Human Proinsulin Research Articles

In Vivo Misfolding of Proinsulin Below the Threshold of Frank Diabetes

OBJECTIVEEndoplasmic reticulum (ER) stress has been described in pancreatic β-cells after onset of diabetes—a situation in which failing β-cells have exhausted available compensatory mechanisms. Herein we have compared two mouse models expressing equally small amounts of transgenic proinsulin in pancreatic β-cells.RESEARCH DESIGN AND METHODSIn hProCpepGFP mice, human proinsulin (tagged with green fluorescent protein [GFP] within the connecting [C]-peptide) is folded in the ER, exported, converted to human insulin, and secreted. In hProC(A7)Y-CpepGFP mice, misfolding of transgenic mutant proinsulin causes its retention in the ER. Analysis of neonatal pancreas in both transgenic animals shows each β-cell stained positively for endogenous insulin and transgenic protein.RESULTSAt this transgene expression level, most male hProC(A7)Y-CpepGFP mice do not develop frank diabetes, yet the misfolded proinsulin perturbs insulin production from endogenous proinsulin and activates ER stress response. In nondiabetic adult hProC(A7)Y-CpepGFP males, all β-cells continue to abundantly express transgene mRNA. Remarkably, however, a subset of β-cells in each islet becomes largely devoid of endogenous insulin, with some of these cells accumulating large quantities of misfolded mutant proinsulin, whereas another subset of β-cells has much less accumulated misfolded mutant proinsulin, with some of these cells containing abundant endogenous insulin.CONCLUSIONSThe results indicate a source of pancreatic compensation before the development of diabetes caused by proinsulin misfolding with ER stress, i.e., the existence of an important subset of β-cells with relatively limited accumulation of misfolded proinsulin protein and maintenance of endogenous insulin production. Generation and maintenance of such a subset of β-cells may have implications in the avoidance of type 2 diabetes.

Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.

Evidence That Nasal Insulin Induces Immune Tolerance to Insulin in Adults With Autoimmune Diabetes

OBJECTIVEInsulin in pancreatic β-cells is a target of autoimmunity in type 1 diabetes. In the NOD mouse model of type 1 diabetes, oral or nasal administration of insulin induces immune tolerance to insulin and protects against autoimmune diabetes. Evidence for tolerance to mucosally administered insulin or other autoantigens is poorly documented in humans. Adults with recent-onset type 1 diabetes in whom the disease process is subacute afford an opportunity to determine whether mucosal insulin induces tolerance to insulin subsequently injected for treatment.RESEARCH DESIGN AND METHODSWe randomized 52 adults with recent-onset, noninsulin-requiring type 1 diabetes to nasal insulin or placebo for 12 months. Fasting blood glucose and serum C-peptide, glucagon-stimulated serum C-peptide, and serum antibodies to islet antigens were monitored three times monthly for 24 months. An enhanced ELISpot assay was used to measure the T-cell response to human proinsulin.RESULTSβ-Cell function declined by 35% overall, and 23 of 52 participants (44%) progressed to insulin treatment. Metabolic parameters remained similar between nasal insulin and placebo groups, but the insulin antibody response to injected insulin was significantly blunted in a sustained manner in those who had received nasal insulin. In a small cohort, the interferon-γ response of blood T-cells to proinsulin was suppressed after nasal insulin.CONCLUSIONSAlthough nasal insulin did not retard loss of residual β-cell function in adults with established type 1 diabetes, evidence that it induced immune tolerance to insulin provides a rationale for its application to prevent diabetes in at-risk individuals.

Protein disulfide isomerase isomerizes non‐native disulfide bonds in human proinsulin independent of its peptide‐binding activity

Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba'c) where the b' domain was partially deleted, thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba'c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba'c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.

ChemInform Abstract: Total Chemical Synthesis of Human Proinsulin.

AbstractAn efficient convergent synthesis of human proinsulin by chemical ligation of three synthetic peptide segments is presented.

Paternally Inherited Proinsulin Mutations May Result in Earlier Onset of Monogenic Diabetes Mutation Identity Effect in Monogenic Diabetes

Mutations within the human proinsulin gene ( INS ) have been reported to cause neonatal, maturity onset diabetes of the young (MODY), and antibody-negative idiopathic type 1 diabetes (1–3). However, because the expression of maternally or paternally transmitted INS-IGF2 alleles is different as a result of selective methylation (imprinting) (4), we surmised that the effect of INS mutations may depend on the origin of the mutated allele. To verify this hypothesis, pairs comprising an affected child and parent carrying the same heterozygous INS mutation were studied. A literature search was performed and yielded eight relevant articles reporting heterozygous INS mutations in neonatal, MODY, or antibody-negative type 1 diabetes (supplementary Table 1, available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc10-1142/DC1). We also included an unpublished case …

Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide.

Current treatment for type I diabetes includes delivery of insulin via injection or pump, which is highly invasive and expensive. The production of chloroplast-derived proinsulin should reduce cost and facilitate oral delivery. Therefore, tobacco and lettuce chloroplasts were transformed with the cholera toxin B subunit fused with human proinsulin (A, B, C peptides) containing three furin cleavage sites (CTB-PFx3). Transplastomic lines were confirmed for site-specific integration of transgene and homoplasmy. Old tobacco leaves accumulated proinsulin up to 47% of total leaf protein (TLP). Old lettuce leaves accumulated proinsulin up to 53% TLP. Accumulation was so stable that up to ~40% proinsulin in TLP was observed even in senescent and dried lettuce leaves, facilitating their processing and storage in the field. Based on the yield of only monomers and dimers of proinsulin (3 mg/g leaf, a significant underestimation), with a 50% loss of protein during the purification process, one acre of tobacco could yield up to 20 million daily doses of insulin per year. Proinsulin from tobacco leaves was purified up to 98% using metal affinity chromatography without any His-tag. Furin protease cleaved insulin peptides in vitro. Oral delivery of unprocessed proinsulin bioencapsulated in plant cells or injectable delivery into mice showed reduction in blood glucose levels similar to processed commercial insulin. C-peptide should aid in long-term treatment of diabetic complications including stimulation of nerve and renal functions. Hyper-expression of functional proinsulin and exceptional stability in dehydrated leaves offer a low-cost platform for oral and injectable delivery of cleavable proinsulin.

Delivery of a Therapeutic Protein by Immune-Privileged Sertoli Cells

Immune-privileged Sertoli cells survive long term after allogeneic or xenogeneic transplantation without the use of immunosuppressive drugs, suggesting they could be used as a vehicle to deliver therapeutic proteins. As a model to test this, we engineered Sertoli cells to transiently produce basal levels of insulin and then examined their ability to lower blood glucose levels after transplantation into diabetic SCID mice. Mouse and porcine Sertoli cells transduced with a recombinant adenoviral vector containing furin-modified human proinsulin cDNA expressed insulin mRNA and secreted insulin protein. Transplantation of 5-20 million insulin-expressing porcine Sertoli cells into diabetic SCID mice significantly decreased blood glucose levels in a dose-dependent manner, with 20 million Sertoli cells decreasing blood glucose levels to 9.8 ± 2.7 mM. Similar results were obtained when 20 million insulin-positive, BALB/c mouse Sertoli cells were transplanted; blood glucose levels dropped to 6.3 ± 2.4 mM and remained significantly lower for 5 days. To our knowledge, this is the first study to demonstrate Sertoli cells can be engineered to produce and secrete a clinically relevant factor that has a therapeutic effect, thus supporting the concept of using immune-privileged Sertoli cells as a potential vehicle for gene therapy.

Interaction of histidine-tagged human proinsulin with immobilized nickel ion: Effect of chelating ligand and thermodynamics analysis

The nature of the chelating ligand is one of various factors affecting the performance of IMAC. In this work we studied the effect of three chelating ligands on the adsorption of recombinant human proinsulin with His-tag (proinsulin-(His) 6). The chelating ligands (complexed with Ni(II)) were two tetradentates (carboxymethylated aspartic acid, CM-Asp, and tris(2-aminoethyl)amine, TREN) and one pentadentate (tris (carboxymethyl) ethylenediamine, TED). Their performance were compared with the performance of one tridentate ligand (iminodiacetic acid, IDA) and the commercial adsorbent HisTrap both also complexed with Ni(II). Higher selectivities were achieved with Ni(II)-CM-Asp and Ni(II)-TED. The adsorption capacity decreased according to the order: TREN, HisTrap, IDA, CM-Asp, and TED (172.82, 167.98, 133.09, 110.18, and 69.22 mg of proinsulin/mL of gel, respectively, at 25 °C). Although TREN-agarose had the highest capacity, the ligand with the second highest capacity – HisTrap – showed better performance since proinsulin was eluted in a single, concentrated peak with imidazole, while for Ni(II)-TREN proinsulin was eluted with imidazole and EDTA, requiring an extra step to free the product from the EDTA. Therefore, the choice of an adsorbent for this separation depends on the priority: capacity (TREN or HisTrap for one step process) or selectivity (CM-Asp). Adsorption isotherms were determined for temperatures from 4 to 45 °C and the Langmuir model was fitted to the experimental data. The thermodynamics analysis showed positive Δ H° (from 18.83 to 86.05 kJ/mol), indicating that the adsorption of proinsuin-(His) 6 in all chelating ligands is an endothermic process. The negative change in Gibbs free energy (from −26.30 to −35.11 kJ/mol) indicated that the adsorption of the proinsulin-(His) 6 on all the adsorbents studied was a thermodynamically favorable process.

Conformational transmission in proinsulin and its derivatives: A study using H/D exchange

Buffalo proinsulin cDNA was isolated, sequenced and shown to differ from that of its bovine counterpart in six nucleotides. However, at the protein level the predicted sequences of the two species are identical. Buffalo M-proinsulin, containing the initiation methionine, was produced in Escherichia coli and purified to give Mr of 8812. Following the replacement of 99% of the exchangeable hydrogen atoms with deuterons a preparation containing 131 D atoms was obtained. Buffer exchange of the latter into a protio medium led to, the immediate release of 109 (±1) D atoms into the medium and the retention of 22 (±1) D atoms in the protein. The slow exchange of these D atoms was studied at 0°C/pH 2.8. Insulin derived from buffalo proinsulin as well as bovine when deuteriated and buffer exchanged, similarly, gave the retention of 25 (±1) D atoms. The data show that the secondary structure of the insulin core present within buffalo/bovine proinsulin contains 5 (±1) fewer slow exchanging hydrogen atoms than are present in the final hormone. This effect is attributed, predominantly, to the long range influence of the C-peptide, composed of 26 residues, on the insulin core of buffalo proinsulin. In contrast, in the case of human proinsulin, comprising 31 amino acids in the C-peptide, the secondary structure of the insulin core within human proinsulin is closer to that of insulin itself.

The role of heterologous chloroplast sequence elements in transgene integration and expression.

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5' untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5' UTR and 3' UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5' UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5' UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation.

Total chemical synthesis of human proinsulin

A convergent synthetic strategy based on modern chemical ligation methods was used to make human proinsulin. The synthetic protein was characterized by LCMS, CD spectroscopy, and by 1D- and 2D-NMR spectroscopy. Synthetic human proinsulin had full biochemical activity in a receptor-binding assay.

Short communication Correct targeting of proinsulin in protein storage vacuoles of transgenic soybean seeds

Soybean plants are promising bioreactors for the expression of biochemically complex proteins that cannot be produced in a safe and/or economically viable way in microorganisms, eukaryotic culture cells or secreted by transgenic animal glands. Soybeans present many desirable agronomic characteristics for high scale protein production, such as high productivity, short reproductive cycle, photoperiod sensitivity, and natural organs destined for protein accumulation in the seeds. The significant similarities between plant and human cells in terms of protein synthesis processes, folding, assembly, and post-translational processing are important for efficient accumulation of recombinant proteins. We obtained two transgenic lines using biolystics, incorporating the human proinsulin gene under control of the monocot tissue-specific promoter from sorghum gamma-kafirin seed storage protein gene and the alpha-coixin cotyledonary vacuolar signal peptide from Coix lacryma-jobi (Poaceae). Transgenic plants expressed the proinsulin gene and accumulated the polypeptide in mature seeds. Protein targeting to cotyledonary protein storage vacuoles was successfully achieved and confirmed with immunocytochemistry assays. The combination of different regulatory sequences was apparently responsible for high stability in protein accumulation, since human proinsulin was detected after seven years under room temperature storage conditions.

Misfolded Proinsulin Affects Bystander Proinsulin in Neonatal Diabetes

It has previously been shown that misfolded mutant Akita proinsulin in the endoplasmic reticulum engages directly in protein complexes either with nonmutant proinsulin or with "hProCpepGFP" (human proinsulin bearing emerald-GFP within the C-peptide), impairing the trafficking of these "bystander" proinsulin molecules (Liu, M., Hodish, I., Rhodes, C. J., and Arvan, P. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 15841-15846). Herein, we generated transgenic mice, which, in addition to expressing endogenous proinsulin, exhibit beta-cell-specific expression of hProCpepGFP via the Ins1 promoter. In these mice, hProCpepGFP protein levels are physiologically regulated, and hProCpepGFP is packaged and processed to CpepGFP that is co-stored in beta-secretory granules. Visualization of CpepGFP fluorescence provides a quantifiable measure of pancreatic islet insulin content that can be followed in live animals in states of health and disease. We examined loss of pancreatic insulin in hProCpepGFP transgenic mice mated to Akita mice that develop neonatal diabetes because of the expression of misfolded proinsulin. Loss of bystander insulin in Akita animals is detected initially as a block in CpepGFP/insulin production with intracellular accumulation of the precursor, followed ultimately by loss of pancreatic beta-cells. The data support that misfolded proinsulin perturbs bystander proinsulin in the endoplasmic reticulum, leading to beta-cell failure.

Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted

Mutations in the preproinsulin protein that affect processing of preproinsulin to proinsulin or lead to misfolding of proinsulin are associated with diabetes. We examined the subcellular localization and secretion of 13 neonatal diabetes-associated human proinsulin proteins (A24D, G32R, G32S, L35P, C43G, G47V, F48C, G84R, R89C, G90C, C96Y, S101C and Y108C) in rat INS-1 insulinoma cells. These mutant proinsulin proteins accumulate in the endoplasmic reticulum (ER) and are poorly secreted except for G84R and in contrast to wild-type and hyperproinsulinemia-associated mutant proteins (H34D and R89H) which were sorted to secretory granules and efficiently secreted. We also examined the effect of C96Y mutant proinsulin on the synthesis and secretion of wild-type insulin and observed a dominant-negative effect of the mutant proinsulin on the synthesis and secretion of wild-type insulin due to induction of the unfolded protein response and resulting attenuation of overall translation.

Establishment of electrochemiluminescence immunoassay to detect proinsulin levels in human serum

Objective To establish a electrochemilumineecenee immunoaasay (ECLIA) for detecting proinsulin(PI) levels in human serum. Methods PI ECLIA kit contains biotinylated monoclonal insulin-specificantibody, ruthenium labeled monoclonal C-peptide specific antibody, and streptavidin-coated microparticlea. Its method performance, such as sensitivity, specific, accuracy, was evaluated. Then, the levels of PI in clinical samples were detected by ECLIA, including 44 type 2 diabetes and 60 healthy volunteers. Results Average recovery of ECLIA was 103. 8% with a lower detection limit of 0.38 pmol/L and biologic detection limit between 0.25 pmol/L and 0.5 pmol/L, and the function sensitivity was 0.5 pmol/L. The human insulin and human C-peptide did not cross-react at 500 μU/ml recovery of ECLIA was 103.8% with a lower detection limit of 0.38 pmol/L and biologic detection limit between 0.25 pmol/L and 0.5 pmol/L, and the function sensitivity was 0.5 pmoL/L. The human insulin and human C-peptide did not cross-react at 500 μU/ml and 450 ng/ml respectively. The total coefficient of variation of the ECLIA were leas than 5.0%, and the analytical measure range (AMR) was between 2.22 and 169.68 pmol/L. There was a good correlation (r=0.967,P=0.000) between ECLIA and radio immunoassay (RIA). The 95% reference value for human proinsulin was 1.07-15.54 pmol/L. The plasma levels of PI in type 2 diabetes were significantly higher than those in control healthy group[6.72(3.61-11.92) pmol/L vs 5.72(2.65-8.74) pmol/L, Z=-2.023, P<0.05]. Conclusions The monoclonal-based ECLIA is a sensitive, specific, and rapid method and no radiocontamination. It can be used to detect hanum serum proinsulin in type 2 diabetes. Key words: Proinsulin; Electrochemistry; Luminescence; Immunoassay

Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B

Insulin is a polypeptide hormone which is produced by the beta-cell of pancreas and controls the blood glucose level in the human body. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B generally causes high accumulation of insulin derivatives, leading to more complicated purification processes. A simple method including citraconylation and decitraconylation in the enzymatic modification process was developed for the reduction of a major derivative, des-threonine human insulin. Addition of 3.0 g citraconic anhydride per g protein into the reaction solution led to the citraconylation of lysine residues in human proinsulin and reduction of relative des-threonine insulin content from 13.5 to 1.0%. After the enzymatic hydrolysis of the citraconylated proinsulin, 100% of lysine residues can be decitraconylated and restored by adjusting pH to 2-3 at 25 degrees C. Combination of hydrogen peroxide addition and citraconylation of proinsulin expressed in recombinant Escherichia coli remarkably improved the conversion yield of insulin from 52.7 to 77.7%. Consequently, citraconylation of lysine residues blocked the unexpected cleavage of human proinsulin by trypsin, minimized the formation of des-threonine insulin and hence increased the production yield of active insulin.

Inventory of ‘slow exchanging’ hydrogen atoms in human proinsulin and its derivatives: Observations on the mass spectrometric analysis of deuterio-proteins in D 2O

Secondary structure elements of human proinsulin and of its tryptic products were compared by H/D exchange, in a single-pot, using mass spectrometry. Human proinsulin containing an N-terminal methionine, M-proinsulin, was engineered and converted into a perdeuterio derivative, which using an optimized mass spectrometric protocol and manual calculations gave a mass of 9669.6 (± 1) Da showing the replacement, with deuterium of 146.4 from a total of 149 exchangeable hydrogen atoms (83 from amides and 66 from side-chains). Tryptic digestion of the perdeuterio-M-proinsulin, followed by the transfer of the digest from a deuterio- into a protio-medium showed, at the earliest time of analysis, that of the 27 (± 1) D atoms retained in M-proinsulin, 24 (± 1) were found in the insulin nucleus, M-insulin-RR, and 4.2 (± 1) in the C-peptide-KR. A temporal analysis of the fate of D atoms in these species showed that whereas the C-peptide-KR rapidly exchanged its deuterium, losing all by 6 h, the loss of D atoms from M-proinsulin and M-insulin-RR was gradual and in each case, 12 deuterium atoms survived exchange for 72 h. At all time intervals the loss of D atoms from M-proinsulin mirrored that from M-insulin-RR plus the C-peptide-KR, suggesting that the secondary-structure elements of M-proinsulin are largely conserved in its two component parts.

Chloroplast-derived vaccine antigens and biopharmaceuticals: expression, folding, assembly and functionality.

Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance, and multi-gene expression in a single transformation event. Oral delivery is facilitated by hyperexpression of vaccine antigens against cholera, tetanus, anthrax, plague, or canine parvovirus (4%–31% of total soluble protein, TSP) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato) as well as the availability of antibiotic free selectable markers or the ability to excise selectable marker genes. Hyperexpression of several therapeutic proteins, including human serum albumin (11.1% TSP), somatotropin (7% TSP), interferon-alpha (19% TSP), interferon-gamma (6% TSP), and antimicrobial peptide (21.5% TSP), facilitates efficient and economic purification. Also, the presence of chap-erones and enzymes in chloroplasts facilitates assembly of complex multisubunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLA cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Purification of human proinsulin has been achieved using novel purification strategies (inverse temperature transition property) that do not require expensive column chromatography techniques. Thus, transgenic chloroplasts are ideal bio-reactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.

Recognition of Human Proinsulin Leader Sequence by Class I–Restricted T-Cells in HLA-A*0201 Transgenic Mice and in Human Type 1 Diabetes

OBJECTIVE— A restricted region of proinsulin located in the B chain and adjacent region of C-peptide has been shown to contain numerous candidate epitopes recognized by CD8+ T-cells. Our objective is to characterize HLA class I–restricted epitopes located within the preproinsulin leader sequence.RESEARCH DESIGN AND METHODS— Seven 8- to 11-mer preproinsulin peptides carrying anchoring residues for HLA-A1, -A2, -A24, and -B8 were selected from databases. HLA-A2–restricted peptides were tested for immunogenicity in transgenic mice expressing a chimeric HLA-A*0201/β2-microglobulin molecule. The peptides were studied for binding to purified HLA class I molecules, selected for carrying COOH-terminal residues generated by proteasome digestion in vitro and tested for recognition by human lymphocytes using an ex vivo interferon-γ (IFN-γ) ELISpot assay.RESULTS— Five HLA-A2–restricted peptides were immunogenic in transgenic mice. Murine T-cell clones specific for these peptides were cytotoxic against cells transfected with the preproinsulin gene. They were recognized by peripheral blood mononuclear cells (PBMCs) from 17 of 21 HLA-A2 type 1 diabetic patients. PBMCs from 25 of 38 HLA-A1, -A2, -A24, or -B8 patients produced IFN-γ in response to six preproinsulin peptides covering residues 2–25 within the preproinsulin region. In most patients, the response was against several class I–restricted peptides. T-cells recognizing preproinsulin peptide were characterized as CD8+ T-cells by staining with peptide/HLA-A2 tetramers.CONCLUSIONS— We defined class I–restricted epitopes located within the leader sequence of human preproinsulin through in vivo (transgenic mice) and ex vivo (diabetic patients) assays, illustrating the possible role of preproinsulin-specific CD8+ T-cells in human type 1 diabetes.