Identification of cis- and trans-active factors regulating human islet amyloid polypeptide gene expression in pancreatic beta-cells.

Islet amyloid polypeptide is expressed almost exclusively in pancreatic beta- and delta-cells. Here we report that beta cell-specific expression of the human islet amyloid polypeptide gene is principally regulated by promoter proximal sequences. The sequences that control tissue-specific expression were mapped between nucleotides -2798 and +450 of the human islet amyloid polypeptide (IAPP) gene using transgenic mice. To localize the cis-acting elements involved in this response, we examined the effects of mutations within these sequences using transfected islet amyloid polypeptide promoter expression constructs in pancreatic beta cell lines. The sequences between -222 and +450 bp were found to be necessary for beta cell-specific expression. Linker-scanning mutations of the 5'-promoter proximal region defined several key distinct control elements, including a negative-acting element at -111/-102 base pairs (bp), positive-acting elements like the basic helix-loop-helix-like binding site at -138/-131 bp, and the three A/T-rich, homeobox-like sites at -172/-163, -154/-142, and -91/-84 bp. Mutations within any one of these elements eliminated transcriptional expression by the promoter. Gel mobility shift assays revealed that the PDX-1 homeobox factor, which is required for insulin gene transcription in beta cells, interacted specifically at the -154/-142- and -91/-84-bp sites. Since PDX-1 is highly enriched in beta and delta cells, these results suggest that this factor plays a principal role in defining islet beta cell- and delta cell-specific expression of the IAPP gene.

Type I IL-1 receptor: ligand specific confirmation differences and the role of glycosylation in ligand binding.

The extracellular domain of the type I Interleukin-1 receptor (sIL-1R) was expressed in Drosophila S2 cells as a secreted 43 kDa glycoprotein, as evidenced by its binding to Concanavalin A and enzymatic deglycosylation. sIL-1R bound IL-1 beta with a K(D) of 2 nM as determined by competition ELISA. N-Glycanase treated sIL-1R had a C. 100 fold lower affinity than glycosylated sIL-1R for IL-1 beta, suggesting that glycosylation is a key component of the IL-1 beta/IL-1 receptor interaction. Crosslinking of sIL-1R to (125)I-IL-1 beta could be competed with unlabelled IL-1 alpha, IL-1 beta, IL-1 receptor antagonist (IL-1ra), and a mutant of IL-1 (Th9Gly) which has reduced bioactivity but wild type receptor binding affinity. Limited proteolysis of sIL-1R in the presence of IL-1 alpha, IL-1 beta, IL-1ra, and Thr9Gly IL-1 beta with several different proteases followed by analysis of sIL-1R by Western blot was used to assess the effect of binding on sIL-1R conformation. While some proteases showed no differences in cleavage patterns or sensitivity between free and bound sIL-1R, others showed differences in either cleavage sites or sensitivity with different ligands. This implies that upon ligand binding there is a conformational change in the receptor which is sensitive to the particular ligand bound, and hence has implications for the ability of different ligands to trigger responses after binding to receptor.

Germline manipulation of glucose homeostasis via alteration of glucose transporter levels in skeletal muscle.

Transgenic mice were constructed that overexpress the human Glut1 glucose transporter in skeletal muscle. Transcription of the human Glut1 cDNA was driven by the rat myosin light chain 2 promoter. Soleus and quadriceps muscles from transgenic mice expressed increased levels of Glut1 protein relative to muscles obtained from nontransgenic littermates, but there was no difference in the level of Glut4 protein between the two groups. Skeletal muscles isolated from the transgenic animals exhibited 3-4-fold increases in basal glucose uptake relative to muscles obtained from nontransgenic littermates. Muscles isolated from nontransgenic littermates exhibited 2-3-fold increases in glucose transport after incubation in the presence of insulin, but no insulin-stimulated increase in transport was observed in the muscles of transgenic mice. Plasma glucose levels were reduced by 18 and 30%, respectively, in fed and fasted transgenic mice relative to their nontransgenic siblings, but insulin and glucagon levels were not significantly different between the two groups. Glucose disposal following an oral glucose load was markedly enhanced in the transgenic animals, and plasma lactate and beta-OH-butyrate levels were elevated in both fed and fasted transgenic mice. These data strongly support the hypothesis that glucose transport plays a key role in whole body glucose homeostasis. They also demonstrate that the level of a glucose transporter in skeletal muscle can significantly influence the blood glucose set point and alter the levels of other fuel metabolites in the blood.

Mapping of neutralizing epitopes and the receptor binding site of human interleukin 1 beta.

Antibodies to synthetic peptides of human interleukin 1 beta (IL-1 beta) and to recombinant human IL-1 beta were used to identify epitopes of IL-1 beta associated with the neutralization of its biological activity. Analysis of antisera raised to 17 synthetic peptides derived from the mature IL-1 beta sequence showed that five regions (residues 6-15, 49-80, 58-80, 92-101, and 120-133) were both immunoprecipitating and neutralizing. Using a hexamer epitope mapping method, comparison of the regions recognized by four neutralizing rabbit antisera with those recognized by a rabbit antiserum raised to denatured IL-1 beta suggested two further neutralizing epitopes, residues 39-48 and 83-95. Finally, a neutralizing monoclonal antibody was shown to bind to the peptides 6-11 and 87-95 by peptide binding and mutagenesis. All of these regions appear predominantly on one face of IL-1 beta. The effect of mutations in residues 4-11 and 88-97, which lie within this face, on receptor binding and biological activity was determined. Most of the mutations tested affected both receptor binding and activity, whereas mutations in another face of IL-1 beta (residues 74-80) had no effect. Purification of two of the mutants with reduced bioactivity and receptor binding and analysis by two-dimensional NMR indicated no gross changes in tertiary structure. A third mutant had reduced bioactivity in two different bioassays but no change in receptor binding. Although two-dimensional NMR revealed no gross changes in conformation, small changes did occur at a site distal from that mutated. The data are consistent with other epitope mapping and receptor binding mutagenesis data and suggest that the neutralizing antibodies and receptor recognize different but overlapping regions of IL-1 beta.

Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity.

Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.

Structure-activity studies of human IL-1 beta with mature and truncated proteins expressed in Escherichia coli.

IL-1 beta is synthesized as an inactive 31-kDa intracellular protein, which is then processed upon secretion to an active 17-kDa carboxyl-terminal fragment. To identify the minimal portion of IL-1 beta required for activity, we constructed several deletion mutants of mature IL-1 beta. These included three amino-terminal deletions of 10, 16, and 81 amino acids, two carboxyl-terminal deletions of 17 and 72 amino acids, and one internal fragment between amino acids 17 and 81. Expression of the mutants was monitored by Western blots and immunoprecipitation. With one exception, all of these mutants and the full length 17-kDa IL-1 beta were expressed as soluble protein in Escherichia coli and could be assayed for activity and receptor binding in lysates without further purification. Whereas the intact 17-kDa IL-1 beta retained full biologic activity (greater than 10(7) U/ml of lysate) and competed for binding with 125I-labeled IL-1 beta, none of the lysates containing IL-1 beta deletion mutant proteins had activity or competed for binding to receptor at significantly higher concentrations. The loss of function in the smallest C-terminal deletion mutant does not appear to be due to the direct involvement of these C-terminal residues in receptor binding because both monoclonal and polyclonal antisera directed to this region bind to IL-1 beta but do not neutralize its activity. Therefore, this region is probably indirectly involved in sustaining the structure of the receptor-binding site.

Purification and characterization of human recombinant interleukin-1 beta.

A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli. The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates. The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay. Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes. The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm. NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No initiator Met was observed. Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds. S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay. Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.

Cloning of eukaryotic genes in single-strand phage vectors: the human interferon genes.

Using oligonucleotide probes with defined sequences, we have selected clones from a human lymphocyte cDNA library which represent human leukocyte (HuIFN-alpha) and fibroblast (HuIFN-beta) interferon gene sequences. Double-stranded f1 phage DNA was used as the vector for initial cloning of cDNA. Clones carrying interferon gene sequences were identified by hybridization with the oligonucleotide probes. The same oligonucleotide probes were used as primers for dideoxy chain termination sequencing of the clones. One HuIFN-alpha clone, 201, has a nucleotide sequence different from published HuIFN-alpha sequences. Under control of the lacUV5 promoter, the 201 gene has been used to express biologically active HuIFN-alpha in Escherichia coli.

Post-transcriptional control of protein synthesis in Balb/c-3T3 cells by platelet-derived growth factor and platelet-poor plasma.

Platelet-derived growth factor (PDGF) and platelet-poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density-arrested Balb/c-3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes disaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c-3T3 cells to growth factors, but it is not by itself sufficient.