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Cytochemical analysis of storage materials in cultured skin fibroblasts from patients with I-cell disease
Clinica Chimica Acta 378 (2007) 142 – 146 www.elsevier.com/locate/clinchim
Cytochemical analysis of storage materials in cultured skin fibroblasts from patients with I-cell disease Ikuo Kawashima a , Mai Ohsawa a,b , Tomoko Fukushige c , Yoshihisa Nagayama d , Yo Niida e , Masaharu Kotani a,f , Youichi Tajima a , Takuro Kanekura c , Tamotsu Kanzaki c , Hitoshi Sakuraba a,⁎ a
Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan b Japan Science and Technology Agency, Kawaguchi 332-0012, Japan c Department of Dermatology, Kagoshima University Graduate School of Medicine, Kagoshima 890-8520, Japan d Department of Pediatrics, Niigata City General Hospital, Niigata 950-8739, Japan e Department of Pediatrics, Kanazawa University Graduate School of Medicine, Kanazawa 920-8641, Japan f Department of Molecular Biology, School of Pharmaceutical Science, Ohu University, Koriyama 963-8611, Japan Received 8 August 2006; received in revised form 4 October 2006; accepted 16 November 2006 Available online 28 November 2006
Abstract Background: In cultured fibroblasts from I-cell disease patients the transport of many lysosomal enzymes is defective, and affected cells contain inclusion bodies filled with undegraded substrates. However, the contents of these inclusion bodies have not been well characterized yet. We attempted to identify accumulated substances in cultured I-cell disease fibroblasts cytochemically. Methods: Cultured fibroblasts from I-cell disease patients were double-stained with a monoclonal antibody to lysosome-associated membrane protein-1 (LAMP-1) and that to GM2 ganglioside, or a series of lectins that specifically bind to sugar moieties. Results: The patients' cells were granularly stained with the antibody to GM2 ganglioside and the lectins including Maakia amurensis, Datura stramonium, and concanavalin A. Their localization was coincident with that of LAMP-1. Conclusions: GM2 ganglioside and various kinds of glycoconjugates having sialic acidα2-3galactose, galactoseβ1-4N-acetylglucosamine and mannose residues accumulate in enlarged lysosomes in I-cell disease fibroblasts. © 2006 Elsevier B.V. All rights reserved. Keywords: Cytochemistry; Electronic microscopy; GM2 gangglioside; I-cell disease; Lectin
1. Introduction I-cell disease is a rare genetic disease caused by a deficiency of the enzyme, uridine diphosphate-N-acetylglucosamine: lysosomal enzyme N-acetylglucosaminyl-1-phosphotransferase (phosphotransferase) [1]. Patients with I-cell disease exhibit clinical manifestations including progressive psychomotor Abbreviations: LAMP-1, lysosome-associated membrane protein-1; BSA, bovine serum albumin; PBS, phosphate-buffered saline; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; MAM, Maakia amurensis; DSA, Datura stramonium; Con A, concanavalin A; PNA, Arachis hypogaea agglutinin; SSA, Sambucus sieboldiana; MPS, mucopolysaccharidosis. ⁎ Corresponding author. Tel.: +81 3 3823 2105; fax: +81 3 3823 6008. E-mail address: [email protected] (H. Sakuraba). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.11.019
retardation, coarse facial features, skeletal abnormalities, thick skin, gingival hyperplasia and hernias, which resemble the manifestations in Hurler syndrome but appear earlier and do not include mucopolysacchariduria [1]. As a defect of phosphotransferase results in the abnormal transport of many lysosomal enzymes in cells of mesenchymal origin, these enzyme activities in cultured skin fibroblasts from patients with I-cell disease are decreased, and affected cells contain inclusion bodies filled with undegraded substrates [1]. I-cell disease is pathologically characterized by the presence of these inclusion bodies. However, the contents of such inclusion bodies have not been well characterized, but probably consist of glycoconjugates including oligosaccharides, glycoproteins, glycolipids and mucopolysaccharides [2–5].
I. Kawashima et al. / Clinica Chimica Acta 378 (2007) 142–146 Table 1 Lysosomal enzyme activities in cultured skin fibroblasts Specific activities (nmol/h/mg protein) Patient-1 β-Hexosaminidase A Total β-hexosaminidases α-Galactosidase β-Galactosidase α-Glucosidase α-Fucosidase β-Glucuronidase Lysosomal sialidase α-Mannosidase β-Glucosidase
135 941 2 7 90 2 19 0 9 188
Patient-2 102 594 1 6 39 1 12 0 13 163
Control-1
Control-2
3
2.62 × 103 16.7 × 103 90 1.37 × 103 286 123 127 298 41 327
2.61 × 10 14.9 × 103 84 1.28 × 103 219 81 83 298 45 238
We attempted to identify accumulated substances in cultured fibroblasts from I-cell disease patients by cytochemical staining with antibodies to lysosome-associated membrane protein-1 (LAMP-1) and GM2 ganglioside, and a series of lectins that specifically bind to glycoconjugates. 2. Materials and methods 2.1. Patients Patient-1 was a 17-month-old female child of healthy non-consanguineous Japanese parents. She developed severe psychomotor delay, coarse facies, gingival hypertrophy, thick skin and restricted joint movement. Radiographic examinations revealed a vertebral deformity and widening of the ribs, and
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vacuolated lymphocytes were found in a peripheral blood smear. Urinary mucopolysaccharide was negative. The activities of lysosomal enzymes including β-hexosaminidase A and β-galactosidase in plasma were 10- to 20fold increased compared with those in control subjects. Patient-2 was a 22-month-old Japanese female. Coarse facies and hypotonus were noticed at birth. Then she developed failure to thrive, psychomotor delay, restricted joint movement, hepatosplenomegaly, recurrent respiratory tract infection, vacuolated lymphocytes and dysostosis multiplex. Lysosomal enzyme assaying revealed elevated levels of β-hexosaminidase A, β-galactosidase and β-glucuronidase activities in plasma. Mucopolysacchariduria was not found.
2.2. Biopsying of skin tissues and culture of skin fibroblasts Skin tissues were obtained from the patients with I-cell disease and normal control subjects by means of skin biopsying, and then used for morphological analysis and establishment of cultured fibroblasts. Informed consent was obtained from the corresponding families. This study involving cultured fibroblasts was approved by the Ethical Committee of our institute. The established skin fibroblasts were cultured in Ham's F-10 medium supplemented with 10% fetal calf serum and antibiotics at 37 °C under a 5% CO2–95% air mixture.
2.3. Lysosomal enzyme assays and protein determination Cultured skin fibroblasts from the I-cell disease patients and normal controls were cultured in fresh medium for 2 days. Then, the activities of lysosomal enzymes including β-hexosaminidase A, total β-hexosaminidases (mainly β-hexosaminidases A and B), α-galactosidase, β-galactosidase, α-glucosidase, α-fucosidase, β-glucuronidase, lysosomal sialidase, α-mannosidase and β-glucosidase in cultured fibroblasts were measured fluorometrically with 4-methylumbelliferyl derivatives, as described previously [6]. Protein determination was performed with a Bio-Rad dye-binding assay kit (Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as a standard.
Fig. 1. Electron microscopic analysis. a. Biopsied skin tissue from Patient-1, scale bar = 10 μm). b. A cultured skin fibroblast from Patient-1, scale bar = 1 μm. c. Magnification of a square in b. An arrow head and arrows indicate a nucleus and lysosomes, respectively, scale bar = 0.5 μm. d. A cultured skin fibroblast from Patient2, scale bar = 1 μm. e. A cultured skin fibroblasts from a normal subject, scale bar = 1 μm.
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2.4. Morphological analysis Biopsied skin tissues and skin fibroblasts cultured on 25 mm round plastic Lab-Tek slips (Miles Laboratories, Naperville, IL) were fixed in cold 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/l phosphate buffer, pH 7.4, for 30 min. Then, the specimens were processed as described previously [7], and morphologically examined under an electron microscope (H-7100; Hitachi, Tokyo, Japan).
2.5. Cytochemical analysis of cultured skin fibroblasts Cultured skin fibroblasts from the patients with I-cell disease and controls were cultured on Lab-Tek chamber slides (Nunc, Naperville, IL) for 3 days. Then, the cells were washed with PBS and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, for 10 min at room temperature, washed with PBS, with followed by blocking with 1% BSA in PBS for 30 min as described previously [8]. For double immunostaining with both a mouse monoclonal antibody (mAb) to LAMP-1 as a marker of lysosomes (1:100 diluted, IgG isotype; Southern Biotechnology, Birmingham, AL) and a mouse mAb to GM2 ganglioside (GM2; culture supernatant, IgM isotype) [9], the cells were incubated with a mixture of two mAbs, for 1 h at room temperature. The cells were washed with 1% BSA-PBS and then incubated with a mixture of rhodamine isothiocyanate-conjugated goat anti-mouse IgG F(ab′)2 (1:200 diluted; Jackson Immuno Research, West Grove, PA) and a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM F(ab′)2 (1:100 diluted; Jackson Immuno Research) for 1 h at room temperature.
For double immunostaining with both mAb to LAMP-1 and a FITCconjugated lectins, including Maakia amurensis (MAM; binding specificity, sialic acidα2-3galactose, Seikagaku, Tokyo, Japan), Datura stramonium (DSA; binding specificity, galactoseβ1-4N-acetylgucosamine, Seikagaku), concanavalin A (Con A; binding specificity, mannose, Vector Laboratories, Inc., Burlingame, CA) (each 1 μg/ml), Arachis hypogaea (peanut) agglutinin (PNA; binding specificity, galactoseβ1-3N-acetylgalactosamine > galactose, Seikagaku), and Sambucus sieboldiana (SSA; binding specificity, sialic acidα26galactose/N-acetylgalactosamine, Seikagaku) (each 10 μg/ml), the cells were incubated with a mixture of mAb to LAMP-1 and FITC-conjugated lectins including MAM, DSA, Con A, PNA, and SSA for 1 h at room temperature. The cells were washed with 1%BSA-PBS and then treated with rhodamine isothiocyanate-conjugated goat anti-mouse IgG F(ab′)2 for 1 h at room temperature. After washing with PBS, the slides were embedded with PermaFluor Aqueous Mounting Medium (Thermo Electron Corporation, MA) and mounted with coverglasses. The stained cells were examined under a microscope (Axiovert 135; Carl Zeiss, Oberkochen, Germany) equipped with an Axio Vision 3.1 system.
3. Results 3.1. Enzyme assay As shown in Table 1, intracellular lysosomal enzyme activities including those of β-hexosaminidase A, total β-hexosaminidases,
Fig. 2. Cytochemical analysis of GM2 ganglioside in cultured skin fibroblasts. Cultured skin fibroblasts from a control (C), Patient-1 (P-1), and Patient-2 (P-2) were double-stained with an antibody to lysosome-associated membrane protein-1 (LAMP-1) and that of GM2 ganglioside (GM2). Phase, LAMP-1, GM2, and Merge indicate phase contrast, LAMP-1 single-fluorescence (Red), GM2 single-fluorescence (Green), LAMP-1 and GM2 double-fluorescence (Yellow), respectively. Scale bar = 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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α-galactosidase, β-galactosidase, α-glucosidase, α-fucosidase, β-glucuronidase, lysosomal sialidase and α-mannosidase were decreased, but not that of β-glucosidase, which is one of the membranous lysosomal enzymes.
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tose, galactoseβ1-4N-acetylglucosamine and mannose residues accumulate in increased and/or enlarged lysosomes in the cultured I-cell disease fibroblasts. 4. Discussion
3.2. Electron microscopy Electron microscopy of the I-cell disease skin tissue specimens demonstrated extensive cytoplasmic electron-lucent vacuolation of lysosomes in fibroblasts in the dermis (Fig. 1a). Cells in the epidermis contained many electron-dense inclusion bodies in their cytoplasm (Fig. 1a). The cultured I-cell disease fibroblasts contained numerous membrane bound-vacuoles containing electron-lucent, electron-dense, fibrillogranular and lamellar materials (Fig. 1b, c, and d). 3.3. Cytochemical analysis The results of cytochemical analysis are shown in Figs. 2 and 3. Marked granular fluorescence was observed in fibroblasts from the I-cell disease patients when the cells were stained with antibodies to LAMP-1 and GM2 (Fig. 2), and lectins including MAM, DSA and Con A (Fig. 3). Only weak fluorescence for LAMP-1, GM2, MAM, DSA, and ConA was detected in control cells. Their localization was coincident with that of LAMP-1. There were no differences in the staining patterns of PNA and SSA between the patients' cells and those of controls (data not shown). These results suggest that large amounts of GM2 ganglioside and glycoconjugates having sialic acidα2-3galac-
Lysosomal enzymes are synthesized on endoplasmic reticulum (ER)-bound ribosomes, and modified in the ER through cleavage of the signal peptide and the addition of high-mannose type sugar chains. Then, further modification of the sugar chains occurs and the addition of a mannose 6-phosphate residue, which is a marker for targeting of non-membranous lysosomal enzymes except for membranous enzymes including β-glucosidase, takes place in the Golgi apparatus [1]. The nonmembranous lysosomal enzymes having mannose 6-phosphate residues at the non-reducing ends of sugar chains are transported to endosomes via mannose 6-phosphate receptors. Phosphotransferase is a key enzyme in the generation of the mannose 6-phosphate marker and catalyzes the first step of the process, adding a N-acetylglucosamine 1-phosphate residue to the 6th position of a mannose on sugar chains [1]. Deficient phosphotransferase activity results in defective synthesis of the mannose 6-phosphate marker and causes abnormal transport of non-membranous lysosomal enzymes. As a result, the activities of non-membranous lysosomal enzymes in cultured fibroblasts from I-cell disease patients decrease. The present study revealed that the activities of β-hexosaminidase A, α-galactosidase, βgalactosidase, α-fucosidase, lysosomal sialidase and α-mannosidase were below 10% of the control levels. The results suggest
Fig. 3. Lectin staining of cultured skin fibroblasts. Cultured skin fibroblasts from a control (C), Patient-1 (P-1), and Patient-2 (P-2) were double-stained with an antibody to LAMP-1 and lectins including Maakia amurensis (MAM), Datura stramonium (DSA), and concanavalin A (Con A). LAMP-1, lectins (MAM, DSA, and ConA), and Merge indicate LAMP-1 single-fluorescence (Red), lectin single-fluorescence (Green), LAMP-1 and lectin double-fluorescence (Yellow), respectively. Scale bar = 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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complex accumulation of substrates of these enzymes in lysosomes in cultured I-cell disease fibroblasts. The morphological results suggested storage of oligosaccharides, glycoproteins and glycolipids in lysosomes. In this study, we analyzed storage materials in cultured I-cell disease fibroblasts by means of cytochemistry with antibodies to LAMP-1 and GM2 ganglioside, and lectins that specifically bind to glycoconjugates. Strong granular fluorescence for antibodies to LAMP-1 and GM2, and lectins including MAM, DSA, and ConA was found in the I-cell disease fibroblasts. The results showed increased and/or enlarged lysosomes with excessively accumulated GM2 and glycoconjugates having sialic acidα2-3galactose, galactoseβ1-4N-acetylglucosamine and mannose residues. The accumulation of these substrates is probably due to simultaneously decreased activities of βhexosaminidase A, lysosomal sialidase, β-galactosidase and αmannosidase, respectively. On the other hand, only faint fluorescence was found both in the patient's cells and control cells when the cells were stained with PNA and SSA. The results suggests that the metabolic pathway of glycoconjugates with galactoseβ1-3N-acetylgalactosamine and sialic acidα2-6 galactose/N-acetylgalactosamine residues is not dominant in cultured fibroblasts. Recently, enzyme replacement therapy was introduced for several lysosomal diseases including Gaucher disease [10], Fabry disease [11,12], mucopolysaccharidosis (MPS) I [13], MPS II [14,15], MPS VI [16], and Pompe disease [17]. Therapeutic trials including bone marrow transplantation have also been performed for I-cell disease [18], and efforts are being made to develop effective therapies. For that purpose, establishment of methods for identifying accumulated materials in I-cell disease fibroblasts is very important. We developed a cytochemical method with specific antibodies and lectins for glycoconjugates and identified accumulated glycoconjugates in I-cell disease fibroblasts. The present cytochemical method is sensitive and easy, and will be useful for studying the pathophysiology and evaluation of therapeutic procedures for I-cell disease. Furthermore, cytochemical staining of cultured fibroblasts with MAM, DSA, and ConA and an antibody to GM2 would help diagnosis of I-cell disease. Acknowledgements This work was partly supported by grants from the Japan Science and Technology Agency, the Tokyo Metropolitan Government, the Japan Society for the Promotion of Science, the Ministry of Education, Science, Sports and Culture, and the Ministry of Health, Labor and Welfare of Japan. References [1] Kornfeld S, Sly WS. I-cell disease and pseudo-Hurler polydystrophy: disorders of lysosomal enzyme phosphorylation and localization. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 2001. p. 3469–82.
[2] Hieber V, Distler J, Jourdian GW, et al. Accumulation of 35Smucopolysaccharides in cultured mucolipidosis cells. Birth Defects 1975;6:307–10. [3] Dawson G, Matalon R, Dorfman A. Glycosphingolipids in cultured human skin fibroblasts. II. Characterization and metabolism in fibroblasts from patients with inborn errors of glycosphingolipid and mucopolysaccharide metabolism. J Biol Chem 1972;247:5951–8. [4] Thomas GH, Tiller Jr GE, Reynolds LW, et al. Increased levels of sialic acid associated with sialidase deficiency in I-cell disease (mucolipidosis II) fibroblasts. Biochem Biophys Res Commun 1976;71:188–95. [5] Virtanen I, Ekblom P, Laurila P, et al. Characterization of storage material in cultured fibroblasts by specific lectin binding in lysosomal storage diseases. Pediatr Res 1980;14:1199–203. [6] Suzuki K. Enzymatic diagnosis of sphingolipidoses. Methods Enzymol 1987;138:727–62. [7] Kanzaki T, Kitajima S, Suzumori K. Biological behavior of cloned cells of human malignant fibrous histiocytoma in vivo and in vitro. Cancer Res 1991;51:2133–7. [8] Kotani M, Yamada H, Sakuraba H. Cytochemical and biochemical detection of intracellularly accumulated sialyl glycoconjugates in sialidosis and galactosialidosis fibroblasts with Maakia amurensis. Clin Chim Acta 2004;344:131–5. [9] Kotani M, Kawashima I, Ozawa H, et al. Generation of one set of murine monoclonal antibodies specific for globo-series glycolipids: evidence for differential distribution of the glycolipids in rat small intestine. Arch Biochem Biophys 1994;310:89–96. [10] Barton NW, Brady RO, Dambrosia JM, et al. Replacement therapy for inherited enzyme deficiency — macrophage-targeted glucocerebrosidase for Gaucher's disease. N Engl J Med 1991;324:1464–70. [11] Schiffmann R, Murray GJ, Treco D, et al. Infusion of alpha-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease. Proc Natl Acad Sci U S A 2000;97:365–70. [12] Eng CM, Guffon N, Wilcox WR, et al. Safety and efficacy of recombinant human alpha-galactosidase A replacement therapy in Fabry's disease. N Engl J Med 2001;345:9–16. [13] Wraith JE, Clarke LA, Beck M, et al. Enzyme replacement therapy for mucopolysaccharidosis I: A randomized, double-blinded, placebo-controlled, multinational study of recombinant human alpha-L-iduronidase (laronidase). J Pediatr 2004;144:581–8. [14] Bielicki J, Hopwood JJ, Wilson PJ, et al. Recombinant human iduronate-2-sulphatase: correction of mucopolysaccharidosis-type II fibroblasts and characterization of the purified enzyme. Biochem J 1993;289:241–6. [15] No authors listed. Idursulfase: I2S, iduronato-2-sulfatase. Drugs R D 2006;7:254–258. [16] Harmatz P, Ketteridge D, Giugliani R, et al. Direct comparison of measures of endurance, mobility, and joint function during enzymereplacement therapy of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome): results after 48 weeks in a phase 2 open-label clinical study of recombinant human N-acetylgalactosamine 4-sulfatase. Pediatrics 2005; 115:681–9. [17] Kilinge L, Strab V, Neudorf U, et al. Safety and efficacy of recominant acid alpha-glucosidase (rhGAA) in patients with classical infantile Pompe disease: results of phase II clinical trial. Neuromuscul Disord 2005; 15:24–31. [18] Kurobane I, Inoue S, Gotoh Y, et al. Biochemical improvement after treatment by bone marrow transplantation in I-cell disease. Tohoku J Exp Med 1986;150:63–8.
Cytochemical analysis of storage materials in cultured skin fibroblasts from patients with I-cell disease Ikuo Kawashima a , Mai Ohsawa a,b , Tomoko Fukushige c , Yoshihisa Nagayama d , Yo Niida e , Masaharu Kotani a,f , Youichi Tajima a , Takuro Kanekura c , Tamotsu Kanzaki c , Hitoshi Sakuraba a,⁎ a
Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan b Japan Science and Technology Agency, Kawaguchi 332-0012, Japan c Department of Dermatology, Kagoshima University Graduate School of Medicine, Kagoshima 890-8520, Japan d Department of Pediatrics, Niigata City General Hospital, Niigata 950-8739, Japan e Department of Pediatrics, Kanazawa University Graduate School of Medicine, Kanazawa 920-8641, Japan f Department of Molecular Biology, School of Pharmaceutical Science, Ohu University, Koriyama 963-8611, Japan Received 8 August 2006; received in revised form 4 October 2006; accepted 16 November 2006 Available online 28 November 2006
Abstract Background: In cultured fibroblasts from I-cell disease patients the transport of many lysosomal enzymes is defective, and affected cells contain inclusion bodies filled with undegraded substrates. However, the contents of these inclusion bodies have not been well characterized yet. We attempted to identify accumulated substances in cultured I-cell disease fibroblasts cytochemically. Methods: Cultured fibroblasts from I-cell disease patients were double-stained with a monoclonal antibody to lysosome-associated membrane protein-1 (LAMP-1) and that to GM2 ganglioside, or a series of lectins that specifically bind to sugar moieties. Results: The patients' cells were granularly stained with the antibody to GM2 ganglioside and the lectins including Maakia amurensis, Datura stramonium, and concanavalin A. Their localization was coincident with that of LAMP-1. Conclusions: GM2 ganglioside and various kinds of glycoconjugates having sialic acidα2-3galactose, galactoseβ1-4N-acetylglucosamine and mannose residues accumulate in enlarged lysosomes in I-cell disease fibroblasts. © 2006 Elsevier B.V. All rights reserved. Keywords: Cytochemistry; Electronic microscopy; GM2 gangglioside; I-cell disease; Lectin
1. Introduction I-cell disease is a rare genetic disease caused by a deficiency of the enzyme, uridine diphosphate-N-acetylglucosamine: lysosomal enzyme N-acetylglucosaminyl-1-phosphotransferase (phosphotransferase) [1]. Patients with I-cell disease exhibit clinical manifestations including progressive psychomotor Abbreviations: LAMP-1, lysosome-associated membrane protein-1; BSA, bovine serum albumin; PBS, phosphate-buffered saline; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; MAM, Maakia amurensis; DSA, Datura stramonium; Con A, concanavalin A; PNA, Arachis hypogaea agglutinin; SSA, Sambucus sieboldiana; MPS, mucopolysaccharidosis. ⁎ Corresponding author. Tel.: +81 3 3823 2105; fax: +81 3 3823 6008. E-mail address: [email protected] (H. Sakuraba). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.11.019
retardation, coarse facial features, skeletal abnormalities, thick skin, gingival hyperplasia and hernias, which resemble the manifestations in Hurler syndrome but appear earlier and do not include mucopolysacchariduria [1]. As a defect of phosphotransferase results in the abnormal transport of many lysosomal enzymes in cells of mesenchymal origin, these enzyme activities in cultured skin fibroblasts from patients with I-cell disease are decreased, and affected cells contain inclusion bodies filled with undegraded substrates [1]. I-cell disease is pathologically characterized by the presence of these inclusion bodies. However, the contents of such inclusion bodies have not been well characterized, but probably consist of glycoconjugates including oligosaccharides, glycoproteins, glycolipids and mucopolysaccharides [2–5].
I. Kawashima et al. / Clinica Chimica Acta 378 (2007) 142–146 Table 1 Lysosomal enzyme activities in cultured skin fibroblasts Specific activities (nmol/h/mg protein) Patient-1 β-Hexosaminidase A Total β-hexosaminidases α-Galactosidase β-Galactosidase α-Glucosidase α-Fucosidase β-Glucuronidase Lysosomal sialidase α-Mannosidase β-Glucosidase
135 941 2 7 90 2 19 0 9 188
Patient-2 102 594 1 6 39 1 12 0 13 163
Control-1
Control-2
3
2.62 × 103 16.7 × 103 90 1.37 × 103 286 123 127 298 41 327
2.61 × 10 14.9 × 103 84 1.28 × 103 219 81 83 298 45 238
We attempted to identify accumulated substances in cultured fibroblasts from I-cell disease patients by cytochemical staining with antibodies to lysosome-associated membrane protein-1 (LAMP-1) and GM2 ganglioside, and a series of lectins that specifically bind to glycoconjugates. 2. Materials and methods 2.1. Patients Patient-1 was a 17-month-old female child of healthy non-consanguineous Japanese parents. She developed severe psychomotor delay, coarse facies, gingival hypertrophy, thick skin and restricted joint movement. Radiographic examinations revealed a vertebral deformity and widening of the ribs, and
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vacuolated lymphocytes were found in a peripheral blood smear. Urinary mucopolysaccharide was negative. The activities of lysosomal enzymes including β-hexosaminidase A and β-galactosidase in plasma were 10- to 20fold increased compared with those in control subjects. Patient-2 was a 22-month-old Japanese female. Coarse facies and hypotonus were noticed at birth. Then she developed failure to thrive, psychomotor delay, restricted joint movement, hepatosplenomegaly, recurrent respiratory tract infection, vacuolated lymphocytes and dysostosis multiplex. Lysosomal enzyme assaying revealed elevated levels of β-hexosaminidase A, β-galactosidase and β-glucuronidase activities in plasma. Mucopolysacchariduria was not found.
2.2. Biopsying of skin tissues and culture of skin fibroblasts Skin tissues were obtained from the patients with I-cell disease and normal control subjects by means of skin biopsying, and then used for morphological analysis and establishment of cultured fibroblasts. Informed consent was obtained from the corresponding families. This study involving cultured fibroblasts was approved by the Ethical Committee of our institute. The established skin fibroblasts were cultured in Ham's F-10 medium supplemented with 10% fetal calf serum and antibiotics at 37 °C under a 5% CO2–95% air mixture.
2.3. Lysosomal enzyme assays and protein determination Cultured skin fibroblasts from the I-cell disease patients and normal controls were cultured in fresh medium for 2 days. Then, the activities of lysosomal enzymes including β-hexosaminidase A, total β-hexosaminidases (mainly β-hexosaminidases A and B), α-galactosidase, β-galactosidase, α-glucosidase, α-fucosidase, β-glucuronidase, lysosomal sialidase, α-mannosidase and β-glucosidase in cultured fibroblasts were measured fluorometrically with 4-methylumbelliferyl derivatives, as described previously [6]. Protein determination was performed with a Bio-Rad dye-binding assay kit (Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as a standard.
Fig. 1. Electron microscopic analysis. a. Biopsied skin tissue from Patient-1, scale bar = 10 μm). b. A cultured skin fibroblast from Patient-1, scale bar = 1 μm. c. Magnification of a square in b. An arrow head and arrows indicate a nucleus and lysosomes, respectively, scale bar = 0.5 μm. d. A cultured skin fibroblast from Patient2, scale bar = 1 μm. e. A cultured skin fibroblasts from a normal subject, scale bar = 1 μm.
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2.4. Morphological analysis Biopsied skin tissues and skin fibroblasts cultured on 25 mm round plastic Lab-Tek slips (Miles Laboratories, Naperville, IL) were fixed in cold 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/l phosphate buffer, pH 7.4, for 30 min. Then, the specimens were processed as described previously [7], and morphologically examined under an electron microscope (H-7100; Hitachi, Tokyo, Japan).
2.5. Cytochemical analysis of cultured skin fibroblasts Cultured skin fibroblasts from the patients with I-cell disease and controls were cultured on Lab-Tek chamber slides (Nunc, Naperville, IL) for 3 days. Then, the cells were washed with PBS and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, for 10 min at room temperature, washed with PBS, with followed by blocking with 1% BSA in PBS for 30 min as described previously [8]. For double immunostaining with both a mouse monoclonal antibody (mAb) to LAMP-1 as a marker of lysosomes (1:100 diluted, IgG isotype; Southern Biotechnology, Birmingham, AL) and a mouse mAb to GM2 ganglioside (GM2; culture supernatant, IgM isotype) [9], the cells were incubated with a mixture of two mAbs, for 1 h at room temperature. The cells were washed with 1% BSA-PBS and then incubated with a mixture of rhodamine isothiocyanate-conjugated goat anti-mouse IgG F(ab′)2 (1:200 diluted; Jackson Immuno Research, West Grove, PA) and a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM F(ab′)2 (1:100 diluted; Jackson Immuno Research) for 1 h at room temperature.
For double immunostaining with both mAb to LAMP-1 and a FITCconjugated lectins, including Maakia amurensis (MAM; binding specificity, sialic acidα2-3galactose, Seikagaku, Tokyo, Japan), Datura stramonium (DSA; binding specificity, galactoseβ1-4N-acetylgucosamine, Seikagaku), concanavalin A (Con A; binding specificity, mannose, Vector Laboratories, Inc., Burlingame, CA) (each 1 μg/ml), Arachis hypogaea (peanut) agglutinin (PNA; binding specificity, galactoseβ1-3N-acetylgalactosamine > galactose, Seikagaku), and Sambucus sieboldiana (SSA; binding specificity, sialic acidα26galactose/N-acetylgalactosamine, Seikagaku) (each 10 μg/ml), the cells were incubated with a mixture of mAb to LAMP-1 and FITC-conjugated lectins including MAM, DSA, Con A, PNA, and SSA for 1 h at room temperature. The cells were washed with 1%BSA-PBS and then treated with rhodamine isothiocyanate-conjugated goat anti-mouse IgG F(ab′)2 for 1 h at room temperature. After washing with PBS, the slides were embedded with PermaFluor Aqueous Mounting Medium (Thermo Electron Corporation, MA) and mounted with coverglasses. The stained cells were examined under a microscope (Axiovert 135; Carl Zeiss, Oberkochen, Germany) equipped with an Axio Vision 3.1 system.
3. Results 3.1. Enzyme assay As shown in Table 1, intracellular lysosomal enzyme activities including those of β-hexosaminidase A, total β-hexosaminidases,
Fig. 2. Cytochemical analysis of GM2 ganglioside in cultured skin fibroblasts. Cultured skin fibroblasts from a control (C), Patient-1 (P-1), and Patient-2 (P-2) were double-stained with an antibody to lysosome-associated membrane protein-1 (LAMP-1) and that of GM2 ganglioside (GM2). Phase, LAMP-1, GM2, and Merge indicate phase contrast, LAMP-1 single-fluorescence (Red), GM2 single-fluorescence (Green), LAMP-1 and GM2 double-fluorescence (Yellow), respectively. Scale bar = 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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α-galactosidase, β-galactosidase, α-glucosidase, α-fucosidase, β-glucuronidase, lysosomal sialidase and α-mannosidase were decreased, but not that of β-glucosidase, which is one of the membranous lysosomal enzymes.
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tose, galactoseβ1-4N-acetylglucosamine and mannose residues accumulate in increased and/or enlarged lysosomes in the cultured I-cell disease fibroblasts. 4. Discussion
3.2. Electron microscopy Electron microscopy of the I-cell disease skin tissue specimens demonstrated extensive cytoplasmic electron-lucent vacuolation of lysosomes in fibroblasts in the dermis (Fig. 1a). Cells in the epidermis contained many electron-dense inclusion bodies in their cytoplasm (Fig. 1a). The cultured I-cell disease fibroblasts contained numerous membrane bound-vacuoles containing electron-lucent, electron-dense, fibrillogranular and lamellar materials (Fig. 1b, c, and d). 3.3. Cytochemical analysis The results of cytochemical analysis are shown in Figs. 2 and 3. Marked granular fluorescence was observed in fibroblasts from the I-cell disease patients when the cells were stained with antibodies to LAMP-1 and GM2 (Fig. 2), and lectins including MAM, DSA and Con A (Fig. 3). Only weak fluorescence for LAMP-1, GM2, MAM, DSA, and ConA was detected in control cells. Their localization was coincident with that of LAMP-1. There were no differences in the staining patterns of PNA and SSA between the patients' cells and those of controls (data not shown). These results suggest that large amounts of GM2 ganglioside and glycoconjugates having sialic acidα2-3galac-
Lysosomal enzymes are synthesized on endoplasmic reticulum (ER)-bound ribosomes, and modified in the ER through cleavage of the signal peptide and the addition of high-mannose type sugar chains. Then, further modification of the sugar chains occurs and the addition of a mannose 6-phosphate residue, which is a marker for targeting of non-membranous lysosomal enzymes except for membranous enzymes including β-glucosidase, takes place in the Golgi apparatus [1]. The nonmembranous lysosomal enzymes having mannose 6-phosphate residues at the non-reducing ends of sugar chains are transported to endosomes via mannose 6-phosphate receptors. Phosphotransferase is a key enzyme in the generation of the mannose 6-phosphate marker and catalyzes the first step of the process, adding a N-acetylglucosamine 1-phosphate residue to the 6th position of a mannose on sugar chains [1]. Deficient phosphotransferase activity results in defective synthesis of the mannose 6-phosphate marker and causes abnormal transport of non-membranous lysosomal enzymes. As a result, the activities of non-membranous lysosomal enzymes in cultured fibroblasts from I-cell disease patients decrease. The present study revealed that the activities of β-hexosaminidase A, α-galactosidase, βgalactosidase, α-fucosidase, lysosomal sialidase and α-mannosidase were below 10% of the control levels. The results suggest
Fig. 3. Lectin staining of cultured skin fibroblasts. Cultured skin fibroblasts from a control (C), Patient-1 (P-1), and Patient-2 (P-2) were double-stained with an antibody to LAMP-1 and lectins including Maakia amurensis (MAM), Datura stramonium (DSA), and concanavalin A (Con A). LAMP-1, lectins (MAM, DSA, and ConA), and Merge indicate LAMP-1 single-fluorescence (Red), lectin single-fluorescence (Green), LAMP-1 and lectin double-fluorescence (Yellow), respectively. Scale bar = 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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complex accumulation of substrates of these enzymes in lysosomes in cultured I-cell disease fibroblasts. The morphological results suggested storage of oligosaccharides, glycoproteins and glycolipids in lysosomes. In this study, we analyzed storage materials in cultured I-cell disease fibroblasts by means of cytochemistry with antibodies to LAMP-1 and GM2 ganglioside, and lectins that specifically bind to glycoconjugates. Strong granular fluorescence for antibodies to LAMP-1 and GM2, and lectins including MAM, DSA, and ConA was found in the I-cell disease fibroblasts. The results showed increased and/or enlarged lysosomes with excessively accumulated GM2 and glycoconjugates having sialic acidα2-3galactose, galactoseβ1-4N-acetylglucosamine and mannose residues. The accumulation of these substrates is probably due to simultaneously decreased activities of βhexosaminidase A, lysosomal sialidase, β-galactosidase and αmannosidase, respectively. On the other hand, only faint fluorescence was found both in the patient's cells and control cells when the cells were stained with PNA and SSA. The results suggests that the metabolic pathway of glycoconjugates with galactoseβ1-3N-acetylgalactosamine and sialic acidα2-6 galactose/N-acetylgalactosamine residues is not dominant in cultured fibroblasts. Recently, enzyme replacement therapy was introduced for several lysosomal diseases including Gaucher disease [10], Fabry disease [11,12], mucopolysaccharidosis (MPS) I [13], MPS II [14,15], MPS VI [16], and Pompe disease [17]. Therapeutic trials including bone marrow transplantation have also been performed for I-cell disease [18], and efforts are being made to develop effective therapies. For that purpose, establishment of methods for identifying accumulated materials in I-cell disease fibroblasts is very important. We developed a cytochemical method with specific antibodies and lectins for glycoconjugates and identified accumulated glycoconjugates in I-cell disease fibroblasts. The present cytochemical method is sensitive and easy, and will be useful for studying the pathophysiology and evaluation of therapeutic procedures for I-cell disease. Furthermore, cytochemical staining of cultured fibroblasts with MAM, DSA, and ConA and an antibody to GM2 would help diagnosis of I-cell disease. Acknowledgements This work was partly supported by grants from the Japan Science and Technology Agency, the Tokyo Metropolitan Government, the Japan Society for the Promotion of Science, the Ministry of Education, Science, Sports and Culture, and the Ministry of Health, Labor and Welfare of Japan. References [1] Kornfeld S, Sly WS. I-cell disease and pseudo-Hurler polydystrophy: disorders of lysosomal enzyme phosphorylation and localization. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 2001. p. 3469–82.
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