The nucleotide sequence of a cloned human leukocyte interferon cDNA

We have determined the nucleotide sequence of the human leukocyte interferon cDNA carried in hybrid plasmid Z-pBR322(Pst)/HcIF-2h, which has been shown to direct the formation of a polypeptide with human leukocyte interferon activity (Nagata et al., 1980). The 910 base pair insert contains a 567 (or 543) base pair coding sequence, which determines a putative preinterferon polypeptide consisting of a signal peptide of 23 (or less likely 15) amino acids, followed by an interferon polypeptide of 166 amino acids (calculated molecular weight, 19 390). The coding sequence is preceded by a (most likely incomplete) 56 bp leader and followed by a 242 bp trailer and seven A residues from the poly(A) tail: A comparison of the sequence of 35 amino terminal amino acids of lymphoblastoid interferon (Zoon et al., 1980; M. Hunkapiller and L. Hood, personal communication) and the corresponding sequence deducted for leukocyte interferon revealed 9 differences. This suggests that these two interferons are encoded by two non-allelic genes.     

Hybrid recombinant human leukocyte interferon inhibits differentiation in murine B-16 melanoma cells

We have investigated the effects of recombinant human leukocyte interferons (IFN-alpha A and IFN-alpha D) and various hybrid recombinant human leukocyte interferons on differentiation in B-16 mouse melanoma cells. Inhibition of both spontaneous and melanocyte hormone stimulated differentiation was observed with one hybrid construct, IFN-alpha A/D (Bgl) consisting of amino acids 1 to 62 from IFN-alpha A and amino acids 64 to 166 from IFN-alpha D. In contrast, the parental human interferons, IFN-alpha A and IFN-alpha D, when used alone or in combination, as well as other hybrid human leukocyte interferons, did not cause significant inhibition of melanogenesis in B-16 mouse cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also inhibited B-16 differentiation and the combination of TPA with IFN-alpha A/D (Bgl) or mouse L-cell interferon was synergistic in delaying melanogenesis. These studies indicate that the IFN-alpha A/D (Bgl) hybrid that exhibits antiviral activity on mouse cells can also inhibit differentiation of murine cells.     

Increased nitroblue tetrazolium dye reduction by human neutrophils stimulated by interferon in vitro

Human leukocyte interferon enhanced nitroblue tetrazolium dye (NBT) reduction by human neutrophils (PMNs). Increase in NBT reduction paralleled increase in interferon dose. When human leukocyte interferon was heated to 60 C or 80 C for 30 min, both the antiviral activity and the effect on NBT reduction decreased. Human leukocyte interferon neutralized with anti-human leukocyte interferon serum showed no effect on NBT reduction. A human fibroblast interferon preparation also enhanced NBT reduction. The species dependency of interferon was shown in NBT reduction as well as in antiviral activity.     

Recombinant human leukocyte interferon produced in bacteria has antiproliferative activity

Recombinant human leukocyte interferon synthesized by Escherichia coli possesses antiproliferative activity in addition to antiviral activity. When the ability to inhibit multiplication of lymphoblastoid Daudi cells was examined, the growth-inhibitory capacity of recombinant leukocyte interferon was equivalent to that exhibited by crude human leukocyte interferon or by the homogeneous gamma 2 species of leukocyte interferon synthesized by human cells.     

Purification and characterization of natural human interferon omega 1. Two alternative cleavage sites for the signal peptidase.

Human interferon omega 1 (IFN-omega 1 = IFN-alpha II1) is a recently discovered protein structurally related to IFN-alpha and -beta; the biological activities of IFN-omega 1 and its physiological role are not known to date. We have purified IFN-omega 1 from preparations of human leukocyte IFN, derived from peripheral blood leukocytes induced with Sendai virus, by two sequential cycles of monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase high performance liquid chromatography and showed an Mr of 24,500 upon sodium dodecyl sulfate-gel electrophoresis (theoretical Mr, 19,984). Amino acid sequence analysis revealed that only about 40% of the molecules have the NH2 terminus expected on the basis of the sequence similarity to IFN-alpha, whereas the others contain two additional amino acids. This difference probably results from variable cleavage of the pre-protein by the signal peptidase. No evidence for COOH-terminal heterogeneity was found. Essentially all IFN-omega 1 molecules are glycosylated; enzymatic deglycosylation resulted in a reduction of the Mr to 20,500. Experiments using several plant lectins indicated the presence of biantennary complex oligosaccharides containing neuraminic acid. Two major peaks were observed upon chromatofocusing, with isoelectric points of 8.1 and 8.5. The specific antiviral activity of purified IFN-omega 1 assayed on human cells was determined to be 2.7 x 10(8) IU/mg, similar to that of other human class I IFNs; potent antiviral activity was also observed on cells of bovine and ovine but not of equine or murine origin.     

Human interferon-γ lacking 23 COOH-terminal amino acids is biologically active

We constructed five mutated cDNAs encoding human interferon-γ (IFN-γ) derivatives lacking 19–23 COOH-terminal residues and expressed them in Escherichia coli. All the derivatives were purified to homogeneity. They showed substantially the same order of antiviral activity in vitro as the intact molecule, and behaved as a dimer. The far- and near-UV circular dichroism spectra of the derivatives were quite similar to those of the intact one. These results indicate that the 23 COOH-terminal amino acids at least are not essential for achieving the full antiviral activity and constructing the higher structure of human IFN-γ.     

Human leukocyte interferon: Isolation and characterization of several molecular forms

Purification of human leukocyte interferon by high-performance liquid chromatography yielded eight major interferon species. These were characterized by their molecular weight, amino acid composition, and antiviral activity on human and bovine cells. The molecular weights of the purified species ranged from 16,000 to 21,000. The tryptic peptide profiles of all the pure species were compared. Both amino acid compositions and tryptic peptide profiles revealed a structural similarity among the various species.     

Enzymatic modifications of human fibroblast and leukocyte interferons

The sensitivity of highly purified human fibroblast interferon and partially purified human leukocyte interferon to several proteolytic and glycolytic enzymes was determined with respect to antiviral activity, isoelectric point, molecular weight, and thermal stability. Leucine aminopeptidase altered the distribution of isoelectric points for both interferons but produced little change in molecular weights; this enzyme somewhat reduced the activity of only leukocyte interferon. Treatment of fibroblast interferon with carboxypeptidases A and B did not greatly decrease antiviral activity, but it did slightly reduce the molecular weight of the interferon and substantially altered the distribution of isoelectric point values; similar treatment of leukocyte interferon caused some loss in activity, especially of the 17,000-molecular-weight species. Both interferons were inactivated rapidly by treatment with the endoproteases trypsin, pepsin, bromelain, and subtilisin. Chymotrypsin shifted the isoelectric points of both interferons, but only leukocyte interferon was significantly inactivated. Treatment with neuraminidase and beta-galactosidase changed the isoelectric point distribution but did not affect the activity or thermal stability of either interferon; such a treatment reduced the molecular weight of fibroblast interferon and the size heterogeneity of leukocyte interferon. Treatment with neuraminidase and then leucine aminopeptidase greatly reduced the activity of both interferons, especially leukocyte interferon. The data indicate that biologically active forms of fibroblast and leukocyte interferons can be distinguished by their relative sensitivity to certain proteases.     

Human interferon and cell growth inhibition. III. Separation of activities by treatment with sodium dodecyl sulphate

When human leukocyte interferon was treated with boiling sodium dodecyl sulphate antiviral activity without detectable effect on the growth of human amnion cells could be separated from the growth inhibitory activity by a single gel filtration. Similar results were obtained with mouse L-cell interferon. It is concluded that the two effects of interferon can be separated in distinct molecular entities.     

Role of the carboxy-terminal sequence on the biological activity of human immune interferon (IFN-γ)

The biological activity of human immune interferon depends on its carboxy-terminal structure. New genes coding for mature immune interferon molecules lacking 4, 5, 6, 8, 9, 10 and 11 amino acid residues were constructed, expressed in Escherichia coli and purified to homogeneity. Deletions with 14 and 18 carboxy-terminal amino acids were obtained by limited proteolysis of full-length immune interferon with mouse submaxillary gland ‘Arg-C’ protease and human plasmin, respectively. If a limited number of carboxy-terminal residues are removed, the antiviral and antiproliferative activities and the potential to activate macrophages is drastically enhanced. Maximal enhancement is found with the deletion of 9 or 10 residues. However, removal of 14 or more carboxy-terminal residues results in a sharp decrease of activity. We suggest that human immune interferon is synthesized as a preprotein and that its activity is modulated by proteolytic digestion.     

Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal peroxisomal targeting sequence

We have identified a novel peroxisomal targeting sequence (PTS) at the extreme COOH terminus of human catalase. The last four amino acids of this protein (-KANL) are necessary and sufficient to effect targeting to peroxisomes in both human fibroblasts and Saccharomyces cerevisiae, when appended to the COOH terminus of the reporter protein, chloramphenicol acetyl transferase. However, this PTS differs from the extensive family of COOH-terminal PTS tripeptides collectively termed PTS1 in two major aspects. First, the presence of the uncharged amino acid, asparagine, at the penultimate residue of the human catalase PTS is highly unusual, in that a basic residue at this position has been previously found to be a common and critical feature of PTS1 signals. Nonetheless, this asparagine residue appears to constitute an important component of the catalase PTS, in that replacement with aspartate abolished peroxisomal targeting (as did deletion of the COOH-terminal four residues). Second, the human catalase PTS comprises more than the COOH-terminal three amino acids, in that COOH-terminal-ANL cannot functionally replace the PTS1 signal-SKL in targeting a chloramphenicol acetyl transferase fusion protein to peroxisomes. The critical nature of the fourth residue from the COOH terminus of the catalase PTS (lysine) is emphasized by the fact that substitution of this residue with a variety of other amino acids abolished or reduced peroxisomal targeting. Targeting was not reduced when this lysine was replaced with arginine, suggesting that a basic amino acid at this position is required for maximal functional activity of this PTS. In spite of these unusual features, human catalase is sorted by the PTS1 pathway, both in yeast and human cells. Disruption of the PAS10 gene encoding the S. cerevisiae PTS1 receptor resulted in a cytosolic location of chloramphenicol acetyl transferase appended with the human catalase PTS, as did expression of this protein in cells from a neonatal adrenoleukodystrophy patient specifically defective in PTS1 import. Furthermore, through the use of the two-hybrid system, it was demonstrated that both the PAS10 gene product (Pas10p) and the human PTS1 receptor can interact with the COOH-terminal region of human catalase, but that this interaction is abolished by substitutions at the penultimate residue (asparagine-to- aspartate) and at the fourth residue from the COOH terminus (lysine-to-glycine) which abolish PTS functionality. We have found no evidence of additional targeting information elsewhere in the human catalase protein. An internal tripeptide (-SHL-, which conforms to the mammalian PTS1 consensus) located nine to eleven residues from the COOH terminus has been excluded as a functional PTS. Additionally, in contrast to the situation for S. cerevisiae catalase A, which contains an internal PTS in addition to a COOH-terminal PTS1, human catalase lacks such a redundant PTS, as evidenced by the exclusive cytosolic location of human catalase mutated in the COOH-terminal PTS. Consistent with this species difference, fusions between catalase A and human catalase which include the catalase A internal PTS are targeted, at least in part, to peroxisomes regardless of whether the COOH-terminal human catalase PTS is intact.     

Construction and phosphorylation of a fusion protein Hu-IFN-alpha A/gamma

A protein consisting of human (Hu)-IFN-alpha A to which the COOH-terminal 16 amino acids of Hu-IFN-gamma were fused was prepared by constructing an expression vector by oligonucleotide-directed mutagenesis. The hybrid protein Hu-IFN-alpha A/gamma was expressed under the control of phage lambda PL promoter. The protein was purified with the use of a monoclonal antibody against Hu-IFN-alpha or the COOH-terminal amino acid sequence of Hu-IFN-gamma. The purified protein exhibited a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has antiviral activity on human and bovine cells. Unlike Hu-IFN-alpha A, but similar to Hu-IFN-gamma, the hybrid Hu-IFN-alpha A/gamma can be phosphorylated by [gamma 32P]ATP and cAMP-dependent protein kinase. The phosphorylated molecule binds to the IFN-alpha/beta receptor. The introduction of a phosphorylation site into Hu-IFN-alpha A by fusion of the region of Hu-IFN-gamma which contains the phosphorylation site provides a new reagent for studies of receptor binding, pharmacokinetics, and other studies where labeled interferons are useful. Furthermore, the introduction of phosphorylation sites into proteins provides a new principle for the preparation of a wide variety of reagents for many purposes.     

Genetic transfer of a functional human interferon alpha receptor into mouse cells: cloning and expression of its cDNA

A cDNA coding for the human interferon alpha receptor has been cloned using a gene transfer approach. This consists of transferring human DNA to mouse cells and selecting for cells sensitive to human interferon alpha. The transfected cells expressed the human interferon alpha receptor, and a 5 kb human DNA was isolated from a secondary transfectant. This DNA defects an mRNA present in human cells and was used to clone a 2.7 kb cDNA from a library constructed from human Daudi cells. The sequence of the cDNA is presented. It codes for a glycoprotein of 557 amino acids with an N-terminal hydrophobic region and a single transmembrane-spanning segment. Mouse cells expressing the cDNA become sensitive to the antiviral activity of and express binding sites for human interferon alpha, demonstrating that the cloned cDNA encodes a functional human interferon alpha receptor.     

Molecular size heterogeneity of human leukocyte interferon

Molecular sieving of human leukocyte interferon revealed an apparent molecular weight of 26,000. However, after denaturation by guanidine hydrochloride in the presence of a reducing agent and reactivation by extensive dialysis, a molecular weight of only 21,000 was observed. The reactivated human leukocyte interferon (mol wt 21,000) gave a single peak of activity when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that a single molecular weight species was generated by the denaturation and reactivation procedure. A partial unfolding of the molecule was evident when the interferon preparation was heated to 50 degrees C in the absence or presence of an unfolding agent and then sieved on Sephadex G-100 Superfine. These results suggest that the interferon molecule undergoes a proteolytic cleavage probably by a protease present in extracellular fluid. Thus, a peptide fragment dissociates from the parent molecule when human leukocyte interferon is denatured in the presence of a reducing agent, resulting in a drop of 5,000 in molecular weight; interestingly, the resultant 21,000 molecular weight form still retains its antiviral activity.     

Purification and characterization of interferons from a continuous myeloblastic cell line

Several leukocyte interferon species have been purified from a continuous human myeloblast cell line. The purification procedure involving selective precipitations, gel chromatography, and several steps of high performance liquid chromatography results in interferons with specific activities of 1 to 4 X 10(8) units/mg on bovine MDBK cells. The total yield of interferon is 23%, with the yield of the individual fractions ranging from 0.2 to 11.4%. Five fractions are homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Molecular weights of the interferons were estimated by mobility on the sodium dodecyl sulfate gels and range from 17,600 to 26,200. The species differ in their relative antiviral activities on two cell lines, bovine MDBK and human AG-1732. In addition, the pure species have similar, but distinct, amino acid compositions and tryptic peptide profiles. These result support the conclusion that leukocyte interferon consists of several homologous proteins.     

Chemical characterization of recombinant human leukocyte interferon A using fast atom bombardment mass spectrometry

Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.     

Alveolar macrophage lipoxygenase products of arachidonic acid: isolation and recognition as the predominant constituents of the neutrophil chemotactic activity elaborated by alveolar macrophages

Human immune interferon preparations have anticellular activity on human cell lines (WISH and HEp-2). This anticellular activity copurified with the human immune interferon and appears to be a function of the immune interferon molecule. On the basis of a unit of antiviral activity, purified human immune interferon had about 20 and 100 times more anticellular activity than purified fibroblast or leukocyte interferon, respectively. The possible implications of this finding in the treatment of human neoplasia are discussed.     

Molecular cloning of feline interferon cDNA by direct expression.

A cDNA sequence coding for feline interferon has been cloned for the first time by screening a cDNA library constructed using Okayama-Berg vector and mRNA derived from the feline cells (LSA-I) induced by TPA (12-o-tetradecanoylphorbor 13-acetate) for the ability of transient expression to produce feline interferon in COS1 monkey cells. The amino acid sequence, deduced from the nucleotide sequence by comparing it with the sequences of other mammalian IFNs, consists of 171 amino acids with 6 cysteins and an N-glycosylation site at the amino acid position 79, and has about 60% homology to human IFN alpha 1. The interferon was partially purified through Blue Sepharose, and its molecular weight was estimated to be 2.4 x 10(4). The antiviral activity was acid stable, and glycosylation was suggested.     

Roles of the 29-138 disulfide bond of subtype A of human alpha interferon in its antiviral activity and conformational stability

Human alpha (leukocyte) interferons contain two disulfide bonds between Cys-1 and Cys-98 and between Cys-29 and Cys-138. Reduction of interferon under native conditions leads to irreversible loss of antiviral activity; reduction in denaturant, followed by oxidation in native conditions, leads to restoration of activity. This behavior, unusual for disulfide-containing proteins, was studied by using a thiosulfonate derivative of subtype A of human alpha interferon (IFN-alpha A). The disulfide-free thiosulfonate formed at 25 degrees C has essentially no antiviral activity, while maintaining a conformation related to that of native IFN-alpha A. This species can regain activity after regeneration of its 29-138 disulfide, by thiol-disulfide interchange in native buffer. Incubation of the disulfide-free thiosulfonate under nonreducing conditions at 37 degrees C generates a monomeric species that has lost its native conformation as well as its ability to regain antiviral activity after thiol-disulfide interchange. These results explain the difficulty in obtaining, under native conditions, a reduced species that regains activity upon oxidation; complete reduction of IFN-alpha A in 100 mM 2-mercaptoethanol requires 37 degrees C, a temperature that promotes conformational decay of the disulfide-free form.     

Antibody to staphylococcal enterotoxin A-induced human immune interferon (IFN gamma)

Antiserum to human gamma interferon (IFN gamma) was produced in rabbits immunized with partially purified (10(4.8) to 10(6.2) antiviral U/mg protein) staphylococcal enterotoxin A-induced IFN gamma. Staphylococcal enterotoxins, phytohemagglutinin M, concanavalin A, and pokeweed mitogen-induced antiviral activity in human leukocyte cultures was neutralized to undetectable levels by the antiserum. However, human leukocyte interferon (IFN alpha), human fibroblast interferon (IFN beta), and mouse interferons were not neutralized by the antiserum. After determining the antiserum was specific for IFN gamma and did not neutralize other known types of interferon, it was used with antibody to human IFN alpha to demonstrate the type(s) of interferon stimulated by some new inducers and antigens. Galactose oxidase- and calcium ionophore-induced interferons were neutralized to undetectable levels by the antiserum to IFN gamma. Interferon produced in leukocyte cultures from tuberculin-negative individuals stimulated with tuberculin-purified protein derivative or old tuberculin was IFN alpha, whereas interferon from tuberculin-positive individuals was a combination of alpha and gamma IFN. In addition, the antiserum neutralized the anticellular and natural killer cell enhancement activities of IFN gamma preparations. The specificity of this antiserum for IFN gamma indicates that it is an additional, powerful tool for identifying and classifying known and new interferons produced in vitro or in vivo and for investigating the role(s) of IFN gamma during the course of infectious, neoplastic, and autoimmune diseases.