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We describe a sensitive two-site sandwich enzyme-linked immunosorbent assay for the measurement of intact human proinsulin in 100 microL of serum or plasma. The assay is based on the use of two monoclonal antibodies specific for epitopes at the C-peptide/insulin A chain junction and at the insulin B chain/C-peptide junction, respectively. Cross-reactivities with insulin, C-peptide, and the four proinsulin conversion intermediates were negligible. The detection limit in buffer was 0.2 pmol/L (3 standard deviations from zero). The working range was 0.2-100 pmol/L. The mean intra- and interassay coefficients of variation were 2.4% and 8.9%, respectively. The mean recovery of added proinsulin was 103%. Dilution curves of 40 serum samples are parallel to the proinsulin calibration curve. Proinsulin concentrations in 20 fasting healthy subjects were all above the limit of detection: median (range), 2.7 pmol/L (1.1-6.9 pmol/L). Six fasting non-insulin-dependent diabetes mellitus and five insulinoma patients had proinsulin concentrations significantly higher than healthy subjects: median (range), 7.7 pmol/L (3.2-18 pmol/L) and 153 pmol/L (98-320 pmol/L), respectively.

Isolation of the recombinant protein from a genetically engineered Escherichia coli 1854 producer for further chemical enzymatic transformation into human insulin through proinsulin was studied. Under optimal conditions, the recombinant protein formation was more than 35% of the total cell proteins. Structures of the polypeptides obtained and purified chromatographically were confirmed by amino acid analysis. Human proinsulin was derived from the recombinant protein isolated.

Protein minimization highlights essential determinants of structure and function. Minimal models of proinsulin and insulin-like growth factor I contain homologous A and B domains as single-chain analogues. Such models (designated mini-proinsulin and mini-IGF-I) have attracted wide interest due to their native foldability but complete absence of biological activity. The crystal structure of mini-proinsulin, determined as a T3R3 hexamer, is similar to that of the native insulin hexamer. Here, we describe the solution structure of a monomeric mini-proinsulin under physiologic conditions and compare this structure to that of the corresponding two-chain analogue. The two proteins each contain substitutions in the B-chain (HisB10-->Asp and ProB28-->Asp) designed to destabilize self-association by electrostatic repulsion; the proteins differ by the presence or absence of a peptide bond between LysB29 and GlyA1. The structures are essentially identical, resembling in each case the T-state crystallographic protomer. Differences are observed near the site of cross-linking: the adjoining A1-A8 alpha-helix (variable among crystal structures) is less well-ordered in mini-proinsulin than in the two-chain variant. The single-chain analogue is not completely inactive: its affinity for the insulin receptor is 1500-fold lower than that of the two-chain analogue. Moreover, at saturating concentrations mini-proinsulin retains the ability to stimulate lipogenesis in adipocytes (native biological potency). These results suggest that a change in the conformation of insulin, as tethered by the B29-A1 peptide bond, optimizes affinity but is not integral to the mechanism of transmembrane signaling. Surprisingly, the tertiary structure of mini-proinsulin differs from that of mini-IGF-I (main-chain rms deviation 4.5 A) despite strict conservation of non-polar residues in their respective hydrophobic cores (side-chain rms deviation 4.9 A). Three-dimensional profile scores suggest that the two structures each provide acceptable templates for threading of insulin-like sequences. Mini-proinsulin and mini-IGF-I thus provide examples of homologous protein sequences encoding non-homologous structures., (Copyright 1998 Academic Press Limited.)

A mutant proinsulin gene was constructed through PCR mediated mutagenesis. The code of A19Tyr was deleted. The mutant proinsulin, (deltaY)19-lys-proinsulin [(deltaY)19KPI], was expressed in E. coli and purified. After treatment with trypsin and carboxypeptidase B, and Resource Q separation, (deltaY)19-human insulin [(deltaY)19HI] was obtained. It retains 63.6% of receptor binding activity but only 2.2% of immune activity, and shows a longer retaining time on reverse-phase FPLC and a slower mobility by native PAGE analysis. These results suggest that the deletion of A19Tyr causes some conformational changes on insulin, which plays a minor role on the affinity of insulin to its receptor, and a major role on immunogenicity of the hormone.

Proinsulin C-peptide has been shown to stimulate the activity of Na+ K+ ATPase of rat renal tubule segments. Thirty-six peptides and amino acids, corresponding to parts of the intact rat C-peptide and suitable controls were screened for capacity to stimulate Na+, K+-ATPase in an attempt to determine potential active sites in the C-peptide molecule. The carboxy-terminal tetra and penta peptides were found to elicit 92-103% of the intact molecule's activity, and the remaining segment, des-(27-31) C-peptide, did not possess stimulatory activity. Peptides from the middle C-peptide segment, however, centering around a GGPEAG sequence, stimulated Na+, K+-ATPase activity (36-80% of the intact molecule's effect) but this effect was not balanced by corresponding inactivity of other parts. Furthermore, it was paralleled by activity of a non-native dipeptide D-form. It is concluded that the latter effect and that of the middle segment may represent complex interactions other than the apparently specific effects of the C-terminal segment.

The prohormone convertases PC2 (SPC2) and PC3/PC1 (SPC3) are the major precursor processing endoproteases in a wide variety of neural and endocrine tissues. Both enzymes are normally expressed in the islet beta cells and participate in proinsulin processing. Recently we generated mice lacking active PC2 due to a disruption of the PC2 gene (Furuta, M., Yano, H., Zhou, A., Rouillé, Y., Holst, J. J., Carroll, R. J., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646-6651). Here we report that these PC2 mutant mice have elevated circulating proinsulin, comprising 60% of immunoreactive insulin-like components. Acid ethanol extractable proinsulin from pancreas is also significantly elevated, representing about 35% of total immunoreactive insulin-like components. These increased amounts of proinsulin are mainly stored in secretory granules, giving rise to an altered appearance on electron microscopy. In pulse-chase experiments, the mutant islets incorporate lesser amounts of isotopic amino acids into insulin-related components than normal islets. In both wild-type and mutant islets, proinsulin I was processed more rapidly to insulin, reflecting the preference of both PC2 and PC3 for substrates having a basic amino acid positioned four residues upstream of the cleavage site. The overall half-time for the conversion of proinsulin to insulin is increased approximately 3-fold in the mutant islets and is associated with a 4-5-fold greater elevation of des-31,32 proinsulin, an intermediate that is formed by the preferential cleavage of proinsulin at the B chain-C-peptide junction by PC3 and is C-terminally processed to remove Arg31 and Arg32 by carboxypeptidase E. The constitutive release of newly synthesized proinsulin from both mutant and wild-type islets during the first 1-2 h of chase was normal (<2% of total). These results demonstrate that PC2 plays an essential role in proinsulin processing in vivo, but is quantitatively less important in this regard than PC3, and that its absence does not influence the efficient sorting of proinsulin into the regulated secretory pathway.

Total pancreatic RNA from the holocephalan species Callorhyncus milii (elephantfish) was used to make cDNA as a template for the polymerase chain reaction. Three redundant primers based on the known amino acid sequence of elephantfish insulin were used to amplify a fragment of proinsulin comprising truncated B-chain, complete C-peptide, and complete A-chain. Whereas the C-peptide/A-chain junction contained the expected dibasic cleavage site (-Lys-Arg-), the B-chain/C-peptide junction was found to contain only a single Arg, the first such site to be unequivocally associated with the proteolytic processing of a proinsulin to insulin. Examination of the flanking sequences around this site shows that a typical endocrine/neuroendocrine PC3 conversion enzyme should still be able to cleave, as the general requirements for precursor processing at a monobasic site are satisfied, notably a basic residue (Lys) at the -4 position. An acidic residue (in this case Asp) at the +1 position, which is seen in all known proinsulins, is maintained. The corresponding genomic DNA fragment of elephantfish proinsulin was also amplified by PCR, revealing a 402-bp intron at the conserved IVS-2 position within the C7 codon., (Copyright 1997 Academic Press. Copyright 1997 Academic Press)

A general method for obtaining high-level production of low molecular weight proteins in Escherichia coli is described. This method is based on the use of a novel Met-Xaa-protein construction which is formed by insertion of a single amino acid residue (preferably Arginine or Lysine) between the N-terminal methionine and the protein of interest. The utility of this method is illustrated by examples for achieving high-level production of human insulin-like growth factor-1, human proinsulin, and their analogs. Furthermore, highly produced insulin-like growth factor-1 derivatives and human proinsulin analogs are converted to their natural sequences by removal of dipeptides with cathepsin C.

Psammomys obesus fed a high-calorie diet develops a NIDDM-like syndrome. The use of reverse-phase high-performance liquid chromatography (HPLC) to study Psammomys insulin biosynthesis and release revealed a very delayed elution time for the Psammomys insulin peak appearing near the position of human proinsulin. This unusual peak was initially thought to represent partially processed insulin on the basis of its molecular size and susceptibility to trimming by carboxypeptidase B (CpB). However, the findings of an active carboxypeptidase E (CpE) enzyme and the normal amidated forms of gastrin and cholecystokinin octapeptide (CCK-8) in Psammomys tissues were inconsistent with CpE-related aberrant processing of insulin. Moreover, amino acid sequencing of the delayed peak of Psammomys insulin revealed fully processed insulin with amino acid sequence as predicted by the cDNA. The unique presence of a B-30 phenylalanine residue, resulting in an increased hydrophobicity of the insulin molecule, probably underlies the marked delay in elution time on HPLC. The unusual structure of Psammomys insulin does not appear to contribute to the proinsulinemia observed in diabetic Psammomys since the HPLC-purified molecule did not inhibit PC1 and PC2 convertase activities in an in vitro assay.

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

We have recently developed a method to produce native human proinsulin using a bacterial expression system. A proinsulin fusion protein was recovered from inclusion bodies and cleaved using cyanogen bromide. The released proinsulin polypeptide was S-sulfonated and purified by anion exchange chromatography. Following refolding, proinsulin was purified by reversed-phase high-performance liquid chromatography. Combined peptide mapping and mass spectrometric analysis indicated that the proinsulin contained the correct disulfide bridging pattern. This proinsulin will be used to study the specificity of the furin/PC family of converting enzymes by using it as a substrate in a recently developed assay.

Proteolysis of recombinant human proinsulin by the native trypsin, by trypsin modified with a copolymer of vinylpyrrolidone and acrolein, and by the same modified trypsin immobilized on Silochrom 1.5 was studied by RP HPLC and mass spectrometry. Rate constants of the main stages of proinsulin hydrolysis by the native trypsin were estimated. The values of rate constants of the digestions of the most easily hydrolyzable bonds (those formed by the pairs of the basic amino acid residues) in proinsulin were found to be of the same order as those formed by the separate lysine residues (Lys7) and those formed by the four basic amino acid residues of the C-terminal cluster of melittin. It was established that covalent trypsin binding to the copolymer did not change the ratio of the rate constants of the individual stages of proinsulin hydrolysis, whereas after the immobilization of modified trypsin on the Silochrome, the formation of diarginyl insulin-ArgArg, intermediate forms of hydrolyzed insulin, and desThr-insulin proceeds with comparable rates.

A synthetic DNA encoding human proinsulin was inserted in frame in the bacterial alkaline phosphatase gene. A homogeneous recombinant human proinsulin-alkaline phosphatase conjugate was obtained directly from the periplasm of Escherichia coli transformed with a plasmid carrying the hybrid gene. The recombinant conjugate was stable and could be produced in the bacteria. The immunological properties of the recombinant conjugate and those of the human insulin and human proinsulin were compared using a panel of six different human insulin-specific monoclonal antibodies. Three immunological groups were thus distinguished and one of them indiscriminately recognized all of the insulin-like molecules. One monoclonal antibody from this group was used in combination with the recombinant conjugate to develop successfully a competitive immunoenzymatic assay for detecting insulin.

[B12Thr]human insulin and [B12Leu]human insulin were obtained by means of site-directed random mutagenesis. [B12Thr]human insulin retain total biological activity but [B12Leu]human insulin has much lower biological activity. Receptor binding activities of [B12Thr]human insulin and [B12Leu]human insulin are 56% and 3%, respectively, as that of native porcine insulin. The results suggest that the hydrophobic property of the residue side chain at B12 may not be necessary.

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.

The kinetics for trypsin cleavage of different fusion proteins, consisting of human proinsulin and two IgG-binding domains (ZZ), were investigated. To achieve simultaneous removal of the fusion tag and processing of proinsulin to insulin and free C peptide, three versions of the ZZ-proinsulin fusion protein were generated, having different trypsin-sensitive cleavage sites, Arg, Lys-Arg or Lys. The ZZ-proinsulin fusion proteins which accumulated as inclusion bodies in Escherichia coli cells were solubilized, refolded and purified by IgG affinity chromatography. The yield of ZZ-proinsulin monomers exceeded 90%. The kinetics for the trypsin cleavage revealed unexpected differences when comparing the three linkers and it was found that the single arginine linker was most efficiently processed. Characterization of the cleavage products by reverse-phase chromatography, mass spectrometry and N-terminal sequencing verified that human insulin and C peptide were generated. The results demonstrate that high yields of native insulin, C peptide and affinity tag can be achieved by simultaneous cleavage of a fusion protein at three different trypsin-sensitive sites in a single step. The implications for production and recovery of various recombinant proteins are discussed.

We report the expression in E. coli of a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide linked to the N-terminus of proinsulin by a lysine residue. The key aspects investigated were: (a) the expression of the fused IL2-PI gene, (b) the folding efficiency of the insulin precursor when still carrying the N-fused peptide and (c) the selectivity of the enzymatic cleavage reaction with trypsin in order to remove simultaneously the C-peptide and the N-terminal extension. It was found that this construction expresses the chimeric proinsulin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200 micrograms/ml to yield about 80% of correctly folded proinsulin and then it was converted into insulin by prolonged reaction (5 h) with trypsin and carboxypeptidase B at a low enzyme/substrate rate (1:600). This approach is based on a single enzymatic reaction for the removal of both the N-terminal fused peptide and the C-peptide and avoids the use of toxic cyanogen bromide.

Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain-C-peptide-A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitute pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.

Proinsulin is converted into insulin by the action of two endoproteases. Type I (PC1/PC3) is thought to cleave between the B-chain and the connecting peptide (C-peptide) and type II (PC2) between the C-peptide and the A-chain. An acidic region immediately C-terminal to the point of cleavage at the B-chain/C-peptide junction is well conserved throughout evolution and has been suggested to be important for proinsulin conversion [Gross, Villa-Komaroff, Kahn, Weir and Halban (1989) J. Biol. Chem. 264, 21486-21490]. We have here compared the precise role of this region as a whole and just the first acidic residue C-terminal to the point of cleavage in processing of proinsulin by PC3. To this end, several mutations were introduced in this region of human proinsulin (native sequence, B-chain RREAEDL C-peptide): RRPAEDL (C1Pro mutant); RRLAEDL (C1Leu mutant); RRL (C1-C4del mutant); RRE (del-C1Glu mutant). Mutant and native cDNAs were stably transfected into AtT20 (pituitary corticotroph) cells, in which PC3 is known to be the major conversion endoprotease, and kinetics of proinsulin conversion were studied (pulse-chase/HPLC analysis of proinsulin-related peptides). The results show that the acidic region following the B-chain/C-peptide junction is indeed important for PC3 cleavage at this site, and that the reduced cleavage observed for the C1-C4del mutant proinsulin can be partially overcome by replacing the acidic region with a single acidic residue (del-C1Glu mutant). Replacing only the first residue of the acidic region with leucine (C1Leu mutant) has no impact on conversion, whereas its replacement with proline (C1Pro mutant) almost completely abolishes cleavage at the B-chain/C-peptide junction without affecting that at the C-peptide/A-chain junction.

Background: Insulin (1) and glutamic acid decarboxylase (GAD) (2) are both autoantigens in insulin-dependent diabetes mellitus (IDDM), but no molecular mechanism has been proposed for their association. We have identified a 13 amino acid peptide of proinsulin (amino acids 24-36) that bears marked similarity to a peptide of GAD65 (amino acids 506-518) (G. Rudy, unpublished). In order to test the hypothesis that this region of similarity is implicated in the pathogenesis of IDDM, we assayed T cell reactivity to these two peptides in subjects at risk for IDDM., Materials and Methods: Subjects at risk for IDDM were islet cell antibody (ICA)-positive, first degree relatives of people with insulin-dependent diabetes. Peripheral blood mononuclear cells from 10 pairs of at-risk and HLA-DR matched control subjects were tested in an in vitro proliferation assay., Results: Reactivity to both proinsulin and GAD peptides was significantly greater among at-risk subjects than controls (proinsulin; p < 0.008; GAD; p < 0.018). In contrast to reactivity to the GAD peptide, reactivity to the proinsulin peptide was almost entirely confined to the at-risk subjects., Conclusions: This is the first demonstration of T cell reactivity to a proinsulin-specific peptide. In addition, it is the first example of reactivity to a minimal peptide region shared between two human autoimmune disease-associated self antigens. Mimicry between these similar peptides may provide a molecular basis for the conjoint autoantigenicity of proinsulin and GAD in IDDM.

In pancreatic islets, formation of beta-secretory granule cores involves early proinsulin homohexamerization and subsequent insulin condensation. We examined proinsulin conformational maturation by monitoring accessibility of protein disulfide bonds. Proinsulin disulfides are intact immediately upon synthesis, but are > or = 90% sensitive to in vivo reduction with 2 mM dithiothreitol; wash out of dithiothreitol leads to reoxidation, proinsulin transport, and conversion to insulin. With t1/2 approximately 10 min, newly synthesized proinsulin becomes resistant to disulfide reduction, correlating with endoplasmic reticulum (ER) export. However, inhibition of ER export with brefeldin A blocks acquisition of resistance to reduction, and once proinsulin arrives in the Golgi, it resists reduction despite brefeldin treatment. Moreover, in vivo, resistance of proinsulin disulfides is overcome after increasing [dithiothreitol] > 10-fold, or in vitro, in islets lysed in a zinc-free, but not a zinc-containing, medium. Employing 30 mM dithiothreitol in vivo, a further decrease in disulfide accessibility is observed following proinsulin conversion to insulin. Incubation of islets with chloroquine or zinc enhances and diminishes accessibility of insulin disulfides, respectively. We hypothesize that two major conformational changes culminating in granule core formation, proinsulin hexamerization and insulin condensation, are sensitive to zinc and occur upon ER exit and arrival in immature secretory granules, respectively.

To evaluate the role of protein aggregation and calcium in the sorting of insulin for regulated vs. constitutive release from the intact pancreas, we targeted the expression of a monomeric mutant form of human (pro)insulin (B9/B27) to the pancreatic beta-cells of transgenic mice. This mutant insulin does not form dimers or hexamers, but can aggregate at high concentration in the presence of calcium. A homozygous line (171) was produced that expressed 55% of the total (pro)insulin message in their beta-cells as the mutant form and had normal pancreatic total (pro)insulin content [measured as immunoreactive insulin (IRI)]. Fasting glucose levels in these transgenics and in homozygous control mice expressing native human (pro)insulin were normal, although levels were abnormally elevated during ip glucose tolerance testing. In the presence of extracellular calcium, regulated IRI release from the isolated perfused pancreas of the transgenic mice was undetectable in the absence of secretagogues and responded with normal phasic kinetics when stimulated with increasing steps of glucose, with glucose plus isobutylmethylxanthine, or with arginine. Without extracellular calcium (0 calcium plus EGTA), normal pancreas did not release IRI in either the presence or absence of secretagogues. In contrast, without calcium or secretagogues, transgenic pancreas spontaneously and constitutively released IRI at high levels equivalent to those elicited by glucose (22 mM) plus calcium from normal pancreas. This release was partially inhibited by glucose or arginine. Constitutive secretion was acutely sensitive to calcium; inhibition occurred within minutes after the addition of calcium and quickly returned to its characteristic level (with overshoot) when calcium was subsequently removed. Somatostatin, at a concentration that caused 50% inhibition of normal glucose-stimulated secretion, did not affect constitutive release. Control pancreas from the transgenic mice, expressing native human (pro)insulin, responded normally to secretagogues and did not constitutively release hormone in the absence of calcium. It is concluded that expression of monomeric human insulin in pancreatic beta-cells from transgenic mice did not interfere with normal phenotypic insulin secretion, indicating that the functional secretory apparatus was not impaired. Constitutive secretion of IRI from the intact pancreas requires both the expression of a monomeric form of insulin and the absence of extracellular calcium, two conditions that reduce aggregation. These results are consistent with the hypothesis that protein aggregation favors sorting to the regulated pathway, whereas suppressed aggregation causes sorting for constitutive release.

The signal for degradation of proteins in the endoplasmic reticulum (ER) is thought to be the exposure of internal domains which are buried when the protein has adopted its correct conformation and which are also exposed in assembly intermediates. This raises the question of why the intermediates are not degraded. We developed a system based on the peroxidase-catalyzed iodination of tyrosine residues which continuously monitors the exposure of internal domains of proinsulin. In CHO cells this system discriminated between assembly intermediates of wild type (wt) proinsulin and misfolded proinsulin, as shown by the exclusive iodination of a misfolded mutant which was finally degraded in the ER. Iodination in vitro showed that the assembly intermediates of wt proinsulin also exposed internal domains. This iodination was inhibited by the addition of the molecular chaperone Bip which was co-immunoprecipitated with proinsulin in CHO cells. The results obtained with the mutant proinsulin support the assumption that exposed internal domains represent the signal for degradation in the ER. Observations of wt proinsulin show that Bip masks internal domains of normal assembly intermediates during the entire assembly process, thereby suppressing their degradation. We propose that internal domains contain co-localized signals for Bip binding and for degradation.

We have determined the structure of a metastable disulphide isomer of human insulin. Although not observed for proinsulin folding or insulin-chain recombination, the isomer retains ordered secondary structure and a compact hydrophobic core. Comparison with native insulin reveals a global rearrangement in the orientation of A- and B-chains. One face of the protein's surface is nevertheless in common between native and non-native structures. This face contains receptor-binding determinants, rationalizing the partial biological activity of the isomer. Structures of native and non-native disulphide isomers also define alternative three-dimensional templates. Threading of insulin-like sequences provide an experimental realization of the inverse protein-folding problem.

The optimization of the separation of recombinant human insulin (rhI) and recombinant human proinsulin (rhP) by free-solution capillary zone electrophoresis in uncoated fused-silica capillaries with ionic and zwitterionic buffers was carried out. The relationship between the selectivity and pH of the buffer was established. The effects of pH and ionic strength of the buffer on protein adsorption on the capillary walls and Taylor diffusion was investigated. The separation of rhI and rhP with an efficiency of 200,000 theoretical plates was achieved using 20 mM Na2HPO4-NaOH buffer (pH 11.2). The proposed method allows the simultaneously determination of rhP and some rhI degradation products (which are also included in pharmacopoeias) in pharmacopoeially limited amounts in rhI.

Four families with hyperproinsulinemia found in Japan were described. The details of the first case, who was investigated by Kanazawa et al., were reported and the similarity of the first case to the following cases was shown. Arginine 65 of the proinsulin molecule might be a hot spot of the insulin gene. A possible abnormality of insulin release in affected individuals was disclosed by investigation of the family members of the first case.

Type 2 diabetes is associated with abnormal lipoprotein levels and altered plasma concentrations of insulin, intact and 32, 33 split proinsulin. To investigate whether these are early features of the disease, we studied 36 normoglycaemic first-degree relatives of patients with Type 2 diabetes (13 European, 15 of Asian (Indian-subcontinent), and 8 of Afro-Caribbean origin) and 36 control subjects with no family history of diabetes. Relatives and controls were matched for age (mean +/- S.E. 33 +/- 2 vs 34 +/- 2 years), body mass index (23.7 +/- 0.5 vs 23.7 +/- 0.6 kg m-2), sex (17 M, 19 F) and ethnic origin. After an overnight fast, blood was sampled for measurement of serum lipids, plasma glucose and insulin, intact and 32, 33 split proinsulin by specific immunoradiometric assays. Relatives and controls had similar fasting concentrations of glucose (5.0 +/- 0.1 vs 4.9 +/- 0.1 mmol l-1), total cholesterol (4.51 +/- 0.13 vs 4.54 +/- 0.17 mmol l-1), HDL-cholesterol (1.21 +/- 0.06 vs 1.10 +/- 0.05 mmol l-1), LDL-cholesterol (2.84 +/- 0.14 vs 2.96 +/- 0.14 mmol l-1) and triglyceride (median (range) 0.78 (0.44-2.45) vs 0.83 (0.41-4.03) mmol l-1). Fasting levels of insulin (50.4 (18.9-174.0) vs 51.6 (10.0-118.0) pmol l-1, intact proinsulin (2.8 (0.1-15.0) vs 2.1 (0.6-6.4) pmol l-1 and 32, 33 split proinsulin (2.0(0-23.7) vs 1.6 (0.3-6.0) pmol l-1) were not significantly different between relatives and controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Three types of structurally abnormal insulin "Insulin Chicago, Los Angeles and Wakayama" has been identified in 6 families by the recent development of molecular biology. It has been clarified that a point mutation in the coding region of each patient's insulin gene allele give rise to a substitution of one amino acid of each insulin, resulting in reduced biological activities of these insulins. These abnormal insulin have slow metabolic clearance rate which cause typical hyperinsulinemia accompanying normal level of plasma C-peptide. In this paper, we review insulin gene mutations and discuss its participation to the pathogenesis of NIDDM.

The insulin storage granule of the pancreatic beta cell is assembled within the trans Golgi network from around 50 or so gene products many of which are synthesized coordinately with the major component, proinsulin. An important contribution to our understanding of the regulation of this process has come from studies of the post-translational processing of proinsulin and of other proteins which are stored in the granule, particularly the processing enzymes themselves. The present review focusses on recent insights into the molecular nature of the processing machinery, and the granule Ca(2+)-dependent subtilisin-related endopeptidases which catalyse the initial rate-limiting step in the enzymic conversion of proinsulin.

The mutant proinsulin gene was constructed with the codons for A6 and A11 Cys changed to Ser to delete intra-A chain disulfide bond. After expression and purification, the mutations in the protein were further confirmed by amino acid composition. Electrophoretic mobility of the mutant proinsulin is similar to that of human proinsulin, so are the products of tryptic digestions. The mutant proinsulin, which retains its full radioimmuno activity, shows only 5.4% of receptor binding activity of human proinsulin. This suggests that though the intra-A chain disulfide bond disappears, the other two inter-chain disulfide bonds are still correctly paired, and hence the three dimensional structure has not been altered significantly. This intra-chain disulfide bond is essential for insulin displaying its activity.

Pancreatic beta-cell dysfunction is a characteristic of non-insulin-dependent diabetes mellitus (NIDDM). An aspect of this dysfunction is that an increased proportion of proinsulin is secreted, but an actual beta-cell defect that leads to hyperproinsulinemia is unknown. Nevertheless, an impairment in beta-cell proinsulin conversion mechanism has been suggested as the most likely cause. Insulin is produced from its precursor molecule, proinsulin, by limited proteolytic cleavage at two dibasic sequences (Arg31, Arg32 and Lys64, Arg65). Two endopeptidase activities catalyze this cleavage: PC2 and PC3. PC2 endopeptidase cleaves predominately at Lys64, Arg65, and PC3 endopeptidase cleaves at Arg31, Arg32. The recent identification and characterization of these endopeptidases has enabled a better understanding of the human proinsulin-processing mechanism. In particular, experimental evidence suggests that the majority of human proinsulin processing is sequential. PC3 cleaves proinsulin first to generate a proinsulin conversion intermediate that is the preferred substrate of PC2. Both PC2 and PC3 activities are influenced by Ca2+ and pH, but the more stringent Ca2+ and pH requirements of PC3 suggest it as the most likely enzyme to regulate proinsulin conversion, as well as initiate it. When an increased demand is placed on the proinsulin-processing mechanism by a glucose-stimulated increase in proinsulin biosynthesis, there is a coordinate increase in PC3 biosynthesis (but not in PC2). This supports PC3 as the key endopeptidase that regulates proinsulin processing. In this perspective, the current concepts of the enzymology and regulation of proinsulin conversion at a molecular level are reviewed.(ABSTRACT TRUNCATED AT 250 WORDS)

Residues belonging to epitopes on human insulin that were recognized by a panel of three monoclonal antibodies were located using mutated insulins and insulins from a number of different animal species. Epitopes on human proinsulin recognized by two monoclonal antibodies were also identified using partially processed proinsulin species. Epitopes were located on the C-A and B-C junctions of proinsulin and on the N-termini of the A- and B-chains and the central region of the B-chain of human insulin. Antibodies that bound proinsulin were found to induce conformational changes in the prohormone. The presence of a well-defined interaction between the C-peptide portion and the N-terminus of the A-chain of the insulin moiety of intact proinsulin has also been demonstrated. The relevance of these studies to the development of two-site assays for the measurement of partially processed proinsulin species in human sera is also discussed.

Success in modern medical research is achieved when basic and clinical information about a given disorder converges, either intentionally or fortuitously, with the availability of technology or other means to design and apply interventions for the disorder in question. A prime example is the discovery of insulin and its replacement in patients with IDDM in 1923. Seven decades later, the focus of diabetes management is on improvement in metabolic control to forestall the chronic complications of the disease and improve the quality of life of patients with the disease. Metabolic control is being addressed through the development of insulin analogues using sophisticated techniques to understand the chemistry of insulin and to modify it using rDNA technology. The objective of these efforts is to simulate normal insulin secretion with subcutaneously injected agonists. Quality-of-life needs are being addressed with delivery devices, insulin mixtures, and insulin analogues. Although none of these improvements parallel the discovery of insulin, they do provide an optimistic outlook for patients with diabetes mellitus.

Many studies support the concept of insulin synthesis in tissues other than the pancreas. Our previous investigations have demonstrated the presence of insulin immunoreactivity in the adrenal medulla of the rat. This immunoreactivity was found to be associated with the chromaffin granule. This study is directed at isolating the messenger ribonucleic acid that encodes for preproinsulin. Reverse transcription coupled with polymerase chain reaction was used. Complementary deoxyribonucleic acid (cDNA) was amplified, extracted and reamplified. It was then subjected to digestion with four different restriction endonuclease enzymes. Its resemblance to the corresponding cDNA that encodes for preproinsulin I in the rat was established. Our results suggest that insulin is synthesized in the rat adrenal gland for autocrine, paracrine, neuromodulation or local physiologic function.

The metabolism of des(64,65)-human proinsulin was examined in rats after subcutaneous administration. Profiles of circulating insulin-like immunoreactivity in rat plasma 25 min after subcutaneous administration were evaluated by anion exchange fast protein liquid chromatography and reversed-phase high-performance liquid chromatography. Both techniques indicated the presence of circulating immunoreactivity having retention characteristics of human insulin. This metabolite peak comprised 5-10% of circulating immunoreactivity; the remainder had retention characteristics of des(64,65)-human proinsulin. The peaks of immunoreactive material were isolated and their structure determined using reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry. The major circulating component co-eluted with des(64,65)-human proinsulin and had an identical mass spectrum. Two circulating metabolites were identified. These metabolites co-eluted by reversed-phase high-performance liquid chromatography with human insulin and diarginyl(B31,32)-human insulin and had mass spectra identical to the standard compounds. The data indicate proteolytic processing of des(64,65)-human proinsulin involves an initial tryptic cleavage at the carboxy side of ArgB32, with the formation of human insulin by the subsequent action of a carboxypeptidase to remove the ArgB31-ArgB32 dipeptide from diarginyl(B31,32)-human insulin. The results suggest that some of the pharmacological activity of des(64,65)-human proinsulin may be mediated in part by circulating insulin-like metabolites.

Although the structure of insulin is known in great detail, that of proinsulin has been little investigated, except for a few CD and NMR studies. The secondary structures of human proinsulin are now compared with those of insulin by Fourier Transformed Infrared (FTIR) studies. The deconvolved and second derivative spectra of proinsulin and insulin in the amide I' band region are closely similar with peaks corresponding to alpha-helix, irregular helix, and 3(10) helix at nearly identical positions. For both proteins, the relative contents of the above structures as calculated from the peak areas are in good agreement with the values obtained from the known structure of crystalline porcine insulin. However, compared with insulin, proinsulin has markedly more unordered structures as indicated by the area under the peak at 1643.4 cm-1. In addition, both peak positions and relative areas for turn structure of the prohormone are different from those for insulin. It appears from the above that the A-and B-chain segments of proinsulin and insulin are similar in their secondary structures, especially in helices. The C-chain segment is largely unordered except in a few beta-turns.

Furin is a mammalian propeptide-processing endoprotease in nonendocrine cells and has been demonstrated to be present in virtually all nonendocrine cells, including fibroblasts, epithelial cells, and hepatocytes. Furin cleaves the concensus processing site -Arg-4-X-3-Lys/Arg-2-Arg-1 decreases X+1-. Some subunit-containing precursor proteins, including an insulin receptor precursor, possess an additional basic residue at position -3, thus forming a tetrabasic processing site. This implies that a tetrabasic processing site must be easily cleavable in nonendocrine cells. We created a mutant proinsulin DNA with a peptide structure comprised of B- and A-chains linked to the C-peptide by a pair of tetrabasic residues, in the following order: B-chain-Arg-Arg-Lys-Arg-C peptide-Arg-Arg-Lys-Arg-A-chain. The native proinsulin structure was B-chain-Arg-Arg-C-peptide-Lys-Arg-A-chain. Both the native and mutant proinsulins were expressed in the following four cell lines: a monkey kidney-derived cell line (COS-7), a Chinese hamster ovary-derived cell line (CHO), a human liver cancer-derived cell line (HepG2), and a mouse fibroblast-like cell line (NIH3T3). We used these cell lines because they contain different quantities of furin mRNA, ranking as follows: NIH3T3 > HepG2 > COS > CHO. When mutant insulin was expressed in these cells, the conversion of proinsulin to mature insulin was approximately 85% in NIH3T3, 70% in HepG2, 60% in COS, and 50% in CHO. The conversion correlated well with the furin expression in each cell line as measured by the density of its Northern blot band. Moreover, in CHO, the cell line with the lowest furin expression, coexpression of mutant proinsulin with furin resulted in complete conversion of proinsulin to mature insulin.

The capillary zone electrophoresis (CZE) technique was evaluated for separation and quantification of human growth hormone (hGH), human insulin (hI), and proinsulin. Three different molecular forms of biosynthetic hGH (20K, 22K, 44K), methionyl-hGH, biosynthetic hI, and proinsulin, were studied. The hormones were separated with uncoated capillaries, and various analytical conditions were tested (different buffers, ionic strength, pH). The samples were introduced both at positive pressure and electrokinetically, and the voltage applied was varied in each case. Linearity, repeatibility, and limit of detection (0.8 microM for hGH and 1.3 microM for insulin) were determined. Different purification steps were tried in order to find a suitable preanalytical procedure for hGH and hI purification from plasma. Recovery rates from 65 to 80% were obtained.

Objective: To describe the rationale for the preclinical and clinical developmental course of human proinsulin (HPI), the second product after human insulin for the treatment of diabetes mellitus to be manufactured by DNA technology., Research Design and Methods: The relevant and available published and unpublished preclinical and clinical information generated on pork proinsulin and human proinsulin has been integrated to demonstrate how certain clinically attractive features of pork proinsulin (a soluble intermediate-acting and possibly hepatospecific insulin agonist) led to the development of HPI., Results: Clinical pharmacology studies demonstrated that HPI was definitely, although marginally, hepatospecific. More striking was the finding that the intrasubject/patient coefficient of variation of response to HPI was significantly less than that observed with NPH insulin. However, the fact that unique efficacy in controlled multicenter studies was not demonstrated suggested that these pharmacological features were not translated into clinical benefit. In one multicenter new patient study there were six myocardial infarctions, including two deaths, in patients treated for greater than or equal to 1 yr with HPI and none in the control group., Conclusions: To obtain an independent review of the risks and benefits of HPI, in February 1988, Lilly convened a consultant group that examined all relevant information on HPI available. These experts shared our concerns about the safety of HPI in light of the failure to demonstrate unique efficacy. Accordingly, clinical trials with HPI were suspended in February 1988. Experience with HPI demonstrates the challenge associated with the development of new drugs in general and insulin agonists in particular.

The hormone insulin is synthesized in the beta cell of the pancreas as the precursor, proinsulin, where the carboxyl terminus of the B-chain is connected to the amino terminus of the A-chain by a connecting or C-peptide. Proinsulin is a weak insulin agonist that possesses a longer in vivo half-life than does insulin. A form of proinsulin clipped at the Arg65-Gly66 bond has been shown to be more potent than the parent molecule with protracted in vivo activity, presumably as a result of freeing the amino terminal residue of the A-chain. To generate a more active proinsulin-like molecule, we have constructed an "inverted" proinsulin molecule where the carboxyl terminus of the A-chain is connected to the amino terminus of the B-chain by the C-peptide, leaving the critical Gly1 residue free. Transformation of Escherichia coli with a plasmid coding for A-C-B human proinsulin led to the stable production of the protein. By a process of cell disruption, sulfitolysis, anion-exchange chromatography, refolding, and reversed-phase high-performance liquid chromatography, two forms of the inverted proinsulin differing at their amino termini as Gly1 and Met0-Gly1 were identified and purified to homogeneity. Both proteins were shown by a number of analytical techniques to be of the inverted sequence, with insulin-like disulfide bonding. Biological analyses by in vitro techniques revealed A-C-B human proinsulin to be intermediate in potency when compared to human insulin and proinsulin. The time to maximal lowering of blood glucose in the fasted normal rat appeared comparable to that of proinsulin. Additionally, we were able to generate fully active, native insulin from A-C-B human proinsulin by proteolytic transformation. The results of this study lend themselves to the generation of novel insulin-like peptides while providing a simplified route to the biosynthetic production of insulin.

Neurohaemal lobes of corpora cardiaca of Locusta migratoria are an established storage site for neurohormones produced by the neurosecretory cells of the brain. As previously reported [Hietter, H., Van Dorsselaer, A., Green, B., Denoroy, L., Hoffmann, J.A. & Luu, B. (1990) Eur. J. Biochem. 187, 241-247], the isolation and characterization of a novel 5-kDa peptide from these lobes served as the basis for oligonucleotide screening of cDNA libraries prepared from poly(A) RNA from neurosecretory cells of the central nervous system. From subsequent cDNA cloning studies [Lagueux, M., Lwoff, L., Meister, M., Goltzené, F. & Hoffmann, J.A. (1990) Eur. J. Biochem. 187, 249-254], the existence of a 145-residue precursor protein was deduced, which contained, in addition to the 5-kDa peptide, amino-acid sequences with homology to the A and B chains of an insulin-related peptide. In the present study we have isolated the native molecule from corpora cardiaca of Locusta and characterized, by Edman degradation and plasma-desorption mass spectrometry, the two chains as follows: A chain, Gly-Val-Phe-Asp-Glu-Cys-Cys-Arg-Lys-Ser-Cys-Ser-Ile-Ser-Glu-Leu-Gln-Thr- Tyr-Cys - Gly (Ile, isoleucine); B chain, Ser-Gly-Ala-Pro-Gln-Pro-Val-Ala-Arg-Tyr-Cys-Gly-Glu-Lys-Leu-Ser-Asn-Ala- Leu-Lys - Leu-Val-Cys-Arg-Gly-Asn-Tyr-Asn-Thr-Met-Phe. Taken in conjunction with the previous cloning studies, our data lead to a clear picture of the processing of Locusta preproinsulin. They indicate that locusta corpora cardiaca contain remarkably large amounts of one single insulin form, in contrast to multiple insulin isoforms of Bombyx mori, the only other insect species from which insulin-related peptides have been isolated and characterized [Nagasawa, H., Kataoka, H., Isogai, A., Tamura, S., Suzuki, A., Mizoguchi, A., Fujiwara, Y., Suzuki, A., Takahashi, S. & Ishizaki, H. (1986) Proc. Natl Acad. Sci. USA 83, 5840-5843].

Refolding studies show that native insulin can be reformed from A and B chains only. This suggests that the A and B chains contain the necessary structural information and that the C peptide is not required for this process.

Antibodies were raised against the carboxy terminus of the insulin A chain in sheep, goat and rabbit using as antigen the synthetic octapeptide YQLENYCN conjugated to bovine serum albumin (BSA). All of the antisera obtained cross-reacted with molecules containing this peptide sequence. Using these antibodies, we developed a radioimmunoassay that could detect the insulin A chain itself, both denatured and natural mammalian and avian insulins, as well as proinsulins and insulin fusion proteins from microorganisms.

Hypothetically, the formation of abnormal insulin precursors due to genetic and/or acquired disorders would result in autoantigens by virtue of the altered tertiary structure. These are unlikely to be accessible to the converting enzymes which in turn should produce extended exposure to the immune system. Subsequent humoral and cell-mediated immune response might thus initiate or aggravate the autoimmune destruction of B-cells in subjects genetically susceptible to Type I diabetes.

The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg31-Arg32) and a type II activity that cleaves the C-peptide/A-chain junction (Lys64-Arg65). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, we have undertaken comparative 1H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models of prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the 1H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic 1H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. Unexpectedly, nonlocal perturbations are observed in the insulin moiety of proinsulin, as monitored by the resonances of internal aromatic groups. Remarkably, these perturbations are reverted by site-specific cleavage of the connecting peptide at the CA junction but not the BC junction. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. We propose that this structure (designated the "CA knuckle") provides a recognition element for type II proinsulin endopeptidase.