The role of lysosomal sialidase and β-galactosidase in processing the complex carbohydrate chains on lysosomal enzymes and possibly other glycoproteins

Previous studies using the lectin RCA-I from Ricinus communis have indicated that several lysosomal enzymes in the fibroblasts of patients deficient in β-galactosidase carry excess terminal galactose. Electrophoretic studies have shown that the same enzymes and the non-lysosomal adenosine deaminase also show excess terminal sialic acid in patients deficient in sialidase. In this paper we confirm, using Jack-bean β -galactosidase, that the binding to RCA-I of the purified N -acetyl- β - d -hexosaminidase from a patient with G _{m 1} gangliosidosis depends on a terminal β -linked galactose. We provide evidence, using bacterial sialidase and measuring the binding to RCA-I, for excess subterminal galactose on the enzymes of patients deficient in sialidase. We also show that adenosine deaminase from the fibroblasts of patients deficient in β -galactosidase has increased binding to RCA-I. These observations suggest that in healthy individuals the carbohydrate structure of the precursors of lysosomal enzymes and possibly some other glycoproteins also includes extended carbohydrate side chains with terminal sialic acid and subterminal galactose, and that the mature enzyme extracted from tissues is the product of degradation.     

Lysosomal enzyme levels in human amniotic fluid cells in tissue culture: II. α-galactosidase, β-galactosidase and α-arabinosidase

Changes in the activities of α-galactosidase, β-galactosidase and a-arabinosidase in amniotic fluid cells with time in culture were studied. Marked fluctuations in all three enzymes occurred with passage. In certain cell strains, β-galactosidase showed a marked rise in activity correlated with passage. The activity of all three enzymes, in amniotic fluid cells at the third passage, was correlated with the total time taken to reach con-fluency. There was no consistent pattern of enzyme activity associated with the time after subculture. Enzyme levels in cell strains derived from serial samples of amniotic fluid from several women showed large differences in activity unrelated to gestational age.     

Complementation analysis of human sialidase deficiency using natural substrates

Complementation analysis by somatic cell hybridization to produce heterokaryons has shown that at least three complementation groups exist within the disorders in which the enzyme sialidase is deficient. We have confirmed these results by electrophoretic analysis of two glycoprotein enzymes, adenosine deaminase and acid phosphatase, which show aberrant electrophoretic mobilities in these disorders. These abnormal forms, which have excess sialic acid bound, disappear on complementation and are replaced by normal mobility components. It is suggested that the sialidase produced on complementation uses the abnormal forms as natural substrates and that they may represent normal intermediates in the processing of glycoprotein enzymes.     

gamma-Glutamyl transferase isoenzymes in human bile.

The gamma-glutamyl transferase isoenzymes of bile were studied using electrophoretic, gel filtration, and ultracentrifugation techniques. In view of the known association of other biliary enzymes with lipids the effects of butanol extraction were investigated. The results show the presence of four isoenzymes of gamma-glutamyl transferase in bile, differing in electrophoretic mobilities, molecular size, and density. The correlation between the properties of biliary gamma-glutamyl transferase and of alkaline phosphatase is discussed.     

Lysosomal enzymes in human lymphoblastoid lines: unusual characteristics of RAJI and DAUDI

The lysosomal enzymes N-acetyl hexosaminidase, α-galactosidase and α-mannosidase vary in electrophoretic mobility in different human lymphoblastoid (lymphoid) lines. The relative mobilities of these three enzymes and three out of four other lysosomal enzymes tested correlate well with each other. The patterns appear to be relatively stable characteristics of each line. The lines RAJI and DAUDI show a strikingly fast electrophoretic mobility for all of these enzymes. N-acetyl hexosaminidase is also markedly deficient in DAUDI.     

ENZYME HISTOCHEMICAL CHARACTERISTICS OF NON-HODGKIN'S LYMPHOMAS

Fourteen cases of non-Hodgkin's lymphoma were investigated by both light and electron microscopic enzyme histochemical methods. These cases included 3 mycosis fungoides (MF), 1 chronic lymphocytic leukemia (CLL), 1 hairy cell leukemia (HCL), 5 diffuse histiocytic (DH), 1 nodular histiocytic (NH) and 3 diffuse lymphocytic, poorly differentiated (DLPD) lymphomas. All of these cases except 2 MF and 1 DH were further characterized by an identification of surface markers. Enzymes studied were alkaline phosphatase (AlPase), acid phosphatase (AcPase), β-glucuroddase (β-Gase), adenosine triphosphatase (ATPase), 5'-nucleotidase (YNase), α-naphthyl acetate esterase (α-N. acet eat), naphthol AS-D chloroacetate esterase (N. AS-D chlor eat), naphthol AS acetate esterase (N. AS acet eat) and α-naphthyl butyrate esterase (α-N. but eat). Strong ATPase activity was observed in 4 DH bearing B-cell markers. Four lymphomas (lMF, 2DPLD and 1DH) having T-cell markers never revealed such an ATPase activity. However, lymphomas with T-cell character showed stronger eccentrically localized activity of β-Gase and/or AcPase in the majority of neoplastic cells. One DLPD with B-cell markers showed AlPase activity. Localized α-N. acet eat activity was demonstrated in 2MF, 1DH and 1DLPD. No activity of S'Nase, N. AS-D chlor eat, N. AS acet eat and α-N. but eat was found in the neoplastic cells of all cases. It is suggested that ATPase, β-Gase and AcPase are considered to be an easy method for differentiating either T- or B-cell nature and useful in the classification of non-Hodgkin's lymphomas.     

Sialidosis Type 1

Observations have been made on two brothers who had progressive ataxia, intention myoclonus and visual failure starting early in the third decade of life. Their parents were consanguineous. The brothers showed bilateral cherry red spots at the maculae and bilateral perinuclear cataracts; their intelligence was preserved. Urine was found to contain large amounts of sialylated oligosaccharides; cultured skin fibroblasts showed deficiency of the enzyme sialidase (neuraminidase). Studies on leucocytes and cultured skin fibroblasts showed aberrant electrophoretic mobilities of six enzymes all of which are known to be glycoproteins, and this has been attributed to excessive amounts of sialic acid on the enzyme molecules. The clinical features together with the biochemical findings indicate that these are further cases of the newly described condition Sialidosis Type 1 and it is suggested that the electrophoretic findings might be typical of the condition.     

Appearance of biomarkers of in vitro ageing after successive stimulation of WI-38 fibroblasts with IL-1α and TNF-α: senescence associated β-galactosidase activity and morphotype transition

Sublethal oxidative stresses increase the proportions of human fibroblasts positive for senescence associated β-galactosidase activity and accelerate the transition in the fibroblast morphotypes characterising fibroblast ageing. Stimulation of fibroblasts with TNF-α or IL-1α transiently increases the production of reactive oxygen species (ROS) in human fibroblasts. Here we propose that repeated stimulation of WI-38 fibroblasts with TNF-α or IL-1α can generate enough ROS to accelerate the transition in the fibroblast morphotypes and increase the proportion of cells positive for senescence associated β-galactosidase activity. The involvement of ROS is suggested by experiments where the stimulation of fibroblasts with TNF-α or IL-1α are performed in the presence of N-acetylcysteine which increases the intracellular antioxidant potential. It is proposed that the decrease in the proportions of morphotypes I and II, and the increase in the proportions of morphotypes III to VI observed after successive stimulation with TNF-α or IL1-α is attributed to an increased ROS production occurring during the stimulation.     

The immunophenotype of human decidua and extra-uterine decidual reactions

Decidua associated with products of conception from intra-uterine and extra-uterine gestations and decidualized tissue from the appendix, cervix and Fallopian tube were studied using a panel of antibodies and antisera. Immunolocalization of vimentin and desmin intermediate filament proteins and of α-1-antitrypsin and α-1-antichymotrypsin was identified in most of the 43 cases studied. Placental alkaline phosphatase, β human chorionic gonadotrophin, cytokeratin, smooth-muscle actin and leukocyte markers (CD3, CD20, CD68) were also expressed in some cases. Occasional cases reacted for CD45 and S-100 protein. Similar reaction profiles were obtained at both intra-uterine and extra-uterine sites. The results show that extra-uterine mesenchymal cells which have undergone a decidual reaction correspond closely to their counterparts in the endometrial stroma. Since positive immunostaining within decidual cells for cytokeratins, placental alkaline phosphatase and β human chorionic gonadotrophin indicates that trophoblastic cells are not exclusively recognized by these antibodies, their use does not permit the confident diagnosis of an intra-uterine gestation.     

ENZYME HISTOCHEMICAL PROPERTY OF HUMAN LYMPH NODES – ITS IMMUNOLOGICAL INTERPRETATION –

Lymph nodes surgically removed from patients with various diseases were histochemically investigated. Enzymes examined were alkaline phosphatase (A1P), acid phosphatase (AcP), β-glucuronidase (β-G), and N-acetyl-/3-glucosaminidase (NGa). On the other hand, adult albino rabbits and Wistar rats were immunized by horseradish peroxidase (HrP) mixed with Freund's complete adjuvant and the localizations of these enzymes, antigen HrP and its antibody in their regional lymph nodes at various stages of the immunization were investigated.
AIP in the human lymph nodes was mainly localized in the reticulum cells of the paracortical areas in a characteristic network pattern. This pattern was most prominent in the lymph nodes of SLE patients with elevated serum y-globulin. In the regional lymph nodes of the albino rabbits immunized with HrP, both A1P and anti-HrP antibody were localized along the processes of reticulum cells in the paracortical areas showing a similar network pattern.
The β-G activity in the human lymph nodes was very intense and particulated in plasma cells and lymphocytes In the medullary cords and paracortical areas. The proliferation of these β-G positive plasma cells and lymphocytes in the medullary cords and paracortical areas was most remarkable In SLE patients. In addition, the β-G activity in these cell types responded most readily and steadily to the antigenic stimulation of HrP to both albino rabbits and Wistar rats.     

The mucolipidoses: Identification by abnormal electrophoretic patterns of lysosomal hydrolases

The human mucolipidoses (ML) are characterized by abnormal activities and abnormal electrophoretic patterns of fibroblast lysosomal hydrolases. These altered mobility patterns can be used to confirm the clinical diagnosis of the four mucolipidoses. The mobility patterns of one nonlysosomal and seven lysosomal enzymes were tested in fibroblasts from two ML I (sialidosis type 2, infantile), fifteen ML II (I-cell disease), eight ML III (pseudohurler poly-dystrophy), and one ML IV patients. A single sialidosis type 2, juvenile, line was also examined. Characteristic mobility patterns were found which identify each of the four mucolipidoses. Both the ML I and sialidosis type 2 juvenile lines displayed anodal mobility patterns, but distinct differences between the two disorders were observed. Lysosomal hydrolases from ML II lines demonstrated reduced activities or had altered mobilities. Differing electrophoretic patterns demonstrated the presence of at least two groups within the clinical phenotype diagnosed as ML II, indicating heterogeneity. The ML III lines showed normal electrophoretic patterns for most lysosomal hydrolases. The ML IV line expressed normal mobilities for every enzyme studied, with a single exception. The electrophoretic patterns of only β-hexosaminidase, acid phosphatase-2, α-galactosidase, and esterase A₄ were sufficient to identify and distinguish the different mucolipidosis types. Electrophoretic variation was also seen in liver but not kidney extracts from three ML II patients. β-Hexosaminidase and α-mannosidase B secreted into the medium by ML II and ML III fibroblasts had mobility patterns different from normal and from their intracellular patterns. These data suggest that the mucolipidoses are genetically distinct with heterogeneity within them.     

Spondyloepiphyseal dysplasia, corneal clouding, normal intelligence and acid β-galactosidase deficiency

A 14-year-old girl with a unique type of progressive spondyloepiphyseal dysplasia, corneal clouding, and no evidence of neurological abnormality, was found to have a remarkable deficiency of acid β-galactosidase activity in cultured skin fibroblasts and in leucocyte preparations. In fibroblasts, ganglioside GM₁β-galactosidase activity averaged 7 % of the normal mean while asialofetuin β-galactosidase and 4-methylumbelliferyl-β-galactosidase averaged 1.4 % and 3.5 %, respectively. Activities for all three substrates in leucocytes from both her parents were close to 50 % of the normal mean indicating that the patient is homozygous for a mutation (or mutations) affecting GM₁β-galactosidase.     

Molecular genetics of GM1β-galactosidase

The molecular genetics of GM₁β-galactosidase is reviewed. This enzyme exists in two forms, A and B. Form A is monomeric with a molecular weight of 72,000 and appears to be coded by a single autosomal locus. Form B is polymeric and cross-reacts with anti-A antibodies; it is coded wholly or in part by the same locus that codes for A. The simultaneous loss of A and B in GM₁ gangliosidosis is explained. None of the other β-galactosidases, including neutral β-galactosidase, ceramide lactoside β-galactosidase or cerebroside β-galactosidase cross-react with anti-A antibodies, demonstrating that they are coded by loci separate from A. GM₁, β-galactosidase A is heterocatalytic, cleaving β-Dgalactose from ganglioside GM₁, lactose, N-acetyllactosamine, and galactose-containing glycoproteins such as asialofetuin, red cell stromal glycoproteins and keratan sulfate. The pleotropic effects of a single mutation affecting the locus for β-galactosidase A can be explained by a one gene: one polypeptide: many substrates model. Phenotypic variability among β-galactosidase A mutants may result from better residual activity of the mutant enzyme for one substrate than for another. Patients with normal intelligence and severe bony deformities, who are homozygous for a mutation affecting the enzyme, illustrate this point. Thus far all human mutants for GM₁, β-galactosidase studied are structural mutants, synthesizing nearly normal quantities of mutant enzyme; one is a proven Km mutant, the others are very likely so.     

The use of graft copolymers as enzyme supports, 3. The Immobilization of proteins and enzymes on poly(fluorostyrene) and poly(nitrofluorostyrene) — nylon graft copolymers

The radiation induced, graft copolymerization of m- and p-fluorostyrene onto nylon was successfully achieved. Grafting was followed by nitration of the fluorostyrene groups in the copolymer to provide substrates which were active to the coupling of various enzymes and proteins. Bovine serum albumin, ovalbumin, pepsin, β-galactosidase, and alkaline phosphatase proteins all coupled very efficiently but the activities of the coupled enzymes were relatively low.     

Response of lung enzyme activities in rabbits following short-term exposure ton-hexane: Correlation between morphological and biochemical changes

The activity of lactatedehydrogenase, -glucuronidase, glucose-6-phosphate dehydrogenase, acid and alkaline phosphatase was studied in lung homogenate from New Zealand rabbits exposed to 3000 p.p.m. ofn-hexane 8 h per day for 8 days or filtered air.In hydrocarbon-treated animals all enzymes examined, except alkaline phosphatase, were markedly increased.The biochemical changes correlated well with the morphological changes and the results of cytological evaluation of bronchopulmonary lavage. It is suggested that high values in lung lysosomal enzymes from treated rabbits reflect the acute inflammation whilst the increase in lung glueose-6-phosphate dehydrogenase may depend upon reparative process subsequent ton-hexane-induced lung damage.     

The role of cereal and fungal aylases in cereal flour hypersensitivity

Abstract. To investigate the role of cereal α . and β-amylase in bakers' asthma, we have compared the IgE response of 30 wheat-flour-allergic individuals to barley α and β-amylases with that of fungal α-amylase using radioallergosorbenl test (RAST), RAST inhibition assays and Western blotting. RAST analysis showed 29 of the 30 subjects with inhalant induced cereal allergy had positive IgE to cereal amylases, but only 16 were positive to fungal α-amylase. Regression analysis showed an association between specific IgE to wheat-flour and to barley α-amylase ( r = 0.70) and barley β-amylase ( r = 0.92) but a poor association with fungal α-amylase (r = 0–34). RAST inhibition showed minimal crossreactivity between barley α or β-amylase and barley and fungal α-amylase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that non-reduced barley α-amylase had a molecular weight of 54 kDa and barley β-amylase a molecular weight of 64 kDa. Reduced fungal α-amylase had a molecular weight of 54 k Da. Cereal α and β-amylase appear to be important allergens in patients with allergy to flour.     

Integrin distribution in gastric carcinoma: Association of β3 and β5 integrins with tumor invasiveness

Immunolocalization of a variety of integrins using monoclonal antibodies against β3, β5, α3β1 and α6, and polyclonal antibodies against vitronectin receptor (αvβ3) and l were investigated on PLP-fixed paraffin sections of 19 cases of advanced gastric carcinomas. The β5 integrin, which pairs only with the av subunit, was positive in seven cases (37%), and was associated closely with scirrhous invasion (P < 0.05). β5 was positive chiefly in the cytoplasm of carcinoma cells and infrequently in cell membranes. β3, which is another subunit pairing with av, was positive in six cases (32%), and tended to be associated with scirrhous invasion (P < 0.1). β3 was also located chiefly in the cytoplasm. Five of the seven β5-positive cases showed coexpression of β3. Polyclonal antibodies to αvβ3 also showed a significant difference among the amounts of stroma (P < 0.05). Anti-β1 antibodies showed clear positivity in many cases (89%). Of the β1 integrins, α3β1 was positive in a few cases (26%) without any preferential pattern, and laminin receptor subunit α6 stained on cell membranes of neoplastic epithelia in many cases of carcinoma (89%) except for mucinous carcinoma. These distinctive patterns of integrin positivity indicate a close association of β5 and β3 expression with scirrhous invasion in gastric carcinoma.     

Proton modulation of recombinant GABAA receptors: influence of GABA concentration and the β subunit TM2–TM3 domain

Regulation of GABA_A receptors by extracellular pH exhibits a dependence on the receptor subunit composition. To date, the molecular mechanism responsible for the modulation of GABA_A receptors at alkaline pH has remained elusive. We report here that the GABA-activated current can be potentiated at pH 8.4 for both αβ and αβγ subunit-containing receptors, but only at GABA concentrations below the EC₄₀. Site-specific mutagenesis revealed that a single lysine residue, K279 in the β subunit TM2–TM3 linker, was critically important for alkaline pH to modulate the function of both α1β2 and α1β2γ2 receptors. The ability of low concentrations of GABA to reveal different pH titration profiles for GABA_A receptors was also examined at acidic pH. At pH 6.4, GABA activation of αβγ receptors was enhanced at low GABA concentrations. This effect was ablated by the mutation H267A in the β subunit. Decreasing the pH further to 5.4 inhibited GABA responses via αβγ receptors, whereas those responses recorded from αβ receptors were potentiated. Inserting homologous β subunit residues into the γ2 subunit to recreate, in αβγ receptors, the proton modulatory profile of αβ receptors, established that in the presence of β2^H267, the mutation γ2^T294K was necessary to potentiate the GABA response at pH 5.4. This residue, T294, is homologous to K279 in the β subunit and suggests that a lysine at this position is an important residue for mediating the allosteric effects of both acidic and alkaline pH changes, rather than forming a direct site for protonation within the GABA_A receptor.     

Hunter disease (mucopolysaccharidosis type II) in a karyotypically normal girl

A female child of healthy, unrelated parents presented at 12 months of age with a history of moderately severe developmental delay, macrocephaly, dysmorphic facies, hypotonia, hepato-splenomegaly, mild generalized dysostosis multiplex, mucopolysacchariduria (dermatan and heparan sulfates), and Alder-Reilly bodies in peripheral blood leukocytes. Iduronate sulfatase activity in plasma was markedly depressed: 0.11 units/ml/h (normal, 1.75 ±0.56, N = 6). Analyses of arylsulfatases A, B, and C, heparan N-sulfatase, α-mannosidase, β-mannosidase, β-glucuronidase, β-hexosaminidase, β-galactosidase, and α-fucosidase activities in plasma, leukocytes, and/or cultured skin fibroblasts were all normal. Urinary sulfatide excretion was also within normal limits. Karyotypes of peripheral blood leukocytes and cultured skin fibroblasts were normal. Serum iduronate sulfatase activities in the parents were in the normal range (father, 1.63 units/ml/h; mother, 1.25 units/ml/h). The results of analyses of restriction fragment length polymorphisms (RFLP) of DNA from cultured skin fibroblasts with the use of probes for loci extending from Xpter to Xq28 showed X chromosome heterozygosity and confirmed the paternal origin of one of the X chromosomes. Studies on sulfur-35 uptake in mixed fibroblast cultures showed cross-correction of [³⁵S]-glycosaminoglycan accumulation between cells from the patient and normal cells or cells from a patient with Hurler disease; however, there was no cross-correction between cells from the patient and those from boys affected with classical Hunter disease. This represents only the second confirmed case of Hunter disease reported in a karyotypically normal girl.     

Electrophoretic Polymorphism of Human C4 is Due to Charge Differences in the α-Chain, Presumably in the C4d Fragment

Various common C4 gene products were isolated from serum by immunoprecipitation. After reduction the C4 α-, β-, and γ-polypeptide chains were studied by two-dimensional electrophoresis. Isoelectrofocusing was performed in the first dimension and sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis in the second. The charge differences behind the electrophoretic C4 polymorphism were shown to reside in the 95,000-u(atmic mass units) α-chain. Charge variation closely mirroring the α-chain differences were also found in a 49,000-u fragment of the α-chain, most probably C4d. The basic β-chain could not be studied in detail, but no differences were observed with regard to molecular weight or charge of the γ-chains of the different C4 gene products.