Gaucher Spleens Research Articles

Pathologic gene expression in Gaucher disease: up-regulation of cysteine proteinases including osteoclastic cathepsin K

Deficiency of lysosomal acid β-glucosidase induces glycolipid storage in the macrophages of Gaucher disease but the pathways of multisystem tissue injury and destruction are unknown. To investigate the cognate molecular pathology of this inflammatory disorder, genes that were differentially expressed in spleen samples from a patient with Gaucher disease (Gaucher spleen) were isolated. Of 64 complementary DNA (cDNA) fragments sequenced from an enriched Gaucher cDNA library, 5 encode lysosomal proteins (cathepsins B, K, and S, α-fucosidase, and acid lipase), 10 encode other known proteins, and 2 represent novel sequences from human macrophage cell lines. Transcript abundance of the cathepsins, novel genes, pulmonary and activation-regulated chemokine (PARC), and NMB, a putative tumor suppressor gene, was greatly increased. Immunoblotting showed increased mature forms of all 3 cathepsins found in samples of Gaucher spleens. Immunofluorescence microscopy showed strong cathepsin B and K reactions in sinusoidal endothelium and Gaucher cells. The respective means, plus or minus SD, of cathepsin B, K, and S activities were 183 ± 35, 97 ± 39, and 91 ± 45 nmol/min/mg protein in 4 Gaucher spleens, and 26 ± 4, 10.5 ± 2, and 4.0 ± 2.1 nmol/min/mg protein in 3 control spleens. Plasma cathepsin B, K, and S activities were also elevated in Gaucher disease plasma (P < .001), but compared with control plasma samples, neither cathepsin B nor K activities were significantly elevated in 8 patients with nonglycosphingolipid lysosomal storage diseases or in 9 patients with other glycosphingolipidoses, which suggests disease specificity. All 3 cathepsin activities were increased 2-fold to 3-fold in Gaucher sera compared with control sera. In all 6 patients treated by enzyme replacement for 16-22 months, serum cathepsin activities decreased significantly (P < .01). Longitudinal studies confirmed the progressive reduction of proteinase activities during imiglucerase therapy but in 3 Gaucher patients with mild disease not so treated, serum cathepsin activities remained constant or increased during follow-up. Enhanced expression of cysteine proteinases may promote tissue destruction. Moreover, the first identification of aberrant cathepsin K expression in hematopoietic tissue other than osteoclasts implicates this protease in the breakdown of the matrix that characterizes lytic bone lesions in Gaucher disease.

Pathologic gene expression in Gaucher disease: up-regulation of cysteine proteinases including osteoclastic cathepsin K

AbstractDeficiency of lysosomal acid β-glucosidase induces glycolipid storage in the macrophages of Gaucher disease but the pathways of multisystem tissue injury and destruction are unknown. To investigate the cognate molecular pathology of this inflammatory disorder, genes that were differentially expressed in spleen samples from a patient with Gaucher disease (Gaucher spleen) were isolated. Of 64 complementary DNA (cDNA) fragments sequenced from an enriched Gaucher cDNA library, 5 encode lysosomal proteins (cathepsins B, K, and S, α-fucosidase, and acid lipase), 10 encode other known proteins, and 2 represent novel sequences from human macrophage cell lines. Transcript abundance of the cathepsins, novel genes, pulmonary and activation-regulated chemokine (PARC), and NMB, a putative tumor suppressor gene, was greatly increased. Immunoblotting showed increased mature forms of all 3 cathepsins found in samples of Gaucher spleens. Immunofluorescence microscopy showed strong cathepsin B and K reactions in sinusoidal endothelium and Gaucher cells. The respective means, plus or minus SD, of cathepsin B, K, and S activities were 183 ± 35, 97 ± 39, and 91 ± 45 nmol/min/mg protein in 4 Gaucher spleens, and 26 ± 4, 10.5 ± 2, and 4.0 ± 2.1 nmol/min/mg protein in 3 control spleens. Plasma cathepsin B, K, and S activities were also elevated in Gaucher disease plasma (P < .001), but compared with control plasma samples, neither cathepsin B nor K activities were significantly elevated in 8 patients with nonglycosphingolipid lysosomal storage diseases or in 9 patients with other glycosphingolipidoses, which suggests disease specificity. All 3 cathepsin activities were increased 2-fold to 3-fold in Gaucher sera compared with control sera. In all 6 patients treated by enzyme replacement for 16-22 months, serum cathepsin activities decreased significantly (P < .01). Longitudinal studies confirmed the progressive reduction of proteinase activities during imiglucerase therapy but in 3 Gaucher patients with mild disease not so treated, serum cathepsin activities remained constant or increased during follow-up. Enhanced expression of cysteine proteinases may promote tissue destruction. Moreover, the first identification of aberrant cathepsin K expression in hematopoietic tissue other than osteoclasts implicates this protease in the breakdown of the matrix that characterizes lytic bone lesions in Gaucher disease.

In vitro detergent activation of lysosomal acid β-glucosidase in the spleen of normal and type 1 Gaucher patients is not accompanied by change in aggregation state

The genetic defect in Gaucher disease consists in a deficiency of a membrane-bound lysosomal acid β-glucosidase. Using the radiation inactivation method, we have previously reported a subunit coupling of the mutated acid β-glucosidase from Gaucher type 1 spleen in contrast to the normal one (Maret, A., Potier, M., Salvayre, R. and Douste-Blazy, L. (1983) FEBS Lett. 160, 93–97). We have used the same method to determine the effect of detergents on subunit coupling or uncoupling of acid β-glucosidase in normal and Gaucher spleens. The hypothesis that detergent activation of β-glucosidase could be due to subunit association or dissociation has been tested. The radiation inactivation size of β-glucosidase in absence of detergent was 71 000 and 135 500 for normal and Gaucher spleen, respectively, whereas the corresponding values in presence of detergent were 84 000 and 169 000. The higher values obtained in the presence of detergent are incompatible with association or dissociation of subunits but correspond to the increase generally observed for proteins irradiated in the presence of Triton X-100.

The occurrence of two immunologically distinguishable beta-glucocerebrosidases in human spleen.

The beta-glucosidase activity in spleen from control subjects and patients with different clinical phenotypes of Gaucher's disease was characterized. The occurrence of a soluble non-specific beta-glucosidase with a neutral pH optimum and two membrane-associated beta-glucocerebrosidases with an acid pH optimum was demonstrated. The two beta-glucocerebrosidases can be distinguished on the basis of their ability to react with anti-(placental beta-glucocerebrosidase) antibodies bound to protein-A--Sepharose 4B beads. One of the splenic beta-glucocerebrosidases (form I) is precipitated by the immobilized antibodies and the other (form II) is not. The two forms also differ in binding affinity to concanavalin A, degree of stimulation of enzymic activity by taurocholate and isoelectric point. In contrast, the Km values of the two beta-glucocerebrosidases for natural and artificial substrates are similar and both are inhibited by conduritol B-epoxide. In spleen from three patients with type 1, one patient with type 2 and one patient with type 3 Gaucher's disease form I beta-glucocerebrosidase was found to be clearly deficient, whereas the activity of form II was 25-50% of that in control spleen. The non-specific, neutral beta-glucosidase was not deficient in these Gaucher spleens. The distinct biochemical and immunological properties of non-specific beta-glucosidase and the fact that normal levels of the enzyme are present in patients with Gaucher's disease indicate, in confirmation of previous reports, that non-specific beta-glucosidase is not related to beta-glucocerebrosidase.

The cohydrolases in human spleen that stimulate glucosyl ceramide β-glucosidase

A family of β-glucosidase-stimulating proteins (called cohydrolase SPH-I here) was isolated from bovine, Gaucher human and control human spleens. All preparations exhibited a similar pattern of four major electrophoretic bands in polyacrylamide when stained with the cationic dye, Stains-All. The bovine bands migrated more rapidly, while the two types of human cohydrolase migrate very similarly. The two human preparations differend in several respects: the concentration was much higher in Gaucher spleen; the Gaucher factors eluted a little earlier from gel permeation and decyl agarose columns; much more of the cohydrolase was bound by a concanavalin A column; the control bands stained less in gels than the Gaucher bands. Antibodies raised in rabbits to bovine cohydrolase reacted with all three preparations. All four bands evident that the cohydrolases from control and Gaucher spleens are similar in many respects, yet differ in some secondary fashion, possibly in carbohydrate content. It is suggested that Gaucher cohydrolase is formed from normal cohydrolase by the nonenzymatic action of cellular glucose over a period of many years, due to slowed catabolism of the cofactor.

Characterization of lysosomal hydrolases that are elevated in Gaucher's disease

Although Gaucher's disease occurs in three distinct forms with greatly varying degrees of severity, there is no correlation between the clinical course of the disease and levels of residual glucocerebrosidase, the fundamental enzymatic deficiency. In an effort to study secondary changes which might contribute to the pathology of Gaucher's disease, homogenates of spleen, liver, and brain tissue, as well as serum from patients with Gaucher's disease were analyzed for their content of a number of lysosomal enzymes. Extracts of 8 Gaucher spleens contained 3- to 4-fold increases in acid phosphatase activity as well as 5-to 10-fold increases in galactocerebrosidase 5 5 Galactosylceramide β-galactosidase and galactocerebrosidase, as well as lactosylceramide and lactocerebroside, are used interchangeably in the manuscript. activity. The marked elevation in galactocerebrosidase activity in Gaucher spleen was documented using various [ 3H]galactose labeled galactocerebrosides as substrates and with [ 3H]galactose labeled lactocerebroside under the “lactosylceramidase I” 5 assay conditions established by Suzuki (Tanaka, H., and Suzuki, K., 1975, J. Biol. Chem., 250, 2324–2332) that measure galactocerebrosidase activity specifically in the presence of G mi -ganglioside β-galactosidase. Acid phosphatase determinations using extracts of liver from a case of infantile, neuropathic Gaucher's disease revealed a 2-fold elevation in this activity, whereas brain acid phosphatase activity in this case was similar to that of control tissue. Separation of hexosaminidase A and B activities on DEAE-Sephadex columns indicated increases in both forms of the enzyme in Gaucher tissue with the major increase occurring in the hexosaminidase B component. Glucuronidase and nonspecific esterase were observed to be elevated approximately 2-fold. However, not all lysosomal enzyme activities were increased. Levels of splenic arylsulfatase A and B, α-arabinosidase, sphingomyelinase, α-mannosidase, and G mi -ganglioside β-galactosidase activities in Gaucher spleen were unremarkable. G mi -ganglioside β-galactosidase was measured using 4-methylumbelliferyl-β- d-galactopyranoside and [ 3H]galactose labeled lactocerebroside under the specific assay conditions described by Suzuki for the determination of “lactosylceramidase II” activity. Although levels of arylsulfatase A and B in Gaucher spleen were similar to those of control tissue, arylsulfatase A activity was markedly reduced (20% of control) in homogenates of brain from the case of infantile (type 2) Gaucher's disease. The metabolic and pathologic consequences of these changes in lysosomal enzymes in Gaucher's disease are discussed.

Isolation of heat-stable glucocerebrosidase activators from the spleens of three variants of Gaucher's disease

Heat-stable glycoprotein activator substances which stimulate the activity of human liver glucocerebroside:β-glucosidase have been purified extensively from the spleens of five patients representing the clinical extremes of Gaucher's disease ranging from the acute neuropathic form (type 2) to the adult, chronic nonneuropathic form (type 1). Each preparation of Gaucher factor chromatographed as a single coincident peak of protein and activator activity on Sephadex G-150 and each electrophoresed as a single protein band of molecular weight approximately 10,000–12,000 on disc gels in the presence of sodium dodecyl sulfate. With the exception of one preparation, which contained 7.1% carbohydrate, the activator substances contained approximately 17% carbohydrate. The various factors have similar, but not identical, amino acid compositions and are rich in acidic and hydrophobic amino acids. The yield of activator substance ranged from 12.9 to 27.4 mg of protein per 100 g wet wt of tissue, with the highest and lowest yields being obtained from the spleens of two patients with the intermediate form of Gaucher's disease. The amount of glucocerebroside:β-glucosidase-stimulating activity obtained from the spleens of adult and infantile patients was similar. In addition, the purified heat-stable factors from the Gaucher spleens were capable of stimulating the hydrolysis of the galactose-containing ganglioside, G M1, by a partially purified preparation of human liver β-galactosidase. This study documents the presence of a factor that will stimulate the activity of glucocerebroside:β-glucosidase and β-galactosidase in Gaucher spleens but fails to indicate any correlation between the concentration or activity of the stimulatory substances and the extent of pathology or age of onset of the disease; the spleens of patients with the more severe forms of Gaucher's disease do not contain the lowest amounts of stimulatory activity.

Splenic lipids in Gaucher's disease

Column chromatography (on cellulose, silicic acid, and Florisil) and thin-layer chromatography were employed for the separation and purification of lipid fractions from normal and Gaucher spleens. A new hydrolysis procedure, followed by paper chromatography, was used for identification of sugar moieties. A nonhydrolytic combined colorimetric procedure, with anthrone and orcinol, was used for the estimation of glucose and galactose separately in glycolipids. The limitations of this method were examined. Spleens from two control subjects and three patients with Gaucher's disease have been examined in detail. In all Gaucher spleens, the predominant feature was the massive acccumulation of glucocerebroside; neutral ceramide oligohexoside levels were probably within the normal range, as were other neutral lipids and phospholipids. In one case examined for gangliosides, these were increased twentyfold. One Gaucher spleen, in which others had reported that the stored "cerebroside" contained predominantly lactose as the saccharide moiety, has been examined in detail and it has been established that the stored material was, in fact, glucocerebroside, ceramide lactoside levels not being significantly elevated. In a further nine cases glucose was the major sugar detected in the splenic lipids.

Glycolipids isolated from the spleen of Gaucher's disease.

Glycolipids of Gaueher's spleens (three cases) were examined for their content and chemical characterization, comparing with normal control. Apparent accumulation of glucocerebroside was found in all cases. In a case, however, a faster-moving glycolipid fraction than cerebroside on a thin-layer chromatogram, traces of which were detected also in other cases and normal control, was found in a certain amount. From this fraction, a fatty acid ester of glucocerebroside was isolated. Two-thirds of fatty acid of this fraction were composed of the acids below C20. Hematoside which increased in a certain extent, had the same chemical structure, N-acetylneuraminoyl-(2→3)-galactosyl-(1→4)-glucosyl-ceramide, as that of normal human spleen. Other glycolipids separated from Gaucher's spleens showed no significant variation in amounts and were all identical in chemical character with those found already in normal spleen.

Lipids of the spleen in Gaucher's disease

Thin-layer chromatography (TLC) was used to analyze lipids of eight spleens of patients with Gaucher's disease. Four non-Gaucher spleens were also analyzed. Phospholipid concentrations are not increased in Gaucher spleens, while several classes of neutral lipids are moderately increased. Acid hydrolysis followed by hexose determination was applied to total lipids and to separated fractions. Specific oxidase reactions demonstrated that glucose present is primarily in ceramide glucoside and ceramide lactoside, and galactose is primarily in ceramide lactoside. No ceramide galactoside was detected. The two hexoses found account for the anthrone-positive material in the lipid extract. Two TLC spots seen for Gaucher ceramide glucoside differ only with respect to fatty acid chain length. Long-chain base is all C18-sphingosine. Small amounts of hydroxy acids are present in ceramide lactoside, but not in ceramide glucoside. Ceramide lactoside is present in similar amounts in Gaucher and non-Gaucher spleens.

A general method for the preparation of cerebrosides

A new method applicable to spleen and brain tissue for the large-scale preparation of cerebrosides is described. The procedure involves the extraction of total lipides from fresh tissue with a boiling mixture of chloroform-methanol and the isolation of the cerebrosides by virtue of their property of accumulating at the interphase zone when dilute trichloroacetic acid is added to the lipide extract. Cerebroside preparations thus obtained account for 65–75% of the total tissue cerebrosides. Infrared absorption spectra of different cerebroside preparations from three Gaucher spleens and cattle brains are presented.