Human eosinophil arylsulfatase B. Structure and activity of the purified tetrameric lysosomal hydrolase.

Arylsulfatase B from human eosinophils was purified free of contaminating proteins by gel filtration and sequential affinity chromatography on Affi-Gel Blue and zinc chelate Sepharose. 50 micrograms of the purified enzyme presented as a single stained band on alkaline disc gel electrophoresis. In both goats and rabbits, the purified enzyme elicited monospecific antisera that yielded single precipitation arcs on Ouchterlony analysis with a human eosinophil extract and the purified enzyme; the immunoprecipitation lines fused in a pattern of identity, providing immunochemical evidence for the homogeneity of the purified enzyme. On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a dominant lower molecular weight protein and three other bands with molecular weights approximately two, three, and four times that of the major protein band were resolved. The prominence of the less rapidly migrating protein bands increased relative to the major band if the enzyme was maintained under acidic conditions or was reacted with the cross-linking agent dimethyl suberimidate under alkaline conditions before SDS-polyacrylamide gel electrophoresis, supporting the conclusion that the enzyme consists of four subunits. Two stained bands were present on acid disc gel electrophoresis; they were composed of oligomeric forms of enzyme on analysis by SDS-polyacrylamide gel electrophoresis in a second dimension. A minimum molecular weight of 70,190 was determined from amino acid composition analysis for the tetrameric form of the enzyme. The specific functional activity of the purified arylsulfatase B was concentration and time dependent, compatible with its association or dissociation into subunit forms with differing specific activities. Factors that govern subunit interactions of arylsulfatase B, including local enzyme concentration and pH, provide mechanisms for regulating the enzymatic activity of this lysosomal hydrolase.

Lysosomal arylsulfatases of human leukocytes: increment of phosphorylated B variants in chronic myelogenous leukemia.

Lysosomal arylsulfatases A and B of peripheral leukocytes from patients with chronic myelogenous leukemia and from healthy subjects were studied. Two enzyme activities of leukemia cells were significantly higher than those of cells from healthy subjects, irrespective of total and differential counts of leukemic cells. Upon anion-exchange chromatography, the arylsulfatases of chronic myelogenous leukemia cells and normal leukocytes were separated into the basic B enzyme and its anionic variant (B1) and A enzyme. However, the amount of B1 enzyme relative to B enzyme or the activity ratio of B1 enzyme to total arylsulfatase B (B + B1) was higher in chronic myelogenous leukemia cells than in normal cells. The anionic property of the enzyme was found to be due to phosphate groups bound to the carbohydrate moiety of the arylsulfatase, based on the following results. When B1 enzyme was treated with alkaline phosphatase followed by isoelectric focusing, it was changed to a less anionic enzyme with heterogeneous components which are ascribed to phosphodiester groups linked to the heterogeneous carbohydrate moiety of the enzyme; no effect was observed by sialidase treatment. Upon treatment of B1 enzyme with endo-beta-N-acetylglucosaminidase H, which cleaves sugar chains of a high mannose type in glycoproteins, the anionic heterogeneous components were converted to the basic component similar to B enzyme. From our present and previous observations, it can be concluded that the increase of phosphorylated forms of the lysosomal hydrolase represents one characteristic of rapidly proliferating neoplastic cells.

Metachromatic leukodystrophy. Ultrastructural and enzymatic study of a case of variant O form.

A variant of metachromatic leukodystrophy (MLD), Austin disease, is characterized by a multiple isozyme deficiency of arylsulfatase. A 3 1/2-year-old girl with progressive mental and physical deterioration had decreased activities of arylsulfatases A and B in the leukocytes, shown by acylamide gel electrophoresis. Under the electron microscope, biopsy specimens of the brain and the peripheral nerve showed lamellar structures with socalled zebra bodies in the cytoplasmic processes of glial cells, granulo-membranous inclusions with fingerprint configurations in neurons, and myelinlike material in Schwann cells. Results from our study suggest an intricate nature of this dysmetabolic disorder, which shows ultrastructural changes usually seen in classic MLD, a deficiency of arylsulfatase A only, concomitant with those seen in mucopolysaccharidoses such as Hurler and Sanfilippo syndromes.

Bone marrow transplantation in newborn rats with mucopolysaccharidosis type VI: biochemical, pathological, and clinical findings.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is the lysosomal storage disorder caused by the deficient activity of arylsulfatase B (ASB). In this study, we evaluated bone marrow transplantation (BMT) for the treatment of MPS VI and the effects of irradiation on the survival and engraftment of bone marrow-transplanted neonatal rats. METHODS: One- to 2-day-old MPS VI rats were injected with normal bone marrow after irradiation with 200, 400, or 800 cGy. Ninety percent of the animals receiving a single dose of 200 cGy (n=30) survived the procedure, whereas irradiation with 400 cGy (n=23) or 800 cGy (n=12) resulted in significant mortality (78% and 100%, respectively). Engraftment was monitored by determining ASB activities in peripheral white blood cells and by Y chromosome in situ hybridization analysis. Fifty-two percent of the animals from the 200-cGy group engrafted for up to 8 months after BMT; among the five animals that survived the 400-cGy dose, all engrafted. In comparison, only 20% of nonirradiated animals engrafted at low levels. Of the 24 engrafted animals that were monitored for 8 months after BMT, clinical and/or radiographic improvements were noted in only one (BMT animal 3). Enzymatic analysis revealed that the ASB activities in the reticuloendothelial organs of this animal, as well as two other engrafted but clinically unimproved animals (BMT animals 1 and 2), were normal or near normal; correspondingly, the glycosaminoglycan levels in these organs were significantly reduced. Consistent with the clinical and biochemical observations, light and electron microscopic findings were more improved in BMT animal 3 as compared with BMT animals 1 and 2, although a reduction of storage was evident in each of these transplant recipients, particularly in the trachea and aorta, two tissues that are characteristic sites of pathology in human patients. CONCLUSIONS: These results indicate that BMT in newborn MPS VI patients may prevent many of the pathological and clinical findings in this disorder, but is likely to have very limited and unpredictable effects on the skeletal abnormalities.

Arylsulphatase A and B in juvenile metachromatic leukodystrophy.

A series of five living patients with juvenile metachromatic leukodystrophy (MLD), ten first-degree relatives, and a number of controls were subjected to biochemical investigations including quantitative determination of arylsulphatase A (ASA) and B (ASB) activities in peripheral leukocytes and polyacrylamide disc gel elctrophoresis of arylsulphatases. Five relatives were family members of four previously deceased patients with juvenile MLD. The mean ASA activity of the patients was 1.3 nmol of p-nitrocatechol sulphate hydrolysed in 30 min per mg protein. It was 84 nmol in the relatives, 129 nmol in other neurological patients and 136 nmol in normal controls. The corresponding ASB activity was 38 nmol in the patients, 49 nmol in the relatives, and 99 nmol in normal controls. An extremely low ASB activity, 3.4 nmol, was found in one relative. No ASA band could be visualised in the enzyme electrophoretic patterns of the patients' leukocytes but the bands representing ASB appeared normal. Seven relatives showed ASA bands weaker than normal, and the relative with low ASB activity exhibited very weak ASB band. The low ASB activity in the patients and heterozygotes may be a characteristic feature of the slowly progressive juvenile type MLD diagnosed in the present series.

A sensitive fluorescence assay for the simultaneous and separate determination of arylsulphatases A and B.

A sensitive fluorometric assay utilizing 4-methylumbelliferyl sulphate has been developed for the simultaneous determination of arylsulphatases A and B from leucocytes, based on the differential effect of silver ions on the two enzymes. The procedure allows discrimination between normal cases and those with metachromatic leucodystrophy.

[Hypereosinophilias of the blood. I. Eosinophilic granulocytes]. / Le ipereosinofilie del sangue. Nota I. Il granulocita eosinofilo.

A knowledge of eosinophil granulocytes is indispensable for the study of hypereosinophilia. For this reason, the most recent findings relating to eosinophil morphology, production/regulation mechanism, and function are reported. Particular attention is given to enzyme populations, local control mechanisms and eosinophil cell surface receptors. Among the various enzymes present in the eosinophil, major basic protein (MBP), with its capacity to damage the cells of many organs, plays an important part; other enzymes include eosinophil peroxidase (EPO), arylsulphatase B, phospholipase D, histaminase and cationic proteins (ECP). Factors influencing eosinophil tissue concentrations and mode of action are considered. Recent findings agree on the role of eosinophils in immunological reactions and parasitic infestations: eosinophil plays a part in an immunological physiopathological sequence: it may, act as a killer cell with selective action against invading parasites, or it may be an immune modulator, anti-inflammatory cell able to surround inflammatory reactions and prevent them from spreading.

[Arylsulfatase activity of asthmatic children].

Human eosinophil arylsulfatase (AS) is known to inactivate a slow reacting substance of anaphylaxis (SRS-A). Arylsulfatase A (AS-A) and arylsulfatase B (AS-B) activity was assayed by a modification of the method of Inoue using chromatography, and peripheral eosinophil cell counts were obtained to observe the circadian rhythm of 6 healthy controls and 7 children with asthma. There was no significant diurnal variation in AS between the two groups. Eosinophil counts of both groups were lower in the morning and higher at night. Theophylline and beta 2 stimulants did not affect these activities significantly. Forty asthmatic children were selected to evaluate AS activity and eosinophil counts during and after attacks. AS-B activity was significantly higher in children during attacks than at other times, 5.70 +/- 2.00 vs. 3.74 +/- 0.66 4 MUnmol/ml/2hr (p less than 0.05). This result was more evident within 24 hours of the attack (p less than 0.01). Eosinophil counts were significantly lower during attack, and there was a negative correlation between the eosinophil counts and AS-B activity. AS-B activity in mild asthmatic children was greater than in severe cases. A significant rise in AS-B was seen in EIB negative asthmatics (p less than 0.01), but no remarkable change was seen in either AS-A or AS-B in the EIB positive group. The data suggest that higher AS-B activity during asthma attacks could inactivate SRS-A and modulate allergic inflammatory reaction.

[Activity of serum arylsulfatase B in nasal allergy patients].

Although the arylsulfatase B has been reported to inactivate slow reacting substance of anaphylaxis (SRS-A) in vitro there has not been studied about the relation between this enzyme and nasal allergy in vivo. The present study was done to examine the serum level of arylsulfatase B in 73 nasal allergy patients and 13 normal controls. Serum arylsulfatase activity was quantified by measurement of the hydrolysis product (p-nitrocatechol) generated by the interaction of this enzyme and a substrate (p-nitrocatechol sulfate, Sigma). The results are summarised as follows; 1. Arylsulfatase B activity is significantly elevated in sera of nasal allergy patients than in that of normal subjects. 2. There are no correlation between the enzyme activity and the number of peripheral blood eosinophiles. 3. There is tendency the severe the nasal obstruction, the lower the level of the enzyme activity.

[Arylsulfatase A and B activity in acute lymphoblastic leukemia in children]. / Aktywnosc arylosulfataz A i B w ostrej bialaczce limfoblastycznej u dzieci.

The activity of arylsulphatase A and B was determined in peripheral blood lymphoblasts in 23 children with acute lymphoblastic leukaemia before starting treatment. A low level of activity was found in the group of leukaemic children with poor prognosis, while in children with better prognostic indices this reactivity was high. The course of changes in this activity was traced also during induction of remission of leukaemia and during 3-year maintenance treatment.

Fine structural localization of arylsulfatase B activity in the rabbit blood platelets.

Fine structural localization of arylsulfatase in the rabbit blood platelets has been investigated in this study. Among many cell organellae, reaction products were exclusively observed in the alpha granules of the platelets. Within the alpha granules, arylsulfatase activity appeared to localize in variable patterns, i.e. reaction products confined mainly at the peripheral region in many granules, while they deposited heavily throughout the granule matrices in some others. In a blood platelets, each alpha granule showed the different staining pattern which indicated more variable functional heterogeneity in the granules.

Lysosomal arylsulfatases A and B from horse blood leukocytes: purification and physico-chemical properties.

Lysosomal arylsulfatases A and B (aryl-sulfate sulfohydrolases, EC 3.1.6.1) from horse leukocytes were purified about 680-fold and 70-fold, respectively, starting from a crude extract of the azurophil and specific granules of leukocytes, by affinity, ion exchange, and gel filtration chromatography. Purified arylsulfatase A displayed anomalous kinetics, a pH optimum at 5.2, an isoelectric point at 4.3, and a Km value for p-nitrocatechol sulfate (pNCS) of 0.37 mM. This enzyme was found to exist in two association states depending on pH: a high molecular weight form at pH 5.0 and a low molecular weight form at pH 7.5. Arylsulfatase B displayed normal kinetics, a pH optimum at 5.8, two isoelectric points at pH 8.6 and 8.9, and a Km value for pNCS of 3.38 mM. The thermostability of the two enzymes was different: arylsulfatase B was found to be more stable than arylsulfatase A. Arylsulfatase A was inhibited by sulfate, sulfite, silver, magnesium, manganese and calcium ions and arylsulfatase B by chloride, sulfate, sulfite and silver ions.

Report of a mucopolysaccharidosis occurring in Australian aborigines.

The first 2 reported cases of a mucopolysaccharidosis occurring in an Australian aboriginal family are presented. Though these children had the characteristic morphological features of the Hurler syndrome, enzyme assay of cultured fibroblasts showed normal levels of alpha-L-iduronidase and decreased activity of arylsulphatase B. Thus, they represented the Hurler syndrome clinically, while they had the enzyme defect of the Maroteaux-Lamy syndrome, and they may represent a new severe form of the Maroteaux-Lamy syndrome. The parents of these children were first cousins. Though the children were not full blood aborigines, examination of the pedigree indicates that the gene originated in the common aboriginal family.

Arylsulfatases A and B in leukocytes: a comparative statistical study of late infantile and juvenile forms of metachromatic leukodystrophy and controls.

We report a statistical study on the level of aryl-sulfatases A and B in leukocytes of 106 controls, 19 cases of metachromatic leukodystrophy (MLD) infantile and juvenile forms and 25 obligate heterozygotes for MLD. Arylsulfatase A has been found to be similarly deficient in patients of the two forms. Half of the mean of the controls have been found in both types for heterozygotes. Arylsulfatase B (ASB) is slightly higher than normal in late infantile MLD although it is not statistically significant. In the 5 cases of the juvenile forms that were examined, ASB was found to be significantly reduced. This enzyme may play a role in relation to the onset of the disease.

Processing enzymes acting on carbohydrate moiety of lysosomal hydrolases in leukemic cells: elevated activity of N-acetylglucosamine-1-phosphotransferase.

We previously demonstrated that an acidic variant form of lysosomal arylsulfatase B accumulated in chronic myelogenous leukemia (CML) cells was highly phosphorylated at its carbohydrate moiety (Uehara Y, et al, Cancer Res 43:5618, 1983). Since lysosomal hydrolases including the sulfatase underwent the posttranslational phosphorylation processing at the carbohydrate moiety, we investigated two enzymes acting on the processing in peripheral leukocytes from leukemia patients. The activity level of the first enzyme in the processing, an N-acetylglucosamine-1-phosphotransferase to form phosphodiester at the carbohydrates, was significantly higher in CML cells than in normal control. The transferase level in CML cells was also higher compared with that in normal bone marrow cells, which include myeloid progenitor cells. However, the activity of the second processing enzyme, a phosphodiester glycosidase that converts a phosphodiester to a phosphomonoester, showed no consistent change in CML cells. Thus, increment of the sulfatase variant containing phosphomonoesters and diesters in CML cells is most probably associated with elevated activities of the phosphotransferase. In two cases of CML in blastic crisis and a case of acute myelogenous leukemia (AML), activity of the processing enzyme was considerably decreased concomitant with reduction of peripheral blastic cells by chemotherapy.

Expression of arylsulfatase B variant from leukocytes in chronic myelogenous leukemia related to chemotherapy.

Lysosomal arylsulfatase B of human leukocytes consisted of two forms; a basic form (B) and a variant form (B1) which is phosphorylated at the carbohydrate chains of the B form (Uehara, Y., Gasa, S., Makita, A., Sakurada, K., and Miyazaki, T. (1983) Cancer Res. 43, 5618-5622). The amounts of the variant form relative to the basic form were considerably increased in leukocytes of chronic myelogenous leukemia (CML). The present communication demonstrates that, upon chemotherapy of the patients with CML, degree of phosphorylation as well as the relative amounts of the phosphorylated variant form of CML leukocytes are markedly decreased concomitantly with an increase of the basic, less phosphorylated form. This effect of chemotherapy on the variant form preceded to clinical improvement of the CML patients, suggesting that the relative amount of the phosphorylated enzyme will be a potential prognostic indicator for the therapeutic effect of CML.

Prenatal exclusion of late infantile metachromatic leucodystrophy in a late-presenting pregnancy by assay of fetal leucocytes.

Metachromatic leucodystrophy was excluded in a fetus at risk, by assay of fetal blood collected at fetoscopy. Isolated fetal leucocytes were shown to have activities of arylsulphatase A and cerebroside sulphatase in the heterozygous range. The prediction was confirmed in the newborn.

Correction of feline arylsulphatase B deficiency (mucopolysaccharidosis VI) by bone marrow transplantation.

Feline and human mucopolysaccharidosis VI (MPS VI or Maroteaux-Lamy syndrome) are inherited autosomal recessive deficiencies of lysosomal enzyme arylsulphatase B. Affected cats and children exhibit lesions caused by incompetent degradation, retinal atrophy and excessive urinary excretion of dermatan facial dysmorphia, corneal stromal opacities, leukocyte granulation, retinal atrophy and excessive urinary excretion of dermatan sulphate--and usually die before adulthood. Most attempts to treat humans affected with MPS VI or other mucopolysaccharidoses have been ineffective or logistically prohibitive, but allogeneic bone marrow transplantation (BMT) offers promise for cure of certain inborn errors of metabolism. Engraftment of normal donor marrow may endow the enzyme-deficient recipient with a continuous source of enzyme-competent blood cells and tissue macrophages to facilitate degradation of stored substrate and to prevent genesis of further malformations. To test this hypothesis, we performed allogeneic BMT in a 2-year-old male Siamese cat with advanced MPS VI. Here we describe BMT-induced correction of this hereditary enzyme deficiency.