Glycobiology, 2001, Vol. 11, No. 6 473-479
© 2001 Oxford University Press
Sulfatide promotes the folding of proinsulin, preserves insulin crystals, and mediates its monomerization
2Bartholin Instituttet, Kommunehospitalet, DK-1399 Copenhagen K, Denmark, 3Institute of Clinical Neuroscience, University of Göteborg, Mölndal sjukhus, SE-431 80 Mölndal, Sweden, 4Department of Medical Anatomy, Panum Instituttet, University of Copenhagen, DK-2200 Copenhagen N, Denmark, 5Brange Consult, DK-2930 Klampenborg, Denmark, and 6Department of Chemistry, University of York, Heslington, York Y010 5DD, England
Received on November 15, 2000; revised on January 19, 2001; accepted on January 29, 2001.
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Abstract |
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Sulfatide is a glycolipid that has been associated with insulin-dependent diabetes mellitus. It is present in the islets of Langerhans and follows the same intracellular route as insulin. However, the role of sulfatide in the beta cell has been unclear. Here we present evidence suggesting that sulfatide promotes the folding of reduced proinsulin, indicating that sulfatide possesses molecular chaperone activity. Sulfatide associates with insulin by binding to the insulin domain A8–A10 and most likely by interacting with the hydrophobic side chains of the dimer-forming part of the insulin B-chain. Sulfatide has a dual effect on insulin. It substantially reduces deterioration of insulin hexamer crystals at pH 5.5, conferring stability comparable to those in beta cell granules. Sulfatide also mediates the conversion of insulin hexamers to the biological active monomers at neutral pH, the pH at the beta-cell surface. Finally, we report that inhibition of sulfatide synthesis with chloroquine and fumonisine B1 leads to inhibition of insulin granule formation in vivo. Our observations suggest that sulfatide plays a key role in the folding of proinsulin, in the maintenance of insulin structure, and in the monomerization process.
Key words: sulfatide/insulin/molecular chaperone/islets of Langerhans/secretory granules
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by a specific destruction of the insulin-producing beta cells in the islets of Langerhans. Sulfatide (3'-sulfogalactosylceramide) has recently become interesting in the context of IDDM; it is present in the secretory granules and at the surface of the beta cells (Buschard et al., 1993b
, 1994). Like insulin auto-antibodies (Palmer et al., 1983), antibodies in high titers against sulfatide were demonstrated in newly diagnosed IDDM patients; thus, it has been proposed that sulfatide plays a role in the development of IDDM (Buschard et al., 1993a). In the islets of Langerhans, the main pathway of sulfatide synthesis is through recycling (Fredman et al., 2000) involving degradation to galactosylceramide (GalCer) in the lysosomes, which is transported to the Golgi network and resulfated (Benjamins et al., 1982). Not only is sulfatide present in the same cellular compartments as insulin, it also follows insulin trafficking (Fredman et al., 2000).
Proinsulin enters the Golgi network as a single-stranded immature polypeptide followed by formation of disulfide (S-S) bonds and folding into mature proinsulin; from the trans-Golgi network secretory granules are released, and proinsulin associates into hexamers, which are proteolytically converted to mature insulin (Orci, 1986; Dodson and Steiner, 1998). In the mature secretory granules, insulin hexamers reversibly aggregate into zinc-containing crystals and are stored until release (Hutton, 1989; Halban, 1991). In pharmaceutical formulations, zinc and various additives are used to stabilize the insulin hexamer to prevent irreversible protein aggregation known as fibrillation (Brange and Langkjaer, 1993). Once the insulin crystals are delivered to the bloodstream during exocytosis, they dissolve back into hexamers, which further dissociate into the biologically active monomer. It is estimated that insulin is stored for 4–8 days in the beta cells, raising the question of what factors are present in allowing the beta cell to store large amounts of insulin crystals, which are rapidly converted into monomer insulin on exocytosis. Even though insulin has been extensively studied for several decades, important details concerning folding, self-assembly, and dissociation are missing. The objective of the present study was therefore to explore possible interactions between insulin and sulfatide.
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Results |
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Sulfatide binds to insulin
A panel of monoclonal anti-insulin antibodies (mAbs) were used to investigate whether sulfatide, its precursor GalCer, and ganglioside GM1 were able to bind to the insulin molecule. Four different mAbs, designated HUI-001, HUI-018, OXI-004, and OXI-005, directed against insulin subdomains A8–A10, A15, B3, and B25–B30, respectively, were used. Competitive binding between sulfatide, GalCer, GM1, and subdomain-specific antibodies to insulin was investigated by an enzyme-linked immunosorbent assay (ELISA). The insulin A8–A10 region was blocked 42% (p = 0.003) by sulfatide, whereas A15, B3, and B25–B30 domains were not affected compared to control levels. GalCer did not block at all, whereas GM1 reduced the binding of antibody to the A8–A10 domain with 55% (p = 0.007). The residue A8Thr, which showed reduced labeling of mAb (OXI-004) in the presence of sulfatide, is suggested to be involved in binding the sphingosine base of sulfatide. A proposed model of the interaction between sulfatide and human insulin is depicted in Figure 1. In this model the two aliphatic chains of the sulfatide molecule make van der Waals contact with the nonpolar insulin surface involved in dimer and hexamer formation, and the sulfate group is near to the A1
-amino and B29 
-amino groups. The model demonstrates that the sulfatide molecule is compatible with the insulin molecule in terms of both size and chemistry and could prevent insulin aggregation. The conflict between the model and the antibody binding studies with regard to residues B25–B30 suggests that sulfatide binding to the dimer-forming surface is dynamic and exchangeable, able to prevent dimer formation but unable to prevent antibody binding.
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Sulfatide promotes the folding of proinsulin
Inspired by the close molecular interaction suggested by the natural fitting of sulfatide to insulin, we investigated if sulfatide could assist proinsulin folding. Because mature insulin consists of two polypeptide chains, reducing mature insulin would result in an irreversible denaturation; hence, we investigated the refolding of reduced proinsulin. Sulfatide promoted oligomerization of proinsulin, which is seen as distinct bands corresponding to proinsulin dimers, tetramers, and hexamers (Figure 2). Proinsulin oligomers higher than hexamers were not present, indicating that proinsulin had refolded into its native conformation. In the presence of sulfatide, refolding, dimerization, and hexamerization of proinsulin takes place immediately. In contrast, without sulfatide, proinsulin dimers and hexamers are nearly absent even after 20 min. These properties suggest that sulfatide acts as a molecular chaperone. Neither GM1 nor GalCer showed any effect on reduced proinsulin.
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The functional association between insulin and sulfatide
As mentioned previously, sulfatide is present in the secretory granules where insulin is stored as zinc-containing crystals; this raises the question of weather or not a functional interaction exists between sulfatide and insulin crystals. Insulin crystals were suspended in 0.15 M sodium acetate with or without sulfatide at pH 5.5, the pH in mature granules within the beta cell (Hutton, 1982
). In absence of sulfatide, insulin crystals deteriorated within 24 h as determined by light microscopy. In contrast, 90% of the insulin crystals maintained in the presence of sulfatide were preserved (Figure 3). Complete deterioration of insulin crystals incubated with sulfatide was not seen before day 21. Neither GalCer nor GM1 showed the ability to preserve insulin crystals. Scanning electron microscopy of insulin crystals and insulin crystals + sulfatide incubated at pH 5.5 for 2 days confirmed that sulfatide had a dramatic effect on the stability of the insulin crystals. Insulin crystals normally deteriorated extensively displaying a rough surface (Figure 4, top). In contrast, insulin crystals incubated with sulfatide showed no changes to the crystal surface (Figure 4, bottom). These observations do not reveal the mechanism of how sulfatide protects the insulin crystals, but we observed on several scanning electron images that the insulin crystals appeared to be coated with a thin layer of lipid (not shown). It is possible that crystals coated with lipid is shielded from the environment and thus protected from it.
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In sodium acetate at pH 5.5 and in the presence of divalent zinc ions, insulin (150 µg/ml) precipitated as rhombohedral crystals as described (Schlichtkrull, 1961
). However, when sulfatide (250 µg/ml) was added to insulin (150 µg/ml) at neutral pH followed by addition of zinc ions (2.2 zinc/hexamers) and lowering of pH to 5.5, an insulin fibril–like homogeneous precipitate was formed (Figure 5). The precipitate was consisting of extremely thin sheets with a thickness of approximately 100 nm displaying a heterogeneous size distribution of 10–40 µm. These sheets were of a very condensed structure excluding the sodium silicotungstate stain, as shown on the transmission electron microscopy image (Figure 6). These structures were not seen without sulfatide, nor did we observe this precipitate in the presence of GalCer or GM1. Exposure to low pH values, hydrophobic surfaces, and high temperature favors the formation of insoluble insulin precipitates known as insulin fibrils, by an aggregation process that is only partially understood but known to arise from insulin monomers after conformational changes of the tertiary structure (Brange et al., 1997). Insulin fibrils have characteristic solubility properties different from insulin crystals and amorphous insulin precipitates. Therefore, we analysed the solubility of the insulin sheets formed in presence of sulfatide. The insulin sheets were insoluble in 0.1 M hydrochloric acid, DMSO, and 50% acetonitrile, but soluble in 0.05 M sodium hydroxide, DMSO + HCl, and formic acid. High-performance liquid chromatography (HPLC) analysis of the dissolved insulin sheets produced an elution profile identical to that of native insulin. These solubility characteristics are comparable to those of insulin fibrils (Brange et al., 1997); hence, it appears that sulfatide is responsible for pushing the equilibrium toward the insulin monomer.
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To further investigate the monomerization process we looked at the migration patterns of a monomeric human insulin analogue (B28Asp) (Brange et al., 1988
) and human insulin using a modified thin-layer Chromatography (TLC) assay. Radio-iodinated human insulin and B28Asp insulin were applied to the TLC plate at 25 µM, at which human insulin self-associates into dimeric and hexameric molecules (Brange et al., 1990). Migration of the monomeric B28Asp insulin was faster than that of human insulin, which is explained by the difference in molecular size between monomeric (5.8 kDa) dimeric and hexameric (35 kDa) insulin. When incubated with sulfatide, however, human insulin migrated similarly to B28Asp (Figure 7), indicating a conversion of dimers and hexamers into monomers. It is also evident that B28Asp incubated with sulfatide shows a down-shift in mobility. This is explained by the increased molecular mass of the insulin–sulfatide complex compared to insulin, and it shows that sulfatide it self does not increase mobility in an unspecific manner. The monomerization process is a dynamic equilibrium between insulin hexamers, dimers, and monomers, hence, the mobility shift appears as a smear. The smearing of insulin without sulfatide is a result of dilution of the sample as it migrates through the TLC matrix. Although pushing the equilibrium by dilution is substantially less than the effect of sulfatide. The migration pattern of human insulin and B28Asp was unaffected by the presence of GalCer. This corresponds well with our findings that GalCer did not bind to insulin.
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Though it might seem contradictory that sulfatide has a dual role in both preventing insulin crystals from degradation and to promotes insulin monomerization, the duality can be explained by the different conditions maintained throughout the experiments and by the fact that sulfatide, in the islets of Langerhans, consists mainly of two molecular species with a composition of a saturated fatty acid chain of 16 or 24 carbon atoms, respectively (Fredman et al., 2000
). It is possible that the two different species have different functions in insulin processing. The brain-extracted sulfatide used in these experiments has a different composition from that in islets of Langerhans, however, the beta-cell relevant sulfatide species is also represented in the brain, which justify the use of brain derived sulfatide.
In vivo manipulation of sulfatide synthesis affects insulin processing
We have recently described that chloroquine inhibits the major pathway of sulfatide synthesis in islets of Langerhans, which is processed through recycling, and that fumonisine B1 (inhibitor of ceramide production) represses de novo synthesis of sulfatide (Fredman et al., 2000). Resistance to in vivo reduction of insulin with dithiothreitol in the medium is partly lost when islets are treated with chloroquine (Huang and Arvan, 1995) and insulin biosynthesis but not insulin secretion is inhibited by chloroquine (Chatterjee and Schatz, 1988). Therefore, one might expect that our observations may be related to similar phenomena in vivo. To investigate this we treated rat islets with chloroquine or fumonisine B1, respectively, as well as with chloroquine and fumonisine B1 together. We found that islets treated with fumonisine B1 alone did not affect the number of insulin granules presumably due to de novo sulfatide synthesis being the diminutive pathway only. However, in islets treated with chloroquine or both chloroquine and fumonisine B1 the numbers of insulin granules were reduced to 55% (p = 0.0002) and 34% (p = 10–7), respectively, of the nontreated control values. The difference between inhibiting either recycling or de novo synthesis compared to inhibiting both pathways indicates that both pathways are capable of compensating each other. Thus, inhibition of sulfatide synthesis directly affects the insulin granule formation, which strongly indicates that sulfatide does interact with insulin in vivo.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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In the present study, we present observations suggesting a molecular interaction between insulin and sulfatide. We demonstrate that sulfatide, but not its precursor GalCer, is able to compete with monoclonal antibodies directed against the A8–A10 subdomain in binding to the insulin molecule. A model of the interaction suggests that hydrophobic residues in the dimer-forming part of the insulin molecule interact with the fatty acid chain of sulfatide. The sulfate group in sulfatide has the potential to form a salt bridge to the positively charged B29Lys and to bind to zinc ions, which are crucial for the formation of insulin crystals. Due to electrostatic forces, attraction between insulin and sulfatide can take place over long distances, in contrast to attraction between GalCer and insulin, which depend on hydrophobic interaction alone. Furthermore, the sulfate group could be responsible for correct orientation of sulfatide toward insulin, leading to further interaction with hydrophobic residues in insulin and explaining the difference in specificity toward insulin between GalCer and sulfatide.
The sulfatide-assisted folding of reduced proinsulin suggests that sulfatide possesses molecular chaperone activity. Traditionally molecular chaperones have been a class of proteins that binds transiently to hydrophobic surfaces in proteins, thus preventing unwanted self-aggregation or misfolding (Hendrick and Hartl, 1995; Ellis, 1996; Hartl, 1996). However, molecular chaperones do not necessarily have to be proteins (Ellis, 1997). As an example, phosphatidylethanolamine has been described to act as a molecular chaperone required for the folding of lactose permase in Escherichia coli (Bogdanov et al., 1996; Bogdanov and Dowhan, 1998). Furthermore, we demonstrate that sulfatide shows a functional interaction with insulin in both dissolved and crystalline form. Because sulfatide is actually present within the insulin granules, it may well be nature’s way to protect insulin from degradation and to promote its monomerization, thus explaining the monomerization speed of insulin released from the beta cells. The maintenance of insulin crystal structure is documented qualitatively by electron microscopy and quantitatively by determining the ratio of intact insulin crystals. Likewise, the monomerization of insulin is documented by two different methods. The chromatographic data is supported by the fibrillation study, because fibrillation depends on the presence of insulin monomers. Finally, it is demonstrated that sulfatide does play a role in insulin processing in vivo.
In conclusion, we suggest that sulfatide acts as a molecular chaperone to (pro)insulin and is involved in the maintenance of insulin structure and promoting its monomerization. As mentioned, phospholipids (Bogdanov and Dowhan, 1998) as well as glycolipid A (de Cock et al., 1999) has been described posses molecular chaperone activity. However, to our knowledge this is the first description of a molecule both acting as a molecular chaperone on the unfolded precursor of a protein (proinsulin) and showing a functional association with the mature protein later in storage and release processes.
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Materials and methods |
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Glycolipid isolation
Sulfatide, GalCer, and GM1 were isolated from porcine brain by extraction with chloroform/methanol/water (4:8:3; by volume) and phase partition in chloroform/methanol (2:1) as previously described (Svennerholm and Fredman, 1980
; Buschard et al., 1994).
ELISA
MaxiSorb plates, 96-well (NUNC, Roskilde, Denmark), were coated with 100 µl human insulin (2.5 pmol) per well. Sulfatide was sonicated for 20 s, 20 kHz, 25 W (Branson, Danbury, CT) in phosphate buffered saline (PBS) pH 7.1 (Gibco BRL, Gaithersburg, MD), and 25 pmol was added to half the wells coated with insulin and incubated at room temperature overnight. Insulin was detected using an antibody sandwich with four mouse insulin mAbs designated HUI-001 (Kjems et al., 1992), HUI-018 (Andersen et al., 1993), OXI-004, and OXI-005 (Wu et al., 1986). As secondary layer, horseradish peroxidase (HRP) conjugated rabbit anti-mouse (Dako, Glostrup, Denmark) was used. Insulin was quantified using tetramethyl-benzidine-hydrochloride as an HRP substrate. The ratio of the signals obtained from wells with insulin + sulfatide versus insulin was calculated for each insulin antibody. Assays with GalCer and GM1 were performed as with sulfatide.
Analysis of reduced proinsulin
Porcine proinsulin (Novo Nordisk, Bagsvaerd, Denmark) 10 mg/ml was reduced by boiling for 5 min with a threefold molar excess of dithiothreitol (Sigma, St. Louis, MO) in 7 M urea. Reduced proinsulin was then diluted 100-fold into 0.33 M Tris–HCl (Sigma), 0.1 mM zinc acetate, pH 7.0, in the presence or absence of sulfatide (170 µg/ml). Sulfatide was prepared by sonication, as described above, in the diluting medium. Aliquots were taken at 0, 2, 5, 10, 12, 15, 17, and 20 min, and the reoxidation process was stopped by alkylating free cysteine residues in 0.1 M iodoacetamide (Sigma) and analyzing the products by native 4–12% BIS-Tris acrylamide gel electrophoresis (NuPage, Novex, San Diego, CA) followed by silver staining. Similar experiments were performed where sulfatide was replaced with GalCer and GM1.
Crystallization of human insulin
Human insulin (Novo Nordisk) with a content of 5.3 zinc atoms per insulin hexamer was crystallized in a medium containing 6 mM dissolved insulin (1000 IU/ml), 0.1 M sodium acetate, and 1.2 M sodium chloride, pH 5.5, as described (Schlichtkrull, 1961).
Crystal stability
Sulfatide, GalCer, or GM1 (each 250 µg/ml) were sonicated in 0.15 M sodium acetate, pH 7, respectively. Insulin crystals were added to these solutions and a solution free of glycolipids giving a final insulin concentration of 150 µg/ml; then the pH was adjusted to 5.5 and the crystal suspensions were incubated on a Swelab mixer 820 (Boute Medical, Stockholm, Sweden) at room temperature. The numbers of intact and deteriorated crystals were determined in aliquots of the crystal suspensions by two persons using a Bürker-Türk counting chamber.
Electron microscopy
Samples of insulin crystals and insulin crystals+sulfatide incubated in 0.15 M sodium acetate at pH 5.5 for 2 days, as described above, were collected on 10 µm PTFE filters (Millipore, Bedford, MA, USA) and fixed in phosphate buffered 2% Osmium tetra oxide, pH 7.2, for 2 h. The filters were washed with distilled water, dehydrated in acetone, critical point dried from acetone and coated with chromium in a Xenon sputter coater (Edwards, Crawley, UK).
A precipitate consistent of thin sheets was seen when insulin (2.2 zinc/hexamers, 150 µg/ml) added to a solution of sulfatide (250 µg/ml) in 0.15 M sodium acetate at pH 5.5. This precipitate were adsorbed on to carbon coated Formvar films, carried on electron microscope copper grids, and negatively stained with 1% sodium silicotungstate, pH 7.0
125I-labeling of insulin
Human insulin and a monomer human insulin analogue B28Asp (Novo Nordisk) were labelled with 125I using the iodate method (Jorgensen and Larsen, 1980) and purified by reversed-phase HPLC on a C4 column, using an acetonitrile-water–trifluoroacetic acid elution system.
TLC
Sulfatide (250 µg) and GalCer (250 µg) were sonicated in 1 ml PBS (Gibco), pH 7.1, respectively, as described above. 125I-insulin or 125I-B28Asp was added to a final molar ratio glycolipid:insulin of 10:1. Then 0.025 nmol insulin (3 nCi) of each sample was applied to TLC aluminum plates coated with cellulose (Merck, Darmstadt, Germany). The TLC plate was placed in PBS, pH 7.1, using a wick, and the water front was allowed to run to the edge. The TLC plate was dried and covered with X-ray film (NEN-Dupont, Boston, MA).
Chloroquine- and fumonisine B1–treated islets
In each experiment, 250 isolated rat islets were incubated 18 h in RPMI 1640 medium (Gibco) with 10% fetal calf serum. Chloroquine (Sigma) and/or fumonisine B1 (Sigma) were added to the medium to final concentrations of 20 µg/ml and 18.5 µg/ml, respectively. The numbers of insulin granules in chloroquine-, fumonisine B1–, or chloroquine and fumonisine B1–treated islets and nontreated controls were determined by counting granules on electron microscopy images n = 30, 29, 31, and 32, respectively. Preparation and measurements of islets were performed as previously described (Buschard et al., 1999). Student’s t test was used to calculate p values.
Molecular modeling of insulin/sulfatide complex
To gain some understanding of the possible interactions between these two molecules, one molecule of sulfatide was modeled onto an insulin monomer. The atomic coordinates for the insulin molecule were taken from the X-ray crystal structure of the T6 insulin hexamer (Baker et al., 1988). The sulfatide molecule was built using the program Quanta (MSI, 1996) and then manually fitted onto surfaces of the insulin molecule, taking into account chemical compatibility and van der Waals interactions.
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We thank Prof. Guy Dodson and Dr. Anthony Wilkinson for constructive criticism of the manuscript. This study was supported by Swedish Medical Research Council K99-03X-09909-08C
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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ELISA, enzyme-linked immunosorbent assay; GalCer, galactosylceramide; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; IDDM, insulin-dependent diabetes mellitus; mAb, monoclonal anti-insulin antibody; PBS, phosphate buffered saline; TLC, thin-layer chromatography.
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1 To whom correspondence should be addressed
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References |
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