Acid-Modulated Peptide Synthesis for Application on Oxide Biosensor Interfaces.

In this paper we report an acid-modulated strategy for novel peptide microarray production on biosensor interfaces. We initially selected a controlled pore glass (CPG) as a support for solid-phase peptide synthesis (SPPS) to implement a chemistry that can be performed at the interface of multiple field effect transistor (FET) sensors, eventually to generate label-free peptide microarrays for protein screening. Our chemistry uses a temporary protection of the N-terminal amino function of each amino acid building block with a tert-butyloxycarbonyl (Boc) group that can be removed after each SPPS cycle, in combination with semi-permanent protection of the side chains of trifunctional amino acid residues. Such a protection scheme with a well-proven record of application in conventional, batchwise SPPS has been fine-tuned for optimal performance on CPG and, from there, translated to SPR chips that allow layer-by-layer monitoring of amino acid coupling. Our results validate this acid-modulated synthesis as a feasible approach for producing peptides in high yields and purity on flat glass surfaces, such as those in bio-FETs.

Genome-wide epitope mapping across multiple host species reveals significant diversity in antibody responses to Coxiella burnetii vaccination and infection.

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of human infection. Vaccination is a key control measure, and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available for use in livestock and humans. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin antigens. Defined subunit antigen vaccines offer significant advantages, as they can be engineered to reduce reactogenicity and co-designed with serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccination and/or infection in cattle, goats, humans, and sheep through genome-wide linear epitope mapping to identify candidate vaccine and diagnostic antigens within the predicted bacterial proteome. Using high-density peptide microarrays, we analyzed the seroreactivity in 156 serum samples from vaccinated and infected individuals to peptides derived from 2,092 open-reading frames in the C. burnetii genome. We found significant diversity in the antibody responses within and between species and across different types of C. burnetii exposure. Through the implementation of three different vaccine candidate selection methods, we identified 493 candidate protein antigens for protein subunit vaccine design or serodiagnostic evaluation, of which 65 have been previously described. This is the first study to investigate multi-species seroreactivity against the entire C. burnetii proteome presented as overlapping linear peptides and provides the basis for the selection of antigen targets for next-generation Q fever vaccines and diagnostic tests.

Focused B cell response to recurring gluten motif with implications for epitope spreading in celiac disease.

Antibodies to deamidated gluten peptides are accurate diagnostic markers of celiac disease. However, binding of patient antibodies to all possible gluten epitopes has not previously been investigated. Here, we assess serum antibody specificity across the gluten proteome by use of high-density peptide arrays. We confirm the importance of deamidation for antibody binding, and we show that the response is remarkably focused on the known epitope QPEQPFP (where E results from deamidation of Q). In addition, we describe an epitope in native (non-deamidated) gluten, QQPEQII (where E is gene encoded), which is associated with both B cell and T cell reactivity. Antibodies to this native epitope are cross-reactive with the major deamidated epitope due to recognition of the shared PEQ motif. Since cross-reactive B cells can present peptides to different gluten-specific T cells, we propose that such B cells play a role in epitope spreading by engaging T cells with multiple specificities.

A Systematic, Unbiased Mapping of CD8+ and CD4+ T Cell Epitopes in Yellow Fever Vaccinees.

Examining CD8+ and CD4+ T cell responses after primary Yellow Fever vaccination in a cohort of 210 volunteers, we have identified and tetramer-validated 92 CD8+ and 50 CD4+ T cell epitopes, many inducing strong and prevalent (i.e., immunodominant) T cell responses. Restricted by 40 and 14 HLA-class I and II allotypes, respectively, these responses have wide population coverage and might be of considerable academic, diagnostic and therapeutic interest. The broad coverage of epitopes and HLA overcame the otherwise confounding effects of HLA diversity and non-HLA background providing the first evidence of T cell immunodomination in humans. Also, double-staining of CD4+ T cells with tetramers representing the same HLA-binding core, albeit with different flanking regions, demonstrated an extensive diversification of the specificities of many CD4+ T cell responses. We suggest that this could reduce the risk of pathogen escape, and that multi-tetramer staining is required to reveal the true magnitude and diversity of CD4+ T cell responses. Our T cell epitope discovery approach uses a combination of (1) overlapping peptides representing the entire Yellow Fever virus proteome to search for peptides containing CD4+ and/or CD8+ T cell epitopes, (2) predictors of peptide-HLA binding to suggest epitopes and their restricting HLA allotypes, (3) generation of peptide-HLA tetramers to identify T cell epitopes, and (4) analysis of ex vivo T cell responses to validate the same. This approach is systematic, exhaustive, and can be done in any individual of any HLA haplotype. It is all-inclusive in the sense that it includes all protein antigens and peptide epitopes, and encompasses both CD4+ and CD8+ T cell epitopes. It is efficient and, importantly, reduces the false discovery rate. The unbiased nature of the T cell epitope discovery approach presented here should support the refinement of future peptide-HLA class I and II predictors and tetramer technologies, which eventually should cover all HLA class I and II isotypes. We believe that future investigations of emerging pathogens (e.g., SARS-CoV-2) should include population-wide T cell epitope discovery using blood samples from patients, convalescents and/or long-term survivors, who might all hold important information on T cell epitopes and responses.

Unbiased Characterization of Peptide-HLA Class II Interactions Based on Large-Scale Peptide Microarrays; Assessment of the Impact on HLA Class II Ligand and Epitope Prediction.

Human Leukocyte Antigen class II (HLA-II) molecules present peptides to T lymphocytes and play an important role in adaptive immune responses. Characterizing the binding specificity of single HLA-II molecules has profound impacts for understanding cellular immunity, identifying the cause of autoimmune diseases, for immunotherapeutics, and vaccine development. Here, novel high-density peptide microarray technology combined with machine learning techniques were used to address this task at an unprecedented level of high-throughput. Microarrays with over 200,000 defined peptides were assayed with four exemplary HLA-II molecules. Machine learning was applied to mine the signals. The comparison of identified binding motifs, and power for predicting eluted ligands and CD4+ epitope datasets to that obtained using NetMHCIIpan-3.2, confirmed a high quality of the chip readout. These results suggest that the proposed microarray technology offers a novel and unique platform for large-scale unbiased interrogation of peptide binding preferences of HLA-II molecules.

HLA Class II Specificity Assessed by High-Density Peptide Microarray Interactions.

The ability to predict and/or identify MHC binding peptides is an essential component of T cell epitope discovery, something that ultimately should benefit the development of vaccines and immunotherapies. In particular, MHC class I prediction tools have matured to a point where accurate selection of optimal peptide epitopes is possible for virtually all MHC class I allotypes; in comparison, current MHC class II (MHC-II) predictors are less mature. Because MHC-II restricted CD4+ T cells control and orchestrated most immune responses, this shortcoming severely hampers the development of effective immunotherapies. The ability to generate large panels of peptides and subsequently large bodies of peptide-MHC-II interaction data are key to the solution of this problem, a solution that also will support the improvement of bioinformatics predictors, which critically relies on the availability of large amounts of accurate, diverse, and representative data. In this study, we have used rHLA-DRB1*01:01 and HLA-DRB1*03:01 molecules to interrogate high-density peptide arrays, in casu containing 70,000 random peptides in triplicates. We demonstrate that the binding data acquired contains systematic and interpretable information reflecting the specificity of the HLA-DR molecules investigated, suitable of training predictors able to predict T cell epitopes and peptides eluted from human EBV-transformed B cells. Collectively, with a cost per peptide reduced to a few cents, combined with the flexibility of rHLA technology, this poses an attractive strategy to generate vast bodies of MHC-II binding data at an unprecedented speed and for the benefit of generating peptide-MHC-II binding data as well as improving MHC-II prediction tools.

Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.

The human leukocyte antigen-A2 (HLA-A2)-restricted zinc transporter 8186-194 (ZnT8186-194) and other islet epitopes elicit interferon-γ secretion by CD8+ T cells preferentially in type 1 diabetes (T1D) patients compared with controls. We show that clonal ZnT8186-194-reactive CD8+ T cells express private T cell receptors and display equivalent functional properties in T1D and healthy individuals. Ex vivo analyses further revealed that CD8+ T cells reactive to ZnT8186-194 and other islet epitopes circulate at similar frequencies and exhibit a predominantly naïve phenotype in age-matched T1D and healthy donors. Higher frequencies of ZnT8186-194-reactive CD8+ T cells with a more antigen-experienced phenotype were detected in children versus adults, irrespective of disease status. Moreover, some ZnT8186-194-reactive CD8+ T cell clonotypes were found to cross-recognize a Bacteroides stercoris mimotope. Whereas ZnT8 was poorly expressed in thymic medullary epithelial cells, variable thymic expression levels of islet antigens did not modulate the peripheral frequency of their cognate CD8+ T cells. In contrast, ZnT8186-194-reactive cells were enriched in the pancreata of T1D patients versus nondiabetic and type 2 diabetic individuals. Thus, islet-reactive CD8+ T cells circulate in most individuals but home to the pancreas preferentially in T1D patients. We conclude that the activation of this common islet-reactive T cell repertoire and progression to T1D likely require defective peripheral immunoregulation and/or a proinflammatory islet microenvironment.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping.

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

Sulfatide Preserves Insulin Crystals Not by Being Integrated in the Lattice but by Stabilizing Their Surface.

BACKGROUND: Sulfatide is known to chaperone insulin crystallization within the pancreatic beta cell, but it is not known if this results from sulfatide being integrated inside the crystal structure or by binding the surface of the crystal. With this study, we aimed to characterize the molecular mechanisms underlying the integral role for sulfatide in stabilizing insulin crystals prior to exocytosis. METHODS: We cocrystallized human insulin in the presence of sulfatide and solved the structure by molecular replacement. RESULTS: The crystal structure of insulin crystallized in the presence of sulfatide does not reveal ordered occupancy representing sulfatide in the crystal lattice, suggesting that sulfatide does not permeate the crystal lattice but exerts its stabilizing effect by alternative interactions such as on the external surface of insulin crystals. CONCLUSIONS: Sulfatide is known to stabilize insulin crystals, and we demonstrate here that in beta cells sulfatide is likely coating insulin crystals. However, there is no evidence for sulfatide to be built into the crystal lattice.

Automated High-Throughput Mapping of Linear B-Cell Epitopes Using a Statistical Analysis of High-Density Peptide Microarray Data.

Detailed information of antibodies' specificity is often missing or inadequate even for continuous (i.e., linear) epitopes. Recent developments in peptide microarray technology has enabled the synthesis of up to two million peptides per array thereby allowing linear peptide epitopes to be examined by a systematic amino acid substitution and positional scanning approach. This kind of analysis generates a very large body of data, which needs to be analyzed and interpreted in a robust and automated manner. Here, we describe a rational systematic approach to define linear antibody epitopes using ANOVA statistics to identify not only significant but also important residues involved in antibody recognition. This statistical approach can be used to perform a comprehensive linear epitope discovery. For polyclonal antibodies, this could be extended to entire proteins pinpointing critical residues for each epitope. We argue that the ANOVA analysis levels out issues of unknown peptide concentration/quality and unknown antibody titers leading to identification of epitopes that otherwise would be neglected if the evaluation was based merely on signal strength.

Materno-Fetal Transfer of Preproinsulin Through the Neonatal Fc Receptor Prevents Autoimmune Diabetes.

The first signs of autoimmune activation leading to ß-cell destruction in type 1 diabetes (T1D) appear during the first months of life. Thus, the perinatal period offers a suitable time window for disease prevention. Moreover, thymic selection of autoreactive T cells is most active during this period, providing a therapeutic opportunity not exploited to date. We therefore devised a strategy by which the T1D-triggering antigen preproinsulin fused with the immunoglobulin (Ig)G Fc fragment (PPI-Fc) is delivered to fetuses through the neonatal Fc receptor (FcRn) pathway, which physiologically transfers maternal IgGs through the placenta. PPI-Fc administered to pregnant PPIB15-23 T-cell receptor-transgenic mice efficiently accumulated in fetuses through the placental FcRn and protected them from subsequent diabetes development. Protection relied on ferrying of PPI-Fc to the thymus by migratory dendritic cells and resulted in a rise in thymic-derived CD4(+) regulatory T cells expressing transforming growth factor-ß and in increased effector CD8(+) T cells displaying impaired cytotoxicity. Moreover, polyclonal splenocytes from nonobese diabetic (NOD) mice transplacentally treated with PPI-Fc were less diabetogenic upon transfer into NOD.scid recipients. Transplacental antigen vaccination provides a novel strategy for early T1D prevention and, further, is applicable to other immune-mediated conditions.

MHC class II tetramers made from isolated recombinant α and ß chains refolded with affinity-tagged peptides.

Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides. As a general approach to class II tetramer generation, this method should support rational CD4+ T cell epitope discovery as well as enable specific monitoring and manipulation of CD4+ T cell responses.

HLA-B7-restricted islet epitopes are differentially recognized in type 1 diabetic children and adults and form weak peptide-HLA complexes.

The cartography of ß-cell epitopes targeted by CD8(+) T cells in type 1 diabetic (T1D) patients remains largely confined to the common HLA-A2 restriction. We aimed to identify ß-cell epitopes restricted by the HLA-B7 (B*07:02) molecule, which is associated with mild T1D protection. Using DNA immunization on HLA-B7-transgenic mice and prediction algorithms, we identified GAD and preproinsulin candidate epitopes. Interferon-γ (IFN-γ) enzyme-linked immunospot assays on peripheral blood mononuclear cells showed that most candidates were recognized by new-onset T1D patients, but not by type 2 diabetic and healthy subjects. Some epitopes were highly immunodominant and specific to either T1D children (GAD(530-538); 44% T cell-positive patients) or adults (GAD(311-320); 38%). All epitopes displayed weak binding affinity and stability for HLA-B7 compared with HLA-A2-restricted ones, a general feature of HLA-B7. Single-cell PCR analysis on ß-cell-specific (HLA-B7 tetramer-positive) T cells revealed uniform IFN-γ and transforming growth factor-ß (TGF-ß) mRNA expression, different from HLA-A2-restricted T cells. We conclude that HLA-B7-restricted islet epitopes display weak HLA-binding profiles, are different in T1D children and adults, and are recognized by IFN-γ(+)TGF-ß(+)CD8(+) T cells. These features may explain the T1D-protective effect of HLA-B7. The novel epitopes identified should find valuable applications for immune staging of HLA-B7(+) individuals.

T cells recognizing a peptide contaminant undetectable by mass spectrometry.

Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted ß-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(206-214) epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP(206-214) using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP(206-214). If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.

Alcohol facilitates CD1d loading, subsequent activation of NKT cells, and reduces the incidence of diabetes in NOD mice.

BACKGROUND: Ethanol ('alcohol') is a partly hydrophobic detergent that may affect the accessibility of glycolipids thereby influencing immunological effects of these molecules. METHODS: The study included cellular in vitro tests using α-galactosylceramide (αGalCer), and in vivo NOD mice experiments detecting diabetes incidence and performing behavioural and bacterial analyses. RESULTS: Alcohol in concentrations from 0.6% to 2.5% increased IL-2 production from NKT cells stimulated with αGalCer by 60% (p<0.05). CD1d expressed on HeLa cells contained significantly increasing amounts of αGalCer with increasing concentrations of alcohol, suggesting that alcohol facilitated the passive loading of αGalCer to CD1d. NOD mice were found to tolerate 5% ethanol in their drinking water without signs of impairment in liver function. Giving this treatment, the diabetes incidence declined significantly. Higher numbers of CD3+CD49b+ NKT cells were found in spleen and liver of the alcohol treated compared to the control mice (p<0.05), whereas the amount of CD4+Foxp3+ regulator T cells did not differ. Increased concentrations of IFN-γ were detected in 24-hour blood samples of alcohol treated mice. Behavioural studies showed no change in attitude of the ethanol-consuming mice, and bacterial composition of caecum samples was not affected by alcohol, disqualifying these as protective mechanisms. CONCLUSION: Alcohol facilitates the uptake of glycolipids and the stimulation of NKT cells, which are known to counteract Type 1 diabetes development. We propose that this is the acting mechanism by which treatment with alcohol reduces the incidence of diabetes in NOD mice. This is corroborated by epidemiology showing beneficial effect of alcohol to reduce the severity of atherosclerosis and related diseases.

A subset of human pancreatic beta cells express functional CD14 receptors: a signaling pathway for beta cell-related glycolipids, sulfatide and ß-galactosylceramide.

BACKGROUND: T1DM is a T-cell-mediated autoimmune disease targeting insulin-producing beta-cells. Multiple factors may contribute to the development of T1DM. Among these, the metabolic state of beta-cells and pro-inflammatory cytokines, produced by infiltrating immune cells, have been implicated in the precipitation of T1DM. METHODS AND RESULTS: In this study, confocal immunofluorescence microscopy of human pancreata revealed a distinct subset of beta-cells expressing the innate LPS co-receptor CD14. Human islets expressed fully functional CD14 as LPS stimulation led to a dose-dependent secretion of tumour necrosis factor (TNFα), interleukin (IL)-1ß and IL-8, which were substantially inhibited by a blocking anti-CD14 mAb. In addition, LPS stimulation impaired the glucose-mediated insulin secretion in rat islets. ß-GalCer and sulfatide, glycolipids that are related to insulin processing and secretion, are possibly interacting with the CD14 receptor complex. ß-GalCer had an LPS-like, serum- and CD14-dependent effect on the induction of pro-inflammatory cytokines in a human monocyte cell line. In contrast, the LPS-mediated cytokine production was inhibited by sulfatide. Human islets also responded to ß-GalCer (10 µg/mL) by secreting TNFα, IL-1ß and IL-8, whereas sulfatide partly inhibited the effect of LPS. CONCLUSIONS: A subset of human beta-cells expresses functional CD14 receptor and thus is able to recognize both exogenous bacterial (LPS) as well as endogenous ligands (e.g. glycolipids of beta-cell origin). The CD14 expression on a subset of human beta-cells may play a role in the innate surveillance of the endocrine environment but may also contribute to innate immune mechanisms in the early stages of beta-cell aggression.

Multiple tissue-specific isoforms of sulfatide activate CD1d-restricted type II NKT cells.

The glycosphingolipid sulfatide (SO(3)-3Galbeta1Cer) is a demonstrated ligand for a subset of CD1d-restricted NKT cells, which could regulate experimental autoimmune encephalomyelitis, a murine model for multiple sclerosis, as well as tumor immunity and experimental hepatitis. Native sulfatide is a mixture of sulfatide isoforms, i.e. sulfatide molecules with different long-chain bases and fatty acid chain lengths and saturation. Here, we demonstrate that sulfatide-specific CD1d-restricted murine NKT hybridomas recognized several different sulfatide isoforms. These included the physiologically relevant isoforms C24:1 and C24:0, major constituents of the myelin sheet of the nervous system, and C16:0, prominent in the pancreatic islet beta-cells. The most potent sulfatide isoform was lysosulfatide (lacking a fatty acid). Shortened fatty acid chain length (C24:1 versus C18:1), or saturation of the long fatty acid (C24:0), resulted in reduced stimulatory capacity, and fatty acid hydroxylation abolished the response. Moreover, sulfatide was not responsible for the natural autoreactivity toward splenocytes by XV19 T hybridoma cells. Our results reveal a promiscuity in the recognition of sulfatide isoforms by a CD1d-restricted NKT-cell clone, and suggest that sulfatide, a major component of the myelin sheet and pancreatic beta-cells, is one of several natural ligands for type II CD1d-restricted NKT cells.

Uptake of the glycosphingolipid sulfatide in the gastrointestinal tract and pancreas in vivo and in isolated islets of Langerhans.

BACKGROUND: The glycosphingolipid sulfatide has previously been found in several mammalian tissues, but information on the uptake of exogenously administered sulfatide in different organs in vivo is limited. In pancreatic beta cells, sulfatide has been shown to be involved in insulin processing and secretion in vitro. In this study, we examined the uptake of exogenously administered sulfatide and its distribution to the pancreatic beta cells. This might encourage future studies of the function(s) of sulfatide in beta cell physiology in vivo. Radioactive sulfatide was given orally to mice whereafter the uptake of sulfatide in the gastrointestinal tract and subsequent delivery to the pancreas was examined. Sulfatide uptake in pancreas was also studied in vivo by i.p. administration of radioactive sulfatide in mice, and in vitro in isolated rat islets. Isolated tissue/islets were analysed by scintillation counting, autoradiography and thin-layer chromatography-ELISA. RESULTS: Sulfatide was taken up in the gastrointestinal tract for degradation or further transport to other organs. A selective uptake of short chain and/or hydroxylated sulfatide fatty acid isoforms was observed in the small intestine. Exogenously administered sulfatide was found in pancreas after i.p, but not after oral administration. The in vitro studies in isolated rat islets support that sulfatide, independently of its fatty acid length, is endocytosed and metabolised by pancreatic islets. CONCLUSION: Our study supports a selective uptake and/or preservation of sulfatide in the gastrointestinal tract after oral administration and with emphasises on pancreatic sulfatide uptake, i.p. administration results in sulfatide at relevant location.

Selective lack of the C16:0 fatty acid isoform of sulfatide in pancreas of type II diabetic animal models.

Sulfatide (3'-sulfogalactosyl-ceramide) is a glycosphingolipid mainly located in the nervous system, but has also been found in the islets of Langerhans. Previous studies have suggested that sulfatide is involved in insulin processing and secretion. In this study, sulfatide expression and metabolism in pancreas and isolated islets of the type II diabetes models, ob/ob- and db/db mouse, was investigated using TLC-ELISA, metabolic labelling and electron microscopy. As in non-diabetic Lewis rat and human pancreas, sulfatide was located in secretory granules of the beta cells. However, the type II diabetic animal models and their background strains had an altered sulfatide expression, involving the lack of the C16:0 sulfatide fatty acid isoform, compared to non-diabetic Lewis rat, BALB/c mouse and human pancreatic tissue, in which the two dominating pancreatic sulfatide isoforms C16:0 and C24:0 are expressed. Correspondingly, in isolated ob/ob islets, sulfatide synthesis excluded the production of C16:0 sulfatide. Insulin administration to ob/ob mouse, which lowers beta cell activity, resulted in significantly increased sulfatide expression in pancreas (p=0.0003), but still no expression of the C16:0 sulfatide isoform. In vitro, the C16:0 sulfatide was shown to be the isomer involved in the preservation of insulin crystals. Thus, it is hypothesized that the selection of sulfatide isomers in pancreas might be a genetic factor contributing to disease development in type II diabetic animal models.

Sulfatide is associated with insulin granules and located to microdomains of a cultured beta cell line.

Previous studies using pancreas from various mammals and freshly isolated islets from rat pancreas have provided evidence supporting possible involvement of the glycosphingolipid sulfatide in insulin processing and secretion. In this study, sulfatide expression and metabolism in the beta cell line RINr1046-38 (RIN-38), commonly used as a model for beta cell functional studies, were investigated and compared with previous findings from freshly isolated islets. RIN-38 cells expressed similar amounts (2.7 +/- 1.1 nmol/mg protein, n = 19) of sulfatide as isolated rat islets and also followed the same metabolic pathway, mainly through recycling. Moreover, in agreement with findings in isolated islets, the major species of sulfatide isolated from RIN-38 cells contained C16:0 and C24:0 fatty acids. By applying subcellular isolations and electron microscopy and immunocytochemistry techniques, sulfatide was shown to be located to the secretory granules, the plasma membrane and enriched in detergent insoluble microdomains. In the electron microscopy studies, Sulph I staining was also associated with mitochondria and villi structures. In conclusion, RIN-38 cells might be an appropriate model, as a complement to isolated islets where the amount of material often limits the experiments, to further explore the role of sulfatide in insulin secretion and signal transduction of beta cells.