Purification of interleukin-1 beta converting enzyme, the protease that cleaves the interleukin-1 beta precursor.

We have purified the IL-1 beta converting enzyme from the THP-1 cell line using standard chromatographic techniques and obtained the N-terminal amino acid sequence of this novel protein. After stimulation of THP-1 cells with lipopolysaccharide, hydroxyurea, and silica, the protease was solubilized by multiple freeze/thawing. The protein was purified by ion-exchange chromatography, affinity chromatography on blue agarose, gel filtration, and chromatofocusing. The molecular weight of the protein is approximately 22,000 Da and the pI is between 7.1 and 6.8. The overall yield for this procedure was 16% of the activity found in the initial cell lysates. An antiserum raised against a peptide based on the N-terminus was used to precipitate the protease, confirming our identification of the 22,000-Da protein as the IL-1 beta converting enzyme.

Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme.

Cowpox virus effectively inhibits inflammatory responses against viral infection in the chick embryo. This study demonstrates that one of the viral genes necessary for this inhibition, the crmA gene (a cytokine response modifier gene), encodes a serpin that is a specific inhibitor of the interleukin-1 beta converting enzyme. This serpin can prevent the proteolytic activation of interleukin-1 beta, thereby suppressing an interleukin-1 beta response to infection. However, the modification of this single cytokine response is not sufficient to inhibit inflammatory responses. This suggests that cowpox virus encodes several cytokine response modifiers that act together to inhibit the release of pro-inflammatory cytokines in response to infection. These viral countermeasures to host defenses against infection may contribute significantly to the pathology associated with poxvirus infections.

Molecular cloning of the interleukin-1 beta converting enzyme.

Interleukin-1 beta (IL-1 beta) mediates a wide range of immune and inflammatory responses. The active cytokine is generated by proteolytic cleavage of an inactive precursor. A complementary DNA encoding a protease that carries out this cleavage has been cloned. Recombinant expression in COS-7 cells enabled the cells to process precursor IL-1 beta to the mature form. Sequence analysis indicated that the enzyme itself may undergo proteolytic processing. The gene encoding the protease was mapped to chromosomal band 11q23, a site frequently involved in rearrangement in human cancers.

Substrate specificity of the protease that processes human interleukin-1 beta.

The substrate specificity of the protease which generates mature human interleukin-1 beta (IL-1 beta) from pro-interleukin-1 beta was investigated using synthetic peptide substrates and recombinant pro-IL-1 beta. The requirement of an L-aspartate in the P-1 position was confirmed together with the need for a small hydrophobic residue in the P-1' position (Gly or Ala). It was shown that the enzyme can tolerate conservative substitutions in the P-2 and P-2' positions. We found little difference in the enzyme's ability to cleave denatured and native pro-IL-1 beta, indicating that tertiary structure recognition is not involved in binding. The enzyme did, however, require a peptide of more than six amino acids for cleavage to occur. These results conclusively demonstrate the unusual specificity of this protease.

A pre-aspartate-specific protease from human leukocytes that cleaves pro-interleukin-1 beta.

Interleukin-1 beta is a 17.4-kilodalton hormone derived from a 33-kilodalton inactive precursor produced by monocytes. We used the precursor as a substrate to detect proteolytic activities in peripheral blood mono-nuclear cell-conditioned medium that might be involved in interleukin-1 beta processing. We found that the conditioned medium, following passage through DEAE-Sephacel, generates a biologically active fragment from the precursor that runs slightly higher than the mature hormone in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The responsible activity behaved as a single protein in ion exchange chromatography. It was completely inhibited by metal ion chelators and not by inhibitors of serine, cysteine, or aspartate proteases, and it was dependent on both calcium (or magnesium) and zinc. The enzyme was not inhibited by three substrate-based metalloprotease inhibitors, phosphoramidon, benzyloxycarbonyl-Gly-Leu-NH2, and N-(2-carboxy-3-phenylpropionyl)-Leu. NH2-terminal sequence analysis showed that cleavage of the precursor occurred between a histidine and an aspartate residue, and digestion of synthetic peptides indicated that the protease is specific for pre-aspartate cleavages.

Activation of interleukin-1 beta by a co-induced protease.

The proteolytic generation of mature interleukin-1 beta (IL-1 beta) from its inactive precursor does not proceed by a conventional pathway for hormonal processing. Pro-IL-1 beta is found dispersed in the cytoplasm, and there are no basic amino acid residues or other commonly recognized processing sites adjoining the mature N-terminus. Processing appears to occur during release of the hormone. In the present study, we have identified a specific protease that generates mature IL-1 beta from the precursor. This enzyme is co-induced with the hormone, and it differs in its cleavage specificity and inhibitor sensitivity from all known proteases.

Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor.

Interleukin-1 beta (IL-1 beta) is derived from an inactive precursor by proteolytic cleavage. To study IL-1 beta processing, we expressed the precursor in Escherichia coli, partially purified it, and used it as a substrate for various potentially relevant protease preparations. The precursor alone was virtually inactive, but incubation with membranes from human monocytes or myeloid cell lines yielded a 500-fold increase in IL-1 bioactivity. Western blot analysis of the incubated material showed that the 31,000-Da precursor is broken down to three major products, ranging from 17,400 to about 19,000 Da. The most active of these products is the smallest one, and it co-migrates during electrophoresis with mature IL-1 beta. Four purified known proteases were also tested for their effect on precursor IL-1 beta, and none of these products co-migrated with the mature protein. Chymotrypsin and Staphylococcus aureus protease yielded slightly larger products, which were highly active. Elastase and trypsin yielded substantially larger products, and these had little IL-1 activity. The products of three of the known proteases were identified by NH2-terminal sequencing. These results show conclusively that proteolysis of precursor IL-1 beta generates biological activity and that the cleavage must occur close to the mature NH2 terminus.

Expression and purification of native human granulocyte-macrophage colony-stimulating factor from an Escherichia coli secretion vector.

The human granulocyte-macrophage colony stimulating factor (GM-CSF) was expressed and purified from a high-level Escherichia coli secretion vector. A cDNA fragment encoding mature GM-CSF was fused with the aid of a synthetic oligonucleotide to the E. coli outer membrane signal peptide (ompA) of the secretion expression vector pIN-III-ompA3. The primary construction, designated pLB5001, is under transcriptional control of the tandem lipoprotein promoter (lppP) lactose promoter-operator (lacPO), and is regulated by the lactose repressor. Upon induction, a polypeptide of MW = 14,600 was produced which had GM-CSF activity in a human bone marrow colony assay. The linker sequence between the ompA signal peptide and the amino terminus of the mature GM-CSF was removed by oligonucleotide-directed site-specific mutagenesis to produce GM-CSF with an authentic amino terminus. The resulting construct, designated pLB5001-4, expressed authentic GM-CSF with a specific activity similar to that observed for the pLB5001 specified GM-CSF. Both versions of GM-CSF were associated with the membrane fraction after osmotic shock, and were purified to homogeneity by DEAE-Sephacel chromatography, followed by reversed-phase HPLC. Amino acid sequencing from the amino terminus of the purified GM-CSF established that the ompA signal peptide was cleaved at its normal processing site in both cases.

The cell surface receptors for interleukin-1 alpha and interleukin-1 beta are identical.

Interleukin-1 (IL-1) is a factor that can induce proliferation of murine T lymphocytes and can elicit a variety of other biological responses. These include bone resorption, fibroblast proliferation, acute phase protein release from hepatocytes, cartilage breakdown and fever. This spectrum of activities is consistent with a role for IL-1 as a mediator of inflammation. Recently, sequence data have shown that there are at least two members of the IL-1 family; these distantly related proteins have been termed IL-1 alpha and IL-1 beta. We have found previously that both murine T cells and fibroblasts possess a relative molecular mass (Mr) approximately 80,000 (80K) plasma membrane receptor for human IL-1 beta. We show here that the receptor for IL-1 alpha on both murine and human cells is identical to that for IL-1 beta. This result raises the issue of what separation, if any, there might be between the biological activities of IL-1 alpha and IL-1 beta.

Detection and characterization of high affinity plasma membrane receptors for human interleukin 1.

Interleukin 1 (IL-1) is a polypeptide hormone that acts as a central mediator of inflammation. Since IL-1 action is presumably mediated by specific cell surface receptor(s), we have characterized the binding of this hormone to cells. Purified human IL-1 was labeled to high specific activity with 125I, using Bolton-Hunter reagent. The labeled protein binds specifically to LBRM-33-1A5 (a murine T lymphoma line previously shown to produce IL-2 in response to phytohemagglutinin and IL-1) with an affinity of approximately 0.2-2 X 10(10)/M and, at saturation, to approximately 500 receptors per cell, on intact cells at 8 degrees C in the presence of sodium azide. The affinity of unmodified IL-1 for the murine plasma membrane receptor is 0.9-2 X 10(10)/M, as measured by the inhibition of 125I-IL-1 binding. The murine receptor specificity has been confirmed by demonstrating that, among a series of 12 polypeptide hormones, only IL-1 inhibits 125I-IL-1 binding to LBRM-33-1A5 cells. Treatment of surface-bound 125I-IL-1 with bivalent water-soluble crosslinkers identified a membrane polypeptide of Mr 79,500 to which IL-1 is crosslinked. A variety of cell types have been surveyed for the capacity to bind 125I-IL-1 specifically. The presence of specific binding correlates with the capacity of the cells tested to respond to IL-1. Our results indicate that the biological effects of the polypeptide hormone IL-1 are mediated by high affinity plasma membrane receptors. The identification of these receptors should provide valuable insight into the apparently diverse biological activities of IL-1.

Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs.

Two distinct but distantly related complementary DNAs encoding proteins sharing human interleukin-1 (IL-1) activity (termed IL-1 alpha and IL-1 beta), were isolated from a macrophage cDNA library. The primary translation products of the genes are 271 and 269 amino acids long, although expression in Escherichia coli of the carboxy-terminal 159 and 153 amino acids produces IL-1 biological activity.

Human interleukin 1. Purification to homogeneity.

We have purified human interleukin 1 (IL-1) to homogeneity by a simplified procedure that results in excellent yields of pure material that retains a high level of biological activity. IL-1, secreted by human peripheral blood macrophages that have been stimulated with Staphylococcus aureus, was purified by ion exchange chromatography and affinity chromatography on Procion Red agarose. The pure protein has a specific activity of 3.2 X 10(8) U/mg in the thymocyte mitogenesis assay, and is pyrogenic. No molecular weight heterogeneity was observed, in contrast to findings for mouse IL-1 and earlier reports of human IL-1. Purified IL-1, as analyzed by two-dimensional electrophoresis/electrofocusing gels, exhibited a series of charged species with isoelectric points ranging from 6.0 to 4.9, all with a molecular weight of approximately 17,500. Amino acid analysis indicated an abundance of acidic residues, in agreement with the low isoelectric points. There is little or no cysteine in the molecule. No evidence was found for the presence of carbohydrate moieties. The overall yield for this procedure was approximately 31% of the activity contained in the initial culture supernatant.

Characterization of immunoreactive somatostatin released from rat nervous system in vitro.

There is considerable evidence that somatostatin is released from nerve terminals throughout the central nervous system in response to presynaptic stimulation, thus suggesting a neuromodulator role for the peptide. We here report the partial characterization of immunoreactive somatostatin released from rat nervous system in vitro (hypothalamus, spinal cord and hypothalamic, cortical, thalamic and striatal synaptosomes). Serial dilutions of released somatostatin immunoreactivity showed parallelism with dilutions of synthetic somatostatin standard. Somatostatin immunoreactivity released from all tissue areas coeluted with synthetic tetradecapeptide on Sephadex G-25 (fine grade) gel chromatography; more than 85% of this immunoreactivity bound to Sepharose-anti-somatostatin-serum immunoaffinity columns. In addition, immunoreactive material released from hypothalamus, spinal cord and hypothalamic and cortical synaptosomes inhibited somatotropin (growth hormone, 'STH', 'GH') release from perifused anterior pituitary in a dose-related manner, indicating biological similarity to synthetic somatostatin.