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.

Characteristics of arylsulfatase in Russian sturgeon (Acipenser gueldenstaedti) semen.

Spermatozoa of sturgeons (Acipenseriformes), unlike teleosts, possess an acrosome. This paper provides data concerning biochemical characteristics of arylsulfatase (AS), an acrosomal enzyme, found in Russian sturgeon spermatozoa and seminal plasma. The enzymes were purified by a four-step procedure, using n-butanol extraction, ion-exchange chromatography repeated twice and gel filtration. High purity of our enzymes was confirmed by silver staining electrophoresis and an immunological experiment. Kinetic parameters indicated that the purified enzymes belong to arylsulfatase type A. Similarity of the seminal plasma arylsulfatase to the spermatozoan enzyme showed us that arylsulfatase from seminal plasma might originate from damaged spermatozoa. The possible physiological consequences of the presence of arylsulfatase in Russian sturgeon semen are discussed.

The functional consequences of mis-sense mutations affecting an intra-molecular salt bridge in arylsulphatase A.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulphatase A. We describe the functional consequences of three mis-sense mutations in the arylsulphatase A gene (Asp-335-Val, Arg-370-Trp and Arg-370-Gln), affecting an apparent intramolecular Asp-335 to Arg-370 salt bridge, and interpret the effects and clinical consequences on the basis of the three-dimensional structure of arylsulphatase A. Asp-335-Val and Arg-370-Trp substitutions each cause a complete loss of enzyme activity and are associated with the most severe form of the human disease, whereas the Arg-370-Gln-substituted enzyme retains some residual activity, being found in a patient suffering from the milder juvenile form of the disease. Detailed analysis reveals that formation of the apparent salt bridge depends critically on the presence of aspartic acid and arginine residues at positions 335 and 370, respectively. Substitution by various other amino acids, including glutamic acid and lysine, affects enzyme function severely. Biosynthesis and immunoprecipitation studies indicate that the Asp-335-Val substitution affects folding of arylsulphatase A more severely than either the Arg-370-Trp or Arg-370-Gln substitutions. In vitro mutagenesis data show that clinical severity correlates with the space occupied by residue 370. The combination with structural data suggests that the bulky tryptophan residue broadens the cleft held together by the apparent salt bridge, whereas the smaller glutamine residue still allows the cleft to close, yielding a less severely affected enzyme. The position of residue 370 in the three-dimensional structure of the enzyme provides a plausible explanation for the differing severities in loss of enzyme function caused by the mutations and thus the clinical phenotype.

Bone marrow stem cell-based gene transfer in a mouse model for metachromatic leukodystrophy: effects on visceral and nervous system disease manifestations.

Arylsulfatase A (ASA) knockout mice represent an animal model for the lysosomal storage disease metachromatic leukodystrophy (MLD). Stem cell gene therapy with bone marrow overexpressing the human ASA cDNA from a retroviral vector resulted in the expression of high enzyme levels in various tissues. Treatment partially reduces sulfatide storage in livers exceeding 18 ng ASA/mg tissue, while complete reduction was observed in livers exceeding 50 ng ASA/mg tissue. This corresponds to about 80% and 200% of normal enzyme activity. Similar values seem to apply for kidney. A partial correction of the lipid metabolism was detectable in the brain where the galactoerebroside/sulfatide ratio, which is diminished in ASA-deficient mice, increased upon treatment. This partial correction was accompanied by amelioration of neuropathology; axonal cross-sectional areas, which are reduced in deficient mice, were significantly increased in the saphenic and sciatic nerve but not in the optic nerve. Behavioral tests suggest some improvement of neuromotor abilities. The gene transfer did not delay the degeneration occurring in the acoustic ganglion of ASA-deficient animals. The limited success of the therapy appears to be due to the requirement of unexpected high levels of ASA for correction of the metabolic defect.

Interaction of arylsulfatase A with UDP-N-acetylglucosamine:Lysosomal enzyme-N-acetylglucosamine-1-phosphotransferase.

The critical step in lysosomal targeting of soluble lysosomal enzymes is the recognition by an UDP-N-acetylglucosamine:lysosomal enzyme-N-acetylglucosamine-1-phosphotransferase. The structure of the determinant common to all lysosomal enzymes for proper recognition by the phosphotransferase is not completely understood. Our current knowledge is largely based on the introduction of targeted amino acid substitutions into lysosomal enzymes and analysis of their effects on phosphotransferase recognition. We have investigated the effect of eight anti-arylsulfatase A monoclonal antibodies on the interaction of arylsulfatase A with the lysosomal enzyme phosphotransferase in vitro. We also show that a lysine-rich surface area of arylsulfatases A and B is essential for proper recognition by the phosphotransferase. Monoclonal antibodies bind to at least six different epitopes at different locations on the surface of arylsulfatase A. All antibodies bind outside the lysine-rich recognition area, but nevertheless Fab fragments of these antibodies prevent interaction of arylsulfatase A with the phosphotransferase. Our data support a model in which binding of arylsulfatase A to the phosphotransferase is not restricted to a limited surface area but involves the simultaneous recognition of large parts of arylsulfatase A.

Four monoclonal antibodies inhibit the recognition of arylsulphatase A by the lysosomal enzyme phosphotransferase.

The critical step in the sorting of lysosomal enzymes is their recognition by a phosphotransferase in the Golgi apparatus. The topogenic sequences responsible for the recognition by this enzyme have so far only been defined for the lysosomal protease cathepsin D. We have generated four monoclonal antibodies directed against lysosomal arylsulphatase A (ASA). These antibodies inhibit the recognition of ASA by the phosphotransferase in vitro and thus define a region of topogenic sequences in the ASA polypeptide. The antibodies do not interfere with the enzymic activity nor with pH-dependent dimerization of ASA. The epitopes recognized by the antibodies have been located in the second quarter of the ASA polypeptide using chimeric mouse-human ASA molecules. Three of the monoclonal antibodies bind to identical or closely adjacent epitopes, which are formed by the interaction of amino acid residues 165-184 and 202-240. The fourth antibody recognizes a different epitope within amino acids 256-265.

Transport of newly synthesized arylsulfatase A to the lysosome via transferrin receptor-positive compartments.

We have studied the convergence of the biosynthetic lysosomal route marked by the newly synthesized lysosomal enzyme arylsulfatase A (ASA) with the endosomal/prelysosomal compartment in ASA overexpressing baby hamster kidney (BHK) cells. A monoclonal antibody against ASA conjugated to transferrin (Tf-alpha ASA) was used to load the endocytic pathway via the transferrin receptor. Subsequent internalization of [125I]labeled ASA and Tf-alpha ASA conjugates at 18 degrees C followed by rewarming to 37 degrees C showed that immunocomplexes were formed within the recycling pathway and released into the medium. Furthermore, in cells labeled with [35S]methionine for 10 min about 54% of newly synthesized ASA passed into Tf-alpha ASA accessible compartments during a 4 hour chase period and accumulated in the medium. These data indicate that in overexpressing BHK cells the majority of newly synthesized ASA is transported to the lysosome via transferrin receptor-containing early endosomes.

Structural and immunochemical characterization of human urine arylsulfatase A purified by affinity chromatography.

Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was isolated from an ammonium sulfate precipitate of urinary proteins using two different affinity chromatography methods. One method involved the use of concanavalin A-Sepharose affinity chromatography at an early stage of purification, followed by preparative polyacrylamide gel electrophoresis. The other procedure employed arylsulfatase subunit affinity chromatography as the main step and resulted in a remarkably efficient purification. The enzyme had a specific activity of 63 U/mg. The final preparation of arylsulfatase A was homogeneous on the basis of polyacrylamide gel electrophoresis at pH 7.5, and by immunochemical analysis. However, when an enzyme sample obtained by either method of purification was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing or non-reducing conditions, peptide subunits, of 63.5 and 54.5 kDa, were observed. Immunological tests with 125I-labeled enzyme established the presence of a common protein component in both of the electrophoretically separable peptide subunits of human urine arylsulfatase. The amino acid analysis of homogeneous human urine arylsulfatase A showed only a few differences between it and the human liver enzyme. However, immunological cross-reactivity studies using rabbit anti-human urine arylsulfatase revealed immunological difference between the human urine and liver arylsulfatase A enzymes.

Structural and immunological relationships among mammalian arylsulfatase A enzymes.

Structural and immunological properties of numerous arylsulfatase A enzymes (EC 3.1.6) were examined in order to assess the relationships among these enzymes in animals. Arylsulfatase A enzymes from all animals bind to a Concanavalin A-Sepharose column, consistent with the conclusion that they are all glycoproteins. At pH 7.5 the apparent mol. wts of the enzymes are 80-182 kDa, while at pH 4.5 the mammalian arylsulfatase A enzymes dimerize and exhibit apparent mol. wts in the range of 297-348 kDa, but the enzymes from opossum and other lower classes of animals do not aggregate at pH 4.5. The mammalian arylsulfatase A enzymes, which aggregate at pH 4.5, also bind to rabbit liver arylsulfatase A monomers immobilized on an Affi-Gel 10 matrix. The arylsulfatase A enzymes that were studied all exhibit the anomalous kinetic behavior regarded as characteristic of these enzymes. However, not all of the inactivated enzymes are reactivated by sulfate ions. Goat antiserum raised against homogeneous rabbit liver arylsulfatase A cross-reacts with all of the mammalian enzymes in Ouchterlony gel diffusion experiments, whereas the enzymes from lower classes of animals do not cross-react. Quantitative immunoprecipitation experiments demonstrate that the mammalian enzymes are very similar to each other, with greater than 60% primary sequence homology indicated, while arylsulfatase A from opossum and other lower classes of animals show only a partial immunological similarity with the mammalian enzymes. Taken together, the data suggest that the active site of the enzyme and the structural features of the protein are highly conserved during the evolution of the enzyme molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunofluorescence staining and immunological studies of arylsulphatase A of multiple sulphatase deficiency (MSD) and metachromatic leukodystrophy (MLD) fibroblasts.

Multiple sulphatase deficiency (MSD) and metachromatic leukodystrophy (MLD) are both characterized by a deficiency of arylsulphatase A (ARS A) activity, although they are inherited as separate autosomal recessive traits. However, it has been found that the immunologically active substance with anti-ARS A antibody is present in quite normal levels in MLD and in smaller quantities in MSD fibroblasts (Fiddler, 1979). Indirect immunofluorescence staining with anti-ARS A antibody displayed a coarse granular and diffuse distribution of ARS A or cross-reacting material (CRM) in the normal control and MLD fibroblasts, whereas very weak fluorescence staining was observed in MSD fibroblasts proportional to the decrease in the ARS A activity observed in the lysate enzyme assay. These results suggest that ARS A deficiency in MLD cells is due to an enzymatically deficient ARS A molecule, which is immunologically cross-reactive with anti-normal ARS A antibody. ARS A deficiency in MSD cells appears to be due to a reduced amount of normal ARS A.

A variant form of arylsulfatase A in human urine derived directly from the renal pelvis: kinetic and immunological characterization.

Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was examined in voided and in nephrostomic urine. A variant form of the enzyme was found in nephrostomic urine, in addition to the minor form, which is the sole component of arylsulfatase A in voided urine. The nephrostomic enzyme differed from the voided urine enzyme with respect to the kinetic parameters, the isoelectric point, heat stability and immunological reactivity. The isoelectric points of the voided urine and nephrostomic enzymes were 4.7 and 5.3, respectively. The nephrostomic enzyme was more heat-labile at 62.5 degrees C than the voided urine enzyme. Although the Km values of the two enzymes with nitrocatechol sulfate as substrate were almost the same, the V value of the nephrostomic enzyme was approx. one-hundredth that of the voided urine enzyme. The molecular weight (almost 130 000) did not differ between the voided urine and nephrostomic enzymes. It was demonstrated by various methods, using IgG antibody against the purified voided urine enzyme, that the nephrostomic enzyme was antigenically distinct from the voided urine enzyme.

Complementation of arylsulfatase A in somatic hybrids of metachromatic leukodystrophy and multiple sulfatase deficiency disorder fibroblasts.

Metachromatic leukodystrophy and multiple sulfatase deficiency disorder are severe neurodegenerative diseases inherited as separate autosomal recessive traits. Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) activity is deficient in both diseases but in multiple sulfatase deficiency disorder, activities of arylsulfatases B and C and other sulfatases are also reported to be reduced. Somatic hybrid cell clones produced by fusing cultured fibroblasts from patients with these diseases were isolated by a nonselective technique based on unit-gravity sedimentation. Arylsulfatase A activity was restored in these hybrids. The complemented enzyme resembled the normal arylsulfatase A in heat stability, pH optimum, Km, electrophoretic mobility, and immunologic reactivity. Because a structurally normal enzyme can be restored in a hybrid only though intergenic complementation, these results indicate that the mutations responsible for the deficiency of arylsulfatase A activity in metachromatic leukodystrophy and multiple sulfatase deficiency disorder are nonallelic and that at least two genetic loci control the expression of arylsulfatase A activity in the human genome. Furthermore, arylsulfatase C activity was also restored to normal in the hybrids, indicating that a common sulfatase inhibitor is not the cause of the multiple sulfatse deficiency.

Immunologic studies of arylsulfatase A in normal and metachromatic leukodystrophy liver.

Purified human liver arylsulfatase A on polyacrylamide gel electrophoresis at pH 4.0 is separated into two protein forms with enzymatic activity and two distinct inactive subunits. All of these components were immunologically distinguishable using different antisera preparations. In late infantile metachromatic leukodystrophy, only one of the two inactive subunits was immunologically detected, whereas in the juvenile form of metachromatic leukodystrophy, both inactive subunits were antigenically present.