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A Distinct Ganglioside Composition of Rat Pancreatic Islets
Archives of Biochemistry and Biophysics Vol. 376, No. 2, April 15, pp. 371–376, 2000 doi:10.1006/abbi.2000.1729, available online at http://www.idealibrary.com on
A Distinct Ganglioside Composition of Rat Pancreatic Islets Megumi Saito 1 and Kiyoshi Sugiyama Department of Clinical Pharmacology and Therapeutics, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan
Received October 27, 1999, and in revised form January 20, 2000
Gangliosides in rat pancreatic tissue and isolated pancreatic islets were examined by methods including glycolipid-overlay techniques. The content of gangliosides in isolated pancreatic islets was approximately 6-fold higher than that in pancreatic tissue when compared on a protein basis. While N-glycolylneuraminic acid amounted to 7.2% of total lipid-bound sialic acids of pancreatic tissue, this molecular species was not detected in that of pancreatic islets. A remarkable difference in ganglioside composition was observed between pancreatic tissue and pancreatic islets. Pancreatic tissue showed a complex ganglioside pattern with GM3 as the largest ganglioside component, whereas isolated pancreatic islets had a simpler ganglioside profile without detectable amounts of GM3 and some other components. Pancreatic gangliosides were further examined by thin-layer chromatographic immunostaining with a monoclonal antibody A2B5 that reacts specifically with c-series gangliosides. Pancreatic tissue and pancreatic islets showed almost identical ganglioside patterns consisting of GT3, GT2, GQ1c, and GP1c. The concentration of c-series gangliosides in pancreatic islets was calculated to be more than 250-fold higher than that of pancreatic tissue. These results shows that pancreatic islet cells have a distinct ganglioside composition in rat pancreas. © 2000 Academic Press
Key Words: gangliosides; c-series gangliosides; pancreas; pancreatic islets; sialic acids.
Gangliosides are a family of glycosphingolipids containing one or more sialic acid residues and are distributed mainly on the outer surface of plasma membranes of cells. They are found ubiquitously in tissues and 1 To whom correspondence should be addressed. Fax: ⫹81-54-2645764. E-mail: [email protected].
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cells and participate in diverse cellular functions including growth, differentiation, and signal transduction (1–3). Gangliosides are also antigenic and are involved in immunological reactions in various pathological conditions (4). To execute their functions properly, the expression of gangliosides must be regulated precisely in individual tissues and cell types. Pancreatic islets of Langerhans are a specialized structure of assembled cells, scattering through the whole pancreatic tissue. They consist mainly of four different cell types that synthesize and secrete glucagon (A cells), insulin (B cells), somatostatin (D cells), or pancreatic polypeptides (PP cells). Among these cells, B cells are the dominant cell type; in rats, they amount to about 70% of total islet cells (5). Pancreatic islets constitute only 1–2% of the whole tissue; a large portion of the tissue is occupied by exocrine and ductular cells (6, 7). Recently, we investigated c-series gangliosides in rat pancreatic tissue and found that c-series gangliosides were totally lost in the tissue after treatment with streptozotocin. Since streptozotocin selectively destroy pancreatic B cells without affecting other islet or nonislet cells, it was strongly suggested that c-series gangliosides are located exclusively in pancreatic B cells (8). In the present study, we further characterized gangliosides in rat pancreatic tissue and isolated pancreatic islets and demonstrated that rat pancreatic islets have a distinct ganglioside composition in the pancreas. MATERIALS AND METHODS Materials. Seven-week-old male rats of Sprague–Dawley strain were purchased from Japan SLC (Shizuoka, Japan). A2B5-producing hybridomas CRL 1520 were obtained from American Type Culture Collection (Rockville, MD) and cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. The culture medium containing the monoclonal antibody A2B5 (IgM-type) was used for experiments. An antibody against GM1 or asialo-GM1 (both 371
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IgG-type) was prepared by immunizing rabbits with purified glycolipid (9). Other reagents were purchased from the following companies: Clostridium perfringens sialidase, goat peroxidase-conjugated antibodies against mouse IgM or rabbit IgG, N-acetylneuraminic acid, and N-glycolylneuraminic acid (Sigma, USA), TLC plates (nanoplates, E. Merck), and enhanced chemiluminescence (ECL) 2 Western blotting detection kits (Amersham, USA). Preparation of rat pancreatic islets. Isolation of pancreatic islets was carried out by a collagenase digestion method (10). Collagenase (derived from Clostridium histolyticum, Worthington, USA) was dissolved in Krebs–Ringer bicarbonate (KRB) buffer at a concentration of 1.7 mg/ml and injected into pancreatic tissue through the common bile duct. After excision, the pancreatic tissue was kept in at 37°C for 10 to 13 min. The digested tissue was then dispersed in the KRB buffer and pancreatic islets were hand picked under a dissecting microscope. The isolated pancreatic islets were characterized by examining the ability of insulin secretion using a rat insulin EIA kit (Amersham). The secretion rates of insulin at low (2.8 mM) and high (16.7 mM) glucose concentrations were 0.223 ⫾ 0.100 and 1.271 ⫾ 0.391 ng of insulin/h/10 islets, respectively, showing comparable results to earlier reports (11, 12). Ganglioside analysis. Total lipids were extracted from tissues with 20 vol of chloroform/methanol (1:1) and separated into neutral and acidic lipids by DEAE–Sephadex column chromatography. Acidic lipids were treated with 0.2 M methanolic NaOH to destroy phospholipids and then neutralized with acetic acid. Purified gangliosides were obtained after desalting the neutralized mixture by Sephadex LH-20 column chromatography. Quantitation of gangliosides. Lipid-bound sialic acids were measured using a fluorometric high-performance liquid chromatographic (HPLC) method (13). In brief, a portion of total lipid extract was treated in 25 mM sulfuric acid at 80°C for 1 h. The hydrolysate was incubated with a 1,2-diamino-4,5-methylenedioxybenzene (DMB) reagent at 60°C for 2.5 h. The fluorescent derivatives of sialic acids were analyzed by HPLC with an ODS column (Mightysil RP 18, 250 ⫻ 4.6 mm, Kanto Chemicals, Tokyo, Japan). The excitation and emission wave lengths were 373 and 448 nm, respectively. Quantitation was carried out based upon the standard curves obtained with the N-acetylneuraminic acid and N-glycolylneuraminic acid solutions of different concentrations. TLC-immunostaining of gangliosides with A2B5. TLC-immunostaining was carried out based upon a method reported previously (14). Briefly, gangliosides were first developed on a TLC plate. After coating the plate with a 0.4% polyisobutylmethacrylate solution, gangliosides were reacted consecutively with the A2B5 antibody and peroxidase-conjugated second antibody at room temperature for 1.5 h. The reacted band(s) were detected on an X-ray film using an ECL Western blotting detection kit. Gangliosides developed on the plate was then visualized with resorcinol–HCl reagent (15). Structural analysis of gangliosides using TLC-immunostaining. The oligosaccharide structures of gangliosides were analyzed by a method with in situ hydrolysis of gangliosides by sialidase and immunostaining with an anti-glycolipid antibody (16). Gangliosides were developed on TLC and treated in situ with C. perfringens sialidase (250 mU/ml of 0.1 M sodium acetate buffer, pH 4.8) at room temperature for 1.5 h. The modified glycolipid structures were examined with anti-GM1 antibody or anti-asialo-GM1 antibody. Sialidase treatment of gangliosides in tube reaction. Gangliosides from pancreatic tissue were separated on TLC plates. Each ganglioside was extracted from the corresponding area of silica gel and
2 Abbreviations used: ECL, enhanced chemiluminescence; KRB, Krebs–Ringer bicarbonate; DMB, 1,2-diamino-4,5-methylenedioxybenzene; IDDM, insulin-dependent diabetic mellitus.
TABLE I
The Contents of Gangliosides and Molecular Species of Sialic Acids in Rat Pancreatic Tissue and Isolated Pancreatic Islets
Pancreatic tissue Pancreatic islets
Total sialic acids (ng/mg protein)
N-acetylneuraminic acid (%)
N-glycolylneuraminic acid (%)
88.3 ⫾ 28.1 540 ⫾ 179
92.8 ⫾ 28.3 100
7.2 ⫾ 3.6 0*
Note. Sialic acids in total lipid extracts were analyzed by a fluorometric HPLC method with a 1,2-diamino-4,5-methylenedioxybenzene (DMB) reagent (see Materials and Methods). Each value is the mean ⫾ SD (n ⫽ 3). * The amount was below detection limit.
subjected to DEAE–Sephadex and LH20 column chromatography, successively. The purified ganglioside was partially hydrolyzed by incubating with C. perfringens sialidase (200 mU/ml of 0.1 M sodium acetate buffer, pH 4.8) at 37°C for 45 min. The degradation product(s) were analyzed by TLC after desalting of the sample with a small LH-20 column.
RESULTS
Contents of Gangliosides and Molecular Species of Sialic Acids in Pancreatic Tissue and Isolated Pancreatic Islets The contents of gangliosides in pancreatic tissue and isolated pancreatic islets were measured by quantitation of lipid-bound sialic acids. As shown in Table I, the ganglioside content of pancreatic islets was approximately 6-fold higher than that of pancreatic tissue when compared on protein basis. In pancreatic tissue, N-glycolylneuraminic acid accounted for about 7.2% of total lipid-bound sialic acids, whereas this sialic acid species was not detected in pancreatic islets. Compositions of Major Gangliosides in Pancreatic Tissue and Isolated Pancreatic Islets Major gangliosides in rat pancreatic tissue and isolated pancreatic islets were examined. Pancreatic tissue showed a complex ganglioside pattern that consisted of eight major ganglioside components (Fig. 1). Among these gangliosides, gangliosides 1, 3, 4, 7, and 8 had the same chromatographic mobility to GM3, GD3, GD1a, GT1b, and GQ1b in liver tissue, respectively. Gangliosides in pancreatic tissue were analyzed using a method with in situ hydrolysis by sialidase and immunostaining with anti-GM1 antibody or anti-asialoGM1 antibody (Figs. 2A and 2B). Gangliosides 1 and 3 did not react with anti-GM1 antibody or with antiasialo-GM1 antibody. Sialidase treatment rendered gangliosides 4, 7, and 8 susceptible to anti-GM1 antibody, indicating that these gangliosides shared the
MAJOR AND C-SERIES GANGLIOSIDES OF RAT PANCREATIC ISLETS
FIG. 1. Thin-layer chromatogram of gangliosides in rat pancreatic tissue. Gangliosides were isolated from 7-week-old rat pancreas, developed on TLC with a solvent system of chloroform/methanol/ 0.2% CaCl 2 (50:45:10), and visualized by resorcinol–HCl reagent. Lanes 1 and 2, gangliosides in liver tissue (as reference, equivalent to 3 mg protein) and gangliosides in pancreatic tissue (equivalent to 10 mg protein), respectively.
same oligosaccharide structure of GM1. To further characterize these gangliosides, each ganglioside was isolated by preparative TLC and treated with sialidase
FIG. 2. Structural analyses using TLC immunostaining with in situ treatment with sialidase. After development on a TLC plate, gangliosides in pancreatic tissue were treated in situ with Clostridium perfringens sialidase, followed by immunostaining with antiGM1 antibody (A) or anti-asialo-GM1 antibody (B). (A) Lanes 1 and 2, liver tissue; lanes 3 and 4, pancreatic tissue; lanes 1 and 3, visualized by resorcinol–HCl reagent; and lanes 2 and 4, immunostained with anti-GM1 antibody. (B) Lane 1, pancreatic tissue, visualized by resorcinol–HCl reagent; and lane 2, pancreatic tissue, immunostained with anti-asialo-GM1 antibody. In A and B, the amount of gangliosides in pancreatic tissue was equivalent to 10 mg protein per lane.
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FIG. 3. Sialidase treatment of pancreatic gangliosides in tube reaction. Gangliosides 4, 7, and 8 in rat pancreatic tissue were isolated by preparative TLC and treated with Clostridium perfringens sialidase (200 mU/ml of 0.1 M sodium acetate buffer, pH 4.8) at 37°C for 45 min. The degradation product(s) were visualized with resorcinol–HCl reagent after development on TLC with a solvent system of chloroform/methanol/0.2% CaCl 2 (45:40:10). Lanes 2 and 3, ganglioside 4; lanes 4 and 5, ganglioside 7; and lanes 6 and 7, ganglioside 8. Lanes 2, 4, and 6, before sialidase treatment; lanes 3, 5, and 7, after sialidase treatment. Lane 1, rat liver gangliosides. The arrow indicates free sialic acids released during the enzyme reaction.
in tube reaction for analysis of the degradation product(s) (Fig. 3). Hydrolysis of gangliosides 4, 7, and 8 produced a ganglioside with the same mobility of GM1. In the case of ganglioside 7, another degradation product corresponding to GD1b also was observed. Gangliosides 1 and 3 were hydrolyzed by the enzyme, producing a neutral glycolipid corresponding to LacCer (data nor shown). Based upon these findings and their chromatographic mobility, gangliosides 1, 3, 4, 7, and 8 were respectively identified as GM3, GD3, GD1a, GT1b, and GQ1b. Gangliosides 2 and 5 did not react with anti-GM1 antibody, but did react with anti-asialo GM1 antibody, showing that they had the gangliotetraose oligosaccharide core structure. Ganglioside 6 did not react with anti-GM1 antibody nor with anti-asialo-GM1 antibody following sialidase treatment. The present study also demonstrated that pancreatic tissue expressed trace amounts of GM1 and GD1b (Fig. 2A). Isolated pancreatic islets showed a ganglioside pattern that was distinct from that of pancreatic tissue (Fig. 4). Pancreatic islets consisted mainly of di- and polysialo ganglioside species that included GD3, GT1b, and ganglioside 6 without detectable amounts of GM3, GD1a, ganglioside 2, and ganglioside 5.
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FIG. 4. Thin-layer chromatogram of gangliosides in pancreatic tissue and isolated pancreatic islets. Gangliosides in pancreatic tissue and isolated pancreatic islets were developed on TLC and visualized by resorcinol–HCl reagent. Lane 1, pancreatic tissue (equivalent to 10 mg protein); and lane 2, isolated pancreatic islets (equivalent to 0.18 mg protein).
C-series Gangliosides in Pancreatic Tissue and Pancreatic Islets Pancreatic tissue contained four A2B5-reactive gangliosides, showing a composition that was distinguished from that in liver tissue (Fig. 5). Based upon their chromatographic mobility on TLC and the specificity of A2B5, these gangliosides were identified as GT3, GT2, GQ1c, and GP1c. While a remarkable difference was observed between the major ganglioside
FIG. 5. Immunochromatogram of gangliosides in rat pancreatic tissue. Gangliosides in rat pancreatic tissue were developed on TLC and immunostained with A2B5. Lane 1, liver tissue (equivalent to 15 mg wet weight tissue); and lane 2, pancreatic tissue (equivalent to 15 mg wet weight tissue).
FIG. 6. C-series gangliosides in pancreatic tissue and isolated pancreatic islets. Gangliosides in pancreatic tissue and pancreatic islets were developed on TLC and immunostained with A2B5. Lanes 1 and 3, pancreatic tissue (equivalent to 10 mg protein); and lanes 2 and 4, pancreatic islets (equivalent to 0.04 mg protein). Lanes 1 and 2, visualized by resorcinol–HCl reagent; and lanes 3 and 4, immunostained with A2B5.
patterns of pancreatic tissue and pancreatic islets, the compositional profiles of c-series gangliosides were almost identical (Fig. 6). As compared with pancreatic tissue, pancreatic islets had a much higher concentration of c-series gangliosides; the content of c-series gangliosides in pancreatic islets equivalent to 40 ng protein was even higher than that in pancreatic tissue equivalent to 10 mg protein. DISCUSSION
Gangliosides in pancreatic tissues and isolated islets have been characterized by several researchers. Ganglioside GM2-1, which migrates between GM2 and GM1 on TLC, was reported to be present in pancreatic tissues and pancreatic islets of different species (17– 20). The presence of c-series gangliosides in pancreatic islets has been suggested by immunocytochemistry of isolated preparations with A2B5 (21, 22). However, detailed information regarding ganglioside composition of pancreatic tissues and pancreatic islets is still limited. In the present study, we examined gangliosides of rat pancreas and demonstrated that pancreatic islets have a distinct ganglioside composition. Evidence was also provided suggesting the restricted localization of c-series gangliosides in pancreatic islets. Rat pancreatic tissue showed a GM3-dominant ganglioside pattern. Among individual ganglioside species, GM3, GD3, GD1a, GT1b, and GQ1b were identified based upon their chromatographic behaviors and reactivity to anti-GM1 antibody after sialidase treatment. This conclusion was further supported by the response
MAJOR AND C-SERIES GANGLIOSIDES OF RAT PANCREATIC ISLETS
of each ganglioside to sialidase treatment in tube reaction. The hydrolysis profile of ganglioside 7 was essentially the same as observed when GT1b was treated by Arthrobacter ureafaciens sialidase (16), providing further evidence that this ganglioside is GT1b. While gangliosides 2 and 5 did not react with anti-GM1 antibody, they became reactive with anti-asialo-GM1 antibody following treatment with C. perfringens sialidase. It is known that C. perfringens sialidase hardly hydrolyzes a sialic acid residue connected to the inner galactose, but efficiently cleaves the one linked to the terminal galactose or N-acetylgalactosamine in the gangliotetraose structure (23). The present study indicates that both ganglioside 2 and ganglioside 5 have the gangliotetraose oligosaccharide core structure with a sialic acid residue(s) that are not connected to the inner galactose. Although the structures of gangliosides 2 and 5 remain to be determined, they may be one of “asialo-GM1-derived” gangliosides including GM1b, GD1c, and GD1␣ (24 –27). The structures of ganglioside 6 also is unknown. While this ganglioside had chromatographic mobility similar to that of GT3, no correlation was observed between the amounts of the lipid visualized by resorcinol–HCl reagent and immunostained with A2B5 (see Fig. 6). It thus is unlikely that ganglioside 6 is GT3. The composition of gangliosides in isolated pancreatic islets significantly differed from that in pancreatic tissue. In pancreatic islets, GD3, ganglioside 6, and GT1b constitute the major ganglioside components, while GM3, ganglioside 2, GD1a, and ganglioside 5 were hardly detected. These findings suggests that ganglioside metabolism in pancreatic islet cells is regulated in a manner distinct from that in nonislet cells. In the present study, we could not detect GM2-1, which has been demonstrated in pancreatic islets (17). The content of GM2-1 in rat pancreas may not be high enough to detect by ordinary visualizing methods. C-series gangliosides are characterized by a trisialosyl residue at the inner galactose of the hemato- or ganglio-type oligosaccharide structure (28, 29). Since c-series gangliosides exist as minor components in mammalian tissues, they are difficult to analyze by conventional visualization methods after separation on TLC. In the present study, we employed a specific monoclonal antibody A2B5 for examining c-series gangliosides in rat pancreas. A2B5 was originally prepared by immunizing chicken embryonic retinal cells (30). While there was some controversy about specificity of the antibody (31), recent studies have reported its strict specificity to c-series gangliosides including GT3, 9-O-acetyl GT3, GT1c, GQ1c, GP1c, and G⬙H⬙ (32–35). We have recently characterized A2B5-reactive gangliosides in rat cultured hepatocytes and liver tissue and provided further evidence for the specificity of the antibody; A2B5-reactive gangliosides corresponding to
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GT1c, GQ1c, and GP1c were shown to possess the gangliotetraose oligosaccharide core structure (36). Based upon the reactivity of A2B5 to c-series gangliosides, it is assumed that the trisialosyl structure connected to the inner galactose constitutes the epitope recognized by the antibody. Pancreatic tissue contained four A2B5-reactive gangliosides. Among them, three gangliosides migrating near GD1b, below GQ1b, and far below GQ1b on TLC were respectively assigned to GT3, GQ1c, and GP1c. The ganglioside migrating between GT3 and GT1c was identified as GT2 based upon the specificity of A2B5 and chromatographic mobility on TLC (29). No positive staining of other gangliosides or nonganglioside acidic lipids (e.g., sulfatides) was observed. In contrast to the case of major gangliosides, the compositions of c-series gangliosides in pancreatic tissue and isolated pancreatic islets were almost identical. The concentration of c-series gangliosides in pancreatic islets was much higher than that in pancreatic tissue. These results suggest that c-series gangliosides may be highly enriched in pancreatic islets in rat pancreas, which is consistent with our recent study, suggesting that cseries gangliosides may be localized exclusively in rat pancreatic B cells (8). It is assumed that the expression of c-series gangliosides in brain tissues may be cellspecific. A series of immunohistochemical studies with a specific monoclonal antibody Q211 demonstrated restricted localization of Q211-positive cells in brain tissues (37–39). Recent studies have provided further evidence for this assumption by demonstrating the specific localization of c-series gangliosides in stellate neurons in human cerebellum (40) and developmentdependent expression of GT3 and its O-acetyl derivative in rat glial cells (35). Integration of our present study and others suggests that c-series gangliosides may also be expressed in a cell-specific manner in extraneural tissues. In summary, the present study demonstrates that pancreatic islet cells have a distinct ganglioside composition in rat pancreas. This finding suggests that c-series gangliosides may play an important role in physiological and pathological processes of pancreatic islets. It is known that insulin-dependent diabetic mellitus (IDDM) is developed through autoimmune destruction of pancreatic B cells (41). C-series gangliosides, which probably are localized specifically in pancreatic B cells (8), may serve as the target molecules in the autoimmune mechanism of IDDM. ACKNOWLEDGMENT The authors thank Mr. Takeshi Suzuki for his technical assistance in isolation of pancreatic islets.
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A Distinct Ganglioside Composition of Rat Pancreatic Islets Megumi Saito 1 and Kiyoshi Sugiyama Department of Clinical Pharmacology and Therapeutics, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan
Received October 27, 1999, and in revised form January 20, 2000
Gangliosides in rat pancreatic tissue and isolated pancreatic islets were examined by methods including glycolipid-overlay techniques. The content of gangliosides in isolated pancreatic islets was approximately 6-fold higher than that in pancreatic tissue when compared on a protein basis. While N-glycolylneuraminic acid amounted to 7.2% of total lipid-bound sialic acids of pancreatic tissue, this molecular species was not detected in that of pancreatic islets. A remarkable difference in ganglioside composition was observed between pancreatic tissue and pancreatic islets. Pancreatic tissue showed a complex ganglioside pattern with GM3 as the largest ganglioside component, whereas isolated pancreatic islets had a simpler ganglioside profile without detectable amounts of GM3 and some other components. Pancreatic gangliosides were further examined by thin-layer chromatographic immunostaining with a monoclonal antibody A2B5 that reacts specifically with c-series gangliosides. Pancreatic tissue and pancreatic islets showed almost identical ganglioside patterns consisting of GT3, GT2, GQ1c, and GP1c. The concentration of c-series gangliosides in pancreatic islets was calculated to be more than 250-fold higher than that of pancreatic tissue. These results shows that pancreatic islet cells have a distinct ganglioside composition in rat pancreas. © 2000 Academic Press
Key Words: gangliosides; c-series gangliosides; pancreas; pancreatic islets; sialic acids.
Gangliosides are a family of glycosphingolipids containing one or more sialic acid residues and are distributed mainly on the outer surface of plasma membranes of cells. They are found ubiquitously in tissues and 1 To whom correspondence should be addressed. Fax: ⫹81-54-2645764. E-mail: [email protected].
0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
cells and participate in diverse cellular functions including growth, differentiation, and signal transduction (1–3). Gangliosides are also antigenic and are involved in immunological reactions in various pathological conditions (4). To execute their functions properly, the expression of gangliosides must be regulated precisely in individual tissues and cell types. Pancreatic islets of Langerhans are a specialized structure of assembled cells, scattering through the whole pancreatic tissue. They consist mainly of four different cell types that synthesize and secrete glucagon (A cells), insulin (B cells), somatostatin (D cells), or pancreatic polypeptides (PP cells). Among these cells, B cells are the dominant cell type; in rats, they amount to about 70% of total islet cells (5). Pancreatic islets constitute only 1–2% of the whole tissue; a large portion of the tissue is occupied by exocrine and ductular cells (6, 7). Recently, we investigated c-series gangliosides in rat pancreatic tissue and found that c-series gangliosides were totally lost in the tissue after treatment with streptozotocin. Since streptozotocin selectively destroy pancreatic B cells without affecting other islet or nonislet cells, it was strongly suggested that c-series gangliosides are located exclusively in pancreatic B cells (8). In the present study, we further characterized gangliosides in rat pancreatic tissue and isolated pancreatic islets and demonstrated that rat pancreatic islets have a distinct ganglioside composition in the pancreas. MATERIALS AND METHODS Materials. Seven-week-old male rats of Sprague–Dawley strain were purchased from Japan SLC (Shizuoka, Japan). A2B5-producing hybridomas CRL 1520 were obtained from American Type Culture Collection (Rockville, MD) and cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. The culture medium containing the monoclonal antibody A2B5 (IgM-type) was used for experiments. An antibody against GM1 or asialo-GM1 (both 371
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IgG-type) was prepared by immunizing rabbits with purified glycolipid (9). Other reagents were purchased from the following companies: Clostridium perfringens sialidase, goat peroxidase-conjugated antibodies against mouse IgM or rabbit IgG, N-acetylneuraminic acid, and N-glycolylneuraminic acid (Sigma, USA), TLC plates (nanoplates, E. Merck), and enhanced chemiluminescence (ECL) 2 Western blotting detection kits (Amersham, USA). Preparation of rat pancreatic islets. Isolation of pancreatic islets was carried out by a collagenase digestion method (10). Collagenase (derived from Clostridium histolyticum, Worthington, USA) was dissolved in Krebs–Ringer bicarbonate (KRB) buffer at a concentration of 1.7 mg/ml and injected into pancreatic tissue through the common bile duct. After excision, the pancreatic tissue was kept in at 37°C for 10 to 13 min. The digested tissue was then dispersed in the KRB buffer and pancreatic islets were hand picked under a dissecting microscope. The isolated pancreatic islets were characterized by examining the ability of insulin secretion using a rat insulin EIA kit (Amersham). The secretion rates of insulin at low (2.8 mM) and high (16.7 mM) glucose concentrations were 0.223 ⫾ 0.100 and 1.271 ⫾ 0.391 ng of insulin/h/10 islets, respectively, showing comparable results to earlier reports (11, 12). Ganglioside analysis. Total lipids were extracted from tissues with 20 vol of chloroform/methanol (1:1) and separated into neutral and acidic lipids by DEAE–Sephadex column chromatography. Acidic lipids were treated with 0.2 M methanolic NaOH to destroy phospholipids and then neutralized with acetic acid. Purified gangliosides were obtained after desalting the neutralized mixture by Sephadex LH-20 column chromatography. Quantitation of gangliosides. Lipid-bound sialic acids were measured using a fluorometric high-performance liquid chromatographic (HPLC) method (13). In brief, a portion of total lipid extract was treated in 25 mM sulfuric acid at 80°C for 1 h. The hydrolysate was incubated with a 1,2-diamino-4,5-methylenedioxybenzene (DMB) reagent at 60°C for 2.5 h. The fluorescent derivatives of sialic acids were analyzed by HPLC with an ODS column (Mightysil RP 18, 250 ⫻ 4.6 mm, Kanto Chemicals, Tokyo, Japan). The excitation and emission wave lengths were 373 and 448 nm, respectively. Quantitation was carried out based upon the standard curves obtained with the N-acetylneuraminic acid and N-glycolylneuraminic acid solutions of different concentrations. TLC-immunostaining of gangliosides with A2B5. TLC-immunostaining was carried out based upon a method reported previously (14). Briefly, gangliosides were first developed on a TLC plate. After coating the plate with a 0.4% polyisobutylmethacrylate solution, gangliosides were reacted consecutively with the A2B5 antibody and peroxidase-conjugated second antibody at room temperature for 1.5 h. The reacted band(s) were detected on an X-ray film using an ECL Western blotting detection kit. Gangliosides developed on the plate was then visualized with resorcinol–HCl reagent (15). Structural analysis of gangliosides using TLC-immunostaining. The oligosaccharide structures of gangliosides were analyzed by a method with in situ hydrolysis of gangliosides by sialidase and immunostaining with an anti-glycolipid antibody (16). Gangliosides were developed on TLC and treated in situ with C. perfringens sialidase (250 mU/ml of 0.1 M sodium acetate buffer, pH 4.8) at room temperature for 1.5 h. The modified glycolipid structures were examined with anti-GM1 antibody or anti-asialo-GM1 antibody. Sialidase treatment of gangliosides in tube reaction. Gangliosides from pancreatic tissue were separated on TLC plates. Each ganglioside was extracted from the corresponding area of silica gel and
2 Abbreviations used: ECL, enhanced chemiluminescence; KRB, Krebs–Ringer bicarbonate; DMB, 1,2-diamino-4,5-methylenedioxybenzene; IDDM, insulin-dependent diabetic mellitus.
TABLE I
The Contents of Gangliosides and Molecular Species of Sialic Acids in Rat Pancreatic Tissue and Isolated Pancreatic Islets
Pancreatic tissue Pancreatic islets
Total sialic acids (ng/mg protein)
N-acetylneuraminic acid (%)
N-glycolylneuraminic acid (%)
88.3 ⫾ 28.1 540 ⫾ 179
92.8 ⫾ 28.3 100
7.2 ⫾ 3.6 0*
Note. Sialic acids in total lipid extracts were analyzed by a fluorometric HPLC method with a 1,2-diamino-4,5-methylenedioxybenzene (DMB) reagent (see Materials and Methods). Each value is the mean ⫾ SD (n ⫽ 3). * The amount was below detection limit.
subjected to DEAE–Sephadex and LH20 column chromatography, successively. The purified ganglioside was partially hydrolyzed by incubating with C. perfringens sialidase (200 mU/ml of 0.1 M sodium acetate buffer, pH 4.8) at 37°C for 45 min. The degradation product(s) were analyzed by TLC after desalting of the sample with a small LH-20 column.
RESULTS
Contents of Gangliosides and Molecular Species of Sialic Acids in Pancreatic Tissue and Isolated Pancreatic Islets The contents of gangliosides in pancreatic tissue and isolated pancreatic islets were measured by quantitation of lipid-bound sialic acids. As shown in Table I, the ganglioside content of pancreatic islets was approximately 6-fold higher than that of pancreatic tissue when compared on protein basis. In pancreatic tissue, N-glycolylneuraminic acid accounted for about 7.2% of total lipid-bound sialic acids, whereas this sialic acid species was not detected in pancreatic islets. Compositions of Major Gangliosides in Pancreatic Tissue and Isolated Pancreatic Islets Major gangliosides in rat pancreatic tissue and isolated pancreatic islets were examined. Pancreatic tissue showed a complex ganglioside pattern that consisted of eight major ganglioside components (Fig. 1). Among these gangliosides, gangliosides 1, 3, 4, 7, and 8 had the same chromatographic mobility to GM3, GD3, GD1a, GT1b, and GQ1b in liver tissue, respectively. Gangliosides in pancreatic tissue were analyzed using a method with in situ hydrolysis by sialidase and immunostaining with anti-GM1 antibody or anti-asialoGM1 antibody (Figs. 2A and 2B). Gangliosides 1 and 3 did not react with anti-GM1 antibody or with antiasialo-GM1 antibody. Sialidase treatment rendered gangliosides 4, 7, and 8 susceptible to anti-GM1 antibody, indicating that these gangliosides shared the
MAJOR AND C-SERIES GANGLIOSIDES OF RAT PANCREATIC ISLETS
FIG. 1. Thin-layer chromatogram of gangliosides in rat pancreatic tissue. Gangliosides were isolated from 7-week-old rat pancreas, developed on TLC with a solvent system of chloroform/methanol/ 0.2% CaCl 2 (50:45:10), and visualized by resorcinol–HCl reagent. Lanes 1 and 2, gangliosides in liver tissue (as reference, equivalent to 3 mg protein) and gangliosides in pancreatic tissue (equivalent to 10 mg protein), respectively.
same oligosaccharide structure of GM1. To further characterize these gangliosides, each ganglioside was isolated by preparative TLC and treated with sialidase
FIG. 2. Structural analyses using TLC immunostaining with in situ treatment with sialidase. After development on a TLC plate, gangliosides in pancreatic tissue were treated in situ with Clostridium perfringens sialidase, followed by immunostaining with antiGM1 antibody (A) or anti-asialo-GM1 antibody (B). (A) Lanes 1 and 2, liver tissue; lanes 3 and 4, pancreatic tissue; lanes 1 and 3, visualized by resorcinol–HCl reagent; and lanes 2 and 4, immunostained with anti-GM1 antibody. (B) Lane 1, pancreatic tissue, visualized by resorcinol–HCl reagent; and lane 2, pancreatic tissue, immunostained with anti-asialo-GM1 antibody. In A and B, the amount of gangliosides in pancreatic tissue was equivalent to 10 mg protein per lane.
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FIG. 3. Sialidase treatment of pancreatic gangliosides in tube reaction. Gangliosides 4, 7, and 8 in rat pancreatic tissue were isolated by preparative TLC and treated with Clostridium perfringens sialidase (200 mU/ml of 0.1 M sodium acetate buffer, pH 4.8) at 37°C for 45 min. The degradation product(s) were visualized with resorcinol–HCl reagent after development on TLC with a solvent system of chloroform/methanol/0.2% CaCl 2 (45:40:10). Lanes 2 and 3, ganglioside 4; lanes 4 and 5, ganglioside 7; and lanes 6 and 7, ganglioside 8. Lanes 2, 4, and 6, before sialidase treatment; lanes 3, 5, and 7, after sialidase treatment. Lane 1, rat liver gangliosides. The arrow indicates free sialic acids released during the enzyme reaction.
in tube reaction for analysis of the degradation product(s) (Fig. 3). Hydrolysis of gangliosides 4, 7, and 8 produced a ganglioside with the same mobility of GM1. In the case of ganglioside 7, another degradation product corresponding to GD1b also was observed. Gangliosides 1 and 3 were hydrolyzed by the enzyme, producing a neutral glycolipid corresponding to LacCer (data nor shown). Based upon these findings and their chromatographic mobility, gangliosides 1, 3, 4, 7, and 8 were respectively identified as GM3, GD3, GD1a, GT1b, and GQ1b. Gangliosides 2 and 5 did not react with anti-GM1 antibody, but did react with anti-asialo GM1 antibody, showing that they had the gangliotetraose oligosaccharide core structure. Ganglioside 6 did not react with anti-GM1 antibody nor with anti-asialo-GM1 antibody following sialidase treatment. The present study also demonstrated that pancreatic tissue expressed trace amounts of GM1 and GD1b (Fig. 2A). Isolated pancreatic islets showed a ganglioside pattern that was distinct from that of pancreatic tissue (Fig. 4). Pancreatic islets consisted mainly of di- and polysialo ganglioside species that included GD3, GT1b, and ganglioside 6 without detectable amounts of GM3, GD1a, ganglioside 2, and ganglioside 5.
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FIG. 4. Thin-layer chromatogram of gangliosides in pancreatic tissue and isolated pancreatic islets. Gangliosides in pancreatic tissue and isolated pancreatic islets were developed on TLC and visualized by resorcinol–HCl reagent. Lane 1, pancreatic tissue (equivalent to 10 mg protein); and lane 2, isolated pancreatic islets (equivalent to 0.18 mg protein).
C-series Gangliosides in Pancreatic Tissue and Pancreatic Islets Pancreatic tissue contained four A2B5-reactive gangliosides, showing a composition that was distinguished from that in liver tissue (Fig. 5). Based upon their chromatographic mobility on TLC and the specificity of A2B5, these gangliosides were identified as GT3, GT2, GQ1c, and GP1c. While a remarkable difference was observed between the major ganglioside
FIG. 5. Immunochromatogram of gangliosides in rat pancreatic tissue. Gangliosides in rat pancreatic tissue were developed on TLC and immunostained with A2B5. Lane 1, liver tissue (equivalent to 15 mg wet weight tissue); and lane 2, pancreatic tissue (equivalent to 15 mg wet weight tissue).
FIG. 6. C-series gangliosides in pancreatic tissue and isolated pancreatic islets. Gangliosides in pancreatic tissue and pancreatic islets were developed on TLC and immunostained with A2B5. Lanes 1 and 3, pancreatic tissue (equivalent to 10 mg protein); and lanes 2 and 4, pancreatic islets (equivalent to 0.04 mg protein). Lanes 1 and 2, visualized by resorcinol–HCl reagent; and lanes 3 and 4, immunostained with A2B5.
patterns of pancreatic tissue and pancreatic islets, the compositional profiles of c-series gangliosides were almost identical (Fig. 6). As compared with pancreatic tissue, pancreatic islets had a much higher concentration of c-series gangliosides; the content of c-series gangliosides in pancreatic islets equivalent to 40 ng protein was even higher than that in pancreatic tissue equivalent to 10 mg protein. DISCUSSION
Gangliosides in pancreatic tissues and isolated islets have been characterized by several researchers. Ganglioside GM2-1, which migrates between GM2 and GM1 on TLC, was reported to be present in pancreatic tissues and pancreatic islets of different species (17– 20). The presence of c-series gangliosides in pancreatic islets has been suggested by immunocytochemistry of isolated preparations with A2B5 (21, 22). However, detailed information regarding ganglioside composition of pancreatic tissues and pancreatic islets is still limited. In the present study, we examined gangliosides of rat pancreas and demonstrated that pancreatic islets have a distinct ganglioside composition. Evidence was also provided suggesting the restricted localization of c-series gangliosides in pancreatic islets. Rat pancreatic tissue showed a GM3-dominant ganglioside pattern. Among individual ganglioside species, GM3, GD3, GD1a, GT1b, and GQ1b were identified based upon their chromatographic behaviors and reactivity to anti-GM1 antibody after sialidase treatment. This conclusion was further supported by the response
MAJOR AND C-SERIES GANGLIOSIDES OF RAT PANCREATIC ISLETS
of each ganglioside to sialidase treatment in tube reaction. The hydrolysis profile of ganglioside 7 was essentially the same as observed when GT1b was treated by Arthrobacter ureafaciens sialidase (16), providing further evidence that this ganglioside is GT1b. While gangliosides 2 and 5 did not react with anti-GM1 antibody, they became reactive with anti-asialo-GM1 antibody following treatment with C. perfringens sialidase. It is known that C. perfringens sialidase hardly hydrolyzes a sialic acid residue connected to the inner galactose, but efficiently cleaves the one linked to the terminal galactose or N-acetylgalactosamine in the gangliotetraose structure (23). The present study indicates that both ganglioside 2 and ganglioside 5 have the gangliotetraose oligosaccharide core structure with a sialic acid residue(s) that are not connected to the inner galactose. Although the structures of gangliosides 2 and 5 remain to be determined, they may be one of “asialo-GM1-derived” gangliosides including GM1b, GD1c, and GD1␣ (24 –27). The structures of ganglioside 6 also is unknown. While this ganglioside had chromatographic mobility similar to that of GT3, no correlation was observed between the amounts of the lipid visualized by resorcinol–HCl reagent and immunostained with A2B5 (see Fig. 6). It thus is unlikely that ganglioside 6 is GT3. The composition of gangliosides in isolated pancreatic islets significantly differed from that in pancreatic tissue. In pancreatic islets, GD3, ganglioside 6, and GT1b constitute the major ganglioside components, while GM3, ganglioside 2, GD1a, and ganglioside 5 were hardly detected. These findings suggests that ganglioside metabolism in pancreatic islet cells is regulated in a manner distinct from that in nonislet cells. In the present study, we could not detect GM2-1, which has been demonstrated in pancreatic islets (17). The content of GM2-1 in rat pancreas may not be high enough to detect by ordinary visualizing methods. C-series gangliosides are characterized by a trisialosyl residue at the inner galactose of the hemato- or ganglio-type oligosaccharide structure (28, 29). Since c-series gangliosides exist as minor components in mammalian tissues, they are difficult to analyze by conventional visualization methods after separation on TLC. In the present study, we employed a specific monoclonal antibody A2B5 for examining c-series gangliosides in rat pancreas. A2B5 was originally prepared by immunizing chicken embryonic retinal cells (30). While there was some controversy about specificity of the antibody (31), recent studies have reported its strict specificity to c-series gangliosides including GT3, 9-O-acetyl GT3, GT1c, GQ1c, GP1c, and G⬙H⬙ (32–35). We have recently characterized A2B5-reactive gangliosides in rat cultured hepatocytes and liver tissue and provided further evidence for the specificity of the antibody; A2B5-reactive gangliosides corresponding to
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GT1c, GQ1c, and GP1c were shown to possess the gangliotetraose oligosaccharide core structure (36). Based upon the reactivity of A2B5 to c-series gangliosides, it is assumed that the trisialosyl structure connected to the inner galactose constitutes the epitope recognized by the antibody. Pancreatic tissue contained four A2B5-reactive gangliosides. Among them, three gangliosides migrating near GD1b, below GQ1b, and far below GQ1b on TLC were respectively assigned to GT3, GQ1c, and GP1c. The ganglioside migrating between GT3 and GT1c was identified as GT2 based upon the specificity of A2B5 and chromatographic mobility on TLC (29). No positive staining of other gangliosides or nonganglioside acidic lipids (e.g., sulfatides) was observed. In contrast to the case of major gangliosides, the compositions of c-series gangliosides in pancreatic tissue and isolated pancreatic islets were almost identical. The concentration of c-series gangliosides in pancreatic islets was much higher than that in pancreatic tissue. These results suggest that c-series gangliosides may be highly enriched in pancreatic islets in rat pancreas, which is consistent with our recent study, suggesting that cseries gangliosides may be localized exclusively in rat pancreatic B cells (8). It is assumed that the expression of c-series gangliosides in brain tissues may be cellspecific. A series of immunohistochemical studies with a specific monoclonal antibody Q211 demonstrated restricted localization of Q211-positive cells in brain tissues (37–39). Recent studies have provided further evidence for this assumption by demonstrating the specific localization of c-series gangliosides in stellate neurons in human cerebellum (40) and developmentdependent expression of GT3 and its O-acetyl derivative in rat glial cells (35). Integration of our present study and others suggests that c-series gangliosides may also be expressed in a cell-specific manner in extraneural tissues. In summary, the present study demonstrates that pancreatic islet cells have a distinct ganglioside composition in rat pancreas. This finding suggests that c-series gangliosides may play an important role in physiological and pathological processes of pancreatic islets. It is known that insulin-dependent diabetic mellitus (IDDM) is developed through autoimmune destruction of pancreatic B cells (41). C-series gangliosides, which probably are localized specifically in pancreatic B cells (8), may serve as the target molecules in the autoimmune mechanism of IDDM. ACKNOWLEDGMENT The authors thank Mr. Takeshi Suzuki for his technical assistance in isolation of pancreatic islets.
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