+ and at least two to three deficiency alleles (ASA-), a pseudodeficiency allele, ASAp, is known. On SDS-PAGE the ASAp enzyme migrates slightly faster than ASA+. Treatment of extracts from cells with ASA+/ASA+, ASAp/ASAp, or ASA+/ASAp genotypes with endoglycosidase F leads to the same deglycosylated subunit pattern. Presumably the degree of glycosylation is lower in ASAp than in ASA+. In a large-scale screening project we determined a gene frequency of 7.3% for ASAp. Thus, the ASA locus is polymorphic. In seven families, ASAp showed a codominant mode of inheritance with ASA+. Homozygosity for ASAp has no obvious clinical consequences. In subjects with the compound genotype ASA-/ASAp, the residual enzyme activity may fall below a critical threshold, so that the substrate can no longer be hydrolyzed sufficiently. Since these compounds are not so rare (estimated frequency 0.073%), this mechanism could be of importance in neuropsychiatric disorders with late onset.

Mapping of the gene coding for the human GM2 activator protein to chromosome 5.

The gene coding for the GM2 activator protein has been mapped to human chromosome 5, using an enzyme‐linked immunoadsorbant assay (ELISA) to identify the human protein in human‐mouse somatic cell hybrids.

Activator Proteins for Lysosomal Glycolipid Hydrolysis

Publisher Summary The final degradation of glycosphingolipids occurs in the digestive intracellular organelles known as lysosomes and is accomplished by the sequential action of specific hydrolases. Some of these enzymes appear to be membrane associated and interacting with their lycolipid substrates directly. Others, mainly those acting also on water-soluble substrates, are water soluble and cannot attack their lipid substrates directly. Instead, this interaction has to be mediated by some small nonenzymatic proteins, which are called activator proteins. The physiological significance of activator proteins is demonstrated by two fatal lipid storage diseases that are caused by deficiencies of activator proteins rather than of the degrading enzymes. Those activator proteins that promote the hydrolysis of glycolipid substrates by water-soluble hydrolases, which is, the G M2 activator and the sulfatide/G M1 activator, seem to act by similar mechanisms: they bind the glycolipid and extract it from the membrane to form a water-soluble complex, which is the true substrate for the enzyme. The chapter discusses the assay method of sulfatide/G M1 activator protein with Ganglioside G Ml /β-Galactosidase, Sulfatide/Arylsulfatase A, and Globotriaosylceramide/a-Galactosidase A. There is also a discussion about G M2 activator. Its measurement is based on its ability to accelerate the hydrolysis of ganglioside G M2 by β-hexosaminidase A. The procedure for radioactive labeling of the terminal N-acetylgalactosamine moiety with galactose oxidase/NaB 3 H 4 85 is essentially the same as for ganglioside G M1 . The chapter discusses Cofactors for Glucosylceramide β-Glucosidase and Galactosylceramide β-Galactosidase. There is also a description of the purification procedures of the activator proteins.

The influence of low arylsulfatase A activity on neuropsychiatric morbidity: a large-scale screening in patients

SummaryA total of 1728 patients consecutively admitted to a neuropsychiatric hospital and 379 chronically ill inpatients were examined for activity of arylsulphatase A (ASA) in leucocytes. A further 519 healthy individuals served as controls. We did not find evidence for the involvement of low ASA activity in chronic patients. The consecutive admissions showed a slight preponderance in the lower ASA activity classes. This activity range covers persons heterozygous for ASA deficiency alleles. The data are compatible with the hypothesis that carriers of low ASA activity alleles are at a slightly higher risk for neuropsychiatric disorders.

Assay for cerebroside sulfate (sulfatide) sulfatase in cultured skin fibroblasts with the natural activator protein

A simple procedure was developed to assay the ability of arylsulfatase A in extracts of cultured skin fibroblasts to degrade the natural substrate, sulfatide, in the presence of the physiological activator protein but without detergents. Inhibitory substances were removed by dialysis and by batch-wise ion-exchange chromatography. The enzyme recoveries during purification were monitored with a newly developed method that employs the chromogenic substrate 4-nitrocatecholsulfate at an incubation temperature of 4 degrees C. The residual sulfatidase activities determined with this procedure in fibroblasts from patients with various forms of MLD correlated well with the severity of the disease.

Fundamentals of Ganglioside Catabolism

Gangliosides are components of the outer leaflet of animal plasma membranes and are especially enriched in neuronal surfaces (for rev. see 1 – 3). They are anchored in the bilayer of the membrane by their hydrophobic ceramide moiety whereas the negatively charged hydrophilic oligosaccharide chain extends into the extracellular space and covers the cell surface. As evidenced by in vitro studies as well as by studies in cell culture, ganglioside metabolism is confined to different subcellular compartments (for rev. see 4 – 6) (Fig. 1). Biosynthesis is catalyzed by enzymes bound to the membranes of the endoplasmic reticulum and the Golgi stacks whereas the final degradation of these amphiphilic glycosphingolipids (GSL) takes place in secondary lysosomes. Their advancement through major steps of metabolism is accompanied by and strictly correlated to an intracellular movement.

The Physiological Roles of Activator Proteins for Lysosomal Glycolipid Degradation

Glycosphingolipids are degraded in the lysosome by the sequential action of a number of specific glycosidases. It has been shown that several of the water-soluble glycolipid hydrolases cannot attack their membrane-bound substrates directly but require the assistance of small non-enzymic proteins, so-called activator proteins. These activator proteins bind to the glycolipid substrate, extract it from the membrane and form a water-soluble activator/lipid complex which is believed to be the true substrate of the enzymic reaction [1–4].

Glycosphingolipid Activator Proteins

The catabolism of sphingolipids is accomplished in the intracellular digestive vacuoles known as lysosomes by the sequential action of acid exohydrolases, starting at the hydrophilic end of the molecule. For nearly every degradation step an inherited enzyme deficiency is known leading to a severe sphingolipid storage disease. In the last 20 years intensive studies were performed in many laboratories to understand the molecular basis and the increasing heterogeneity of these disorders (1–6). The lysosomal hydrolases involved in glycolipid degradation have been purified and characterized. Some of these enzymes are membrane-bound whereas others are water-soluble. When the degradation of the presumtive glycosphingolipid substrates by purified soluble enzymes was studied in vitro, only low, often negligible degradation rates were observed. Since sphingolipids are amphiphilic molecules, they form micelles (higher glycosylated glycolipids) or liposomes (sphingomyelin) when dispersed in water. In this tightly packed form, they are hardly accessible to the purified hydrolases.An enormous stimulation of the enzymic hydrolysis can be achieved by the addition of suitable detergents, such as bile salts (3, 7). At appropriate concentrations, detergents and glycolipids form small mixed micelles from which the oligosaccharide chains protrude far enough to be attacked by the hydrolases. However, these detergent-based assay mixtures do not reflect the in vivo situation, since lysosomes do not contain detergents. The interaction between lysosomal, water-soluble hydrolases and their membrane-bound glycolipid substrates must be brought about in some other way. Since 1964 several non-enzymic protein factors, called activators, have been described, which perform this function and thus accelerate the enzymic degradation of glycosphingolipids.

CONCEPTS OF GANGLIOSIDE METABOLISM

Gangliosides (Fig. 1),although characteristic components of all mammalian plasma membranes, are highly abundant in nervous tissues only. They are arranged asymmetrically in the outer leaflet of cellular membranes, their carbohydrate chains facing the extracellular space. Little is known about their function and about the regulation of their metabolism, the elucidation of which might be helpful in understanding the pathogenetic mechanisms underlying the clinical symptoms of ganglioside storage diseases.