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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Key words: sulfatide/insulin/molecular chaperone/islets of Langerhans/secretory granules
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Introduction |
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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 48 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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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 = 107), 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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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 natures 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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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 TrisHCl (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 412% 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-watertrifluoroacetic 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 B1treated 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 B1treated 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). Students 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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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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