Four New Members Expand the Interleukin-1 Superfamily
2000; Elsevier BV; Volume: 275; Issue: 2 Linguagem: Inglês
10.1074/jbc.275.2.1169
ISSN1083-351X
AutoresDirk E. Smith, Blair R. Renshaw, Randal R. Ketchem, Marek Kubin, Kirsten E. Garka, John E. Sims,
Tópico(s)interferon and immune responses
ResumoWe report here the cloning and characterization of four new members of the interleukin-1 (IL-1) family (FIL1δ, FIL1ε, FIL1ζ, and FIL1η, with FIL1 standing for "Family of IL-1"). The novel genes demonstrate significant sequence similarity to IL-1α, IL-1β, IL-1ra, and IL-18, and in addition maintain a conserved exon-intron arrangement that is shared with the previously known members of the family. Protein structure modeling also suggests that the FIL1 genes are related to IL-1β and IL-1ra. The novel genes form a cluster with the IL-1s on the long arm of human chromosome 2. We report here the cloning and characterization of four new members of the interleukin-1 (IL-1) family (FIL1δ, FIL1ε, FIL1ζ, and FIL1η, with FIL1 standing for "Family of IL-1"). The novel genes demonstrate significant sequence similarity to IL-1α, IL-1β, IL-1ra, and IL-18, and in addition maintain a conserved exon-intron arrangement that is shared with the previously known members of the family. Protein structure modeling also suggests that the FIL1 genes are related to IL-1β and IL-1ra. The novel genes form a cluster with the IL-1s on the long arm of human chromosome 2. interleukin accessory protein interleukin 1 receptor antagonist polymerase chain reaction probability density function lipopolysaccharide The cytokine interleukin-1 (IL-1)1 elicits a wide array of biological activities that initiate and promote the host response to injury or infection, including fever, sleep, loss of appetite, acute phase protein synthesis, chemokine production, adhesion molecule up-regulation, vasodilatation, the pro-coagulant state, increased hematopoiesis, and production and release of matrix metalloproteinases and growth factors (1.Dinarello C.A. Int. Rev. Immunol. 1998; 16: 457-499Crossref PubMed Scopus (682) Google Scholar). 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We have searched for novel members of the IL-1 family. We report here the sequences and some of the characteristics of four genes that appear to have descended from the same common ancestor as did IL-1α, IL-1β, IL-1ra, and IL-18. We propose that these novel molecules be designated FIL1δ, -ε, -ζ, and -η, with FIL1 being an acronym for Family of IL-1. The following details supplement the general descriptions given under "Results" for the cloning of the individual IL-1 family members. A 469-base pair single-stranded32P-labeled PCR product spanning the entire mouse FIL1δ coding region (found in GenBankTMW08205) was used to probe a human placenta cDNA library (in λUni-ZAP XR; Stratagene number 937225) (hybridization in 40% formamide at 42 °C; wash in 0.3m NaCl at 55 °C). Several clones were isolated, all of which appeared to lack the full open reading frame by comparison with mouse FIL1δ. Vector-anchored PCR on DNA from the same library was used to isolate the remaining coding sequence. A human genomic library (Stratagene catalog number 946205; in λFixII) was screened using a 32P-labeled single-strand DNA probe corresponding to the entire IL-1-like coding sequence present in GenBankTM EST AA030324 (hybridization in 45% formamide at 42 °C; wash in 0.3 m NaCl at 63 °C). The insert from one positive plaque was mapped to locate the hybridizing region, sequencing of which then revealed the 3′-most exon of the human FIL1ε gene. 5.3 kilobases of human genomic DNA to the 5′ side of this exon was isolated using theCLONTECH Human GenomeWalker kit (catalog number K1803-1). Sequencing of this DNA allowed identification of the remaining coding exons. The structure of the gene was confirmed by isolation of a PCR product in which the predicted exons were indeed spliced, using as template first-strand cDNA from the cell lines HL60 and THP1, and from human thymic tissue. Interestingly, while the original genomic DNA sequence coded for glutamine at amino acid 12, cDNA clones from all three sources contain arginine at amino acid 12. The FIL1ζ open reading frame was identified in a cDNA library made from the pancreatic tumor cell line HPT-4. A human genomic DNA sequenced to identify theFIL1η 3′ exon was obtained using theCLONTECH Human GenomeWalker kit (catalog number K1803-1). Template sequences for structure modeling were extracted from the Protein Data Bank (28.Bernstein F.C. Koetzle T.F. Williams G.J. Meyer Jr., E.F. Brice M.D. Rodgers J.R. Kennard O. Shimanouchi T. Tasumi M. Eur. J. Biochem. 1977; 80: 319-324Crossref PubMed Scopus (581) Google Scholar). A sequence alignment for the superfamily was generated based on that proposed by Bazan et al. (19.Bazan J.F. Timans J.C. Kastelein R.A. Nature. 1996; 379: 591Crossref PubMed Scopus (265) Google Scholar) for the IL-1s and IL-18, which appeared valid by examination of both cysteine and real versus predicted sheet alignments. Preliminary analysis using the program Gene Fold (29.Jaroszewski L. Rychlewski L. Zhang B. Godzik A. Protein Sci. 1998; 7: 1431-1440Crossref PubMed Scopus (88) Google Scholar) demonstrated that the experimentally determined structures for IL-1β (Protein Data Bank designations 1hib, 2i1b, 1iob, 1itb, and 21bi) and IL-1ra (Protein Data Bank designations 1ilr, 1ira, and i1rp) were valid templates for the new IL-1 family members FIL1δ, FIL1ε, and FIL1ζ. Although the sequence identity of the new IL-1-like cytokines is greater to IL-1ra, Gene Fold showed a stronger match to the structure of IL-1β. In addition, the various IL-1β structures appear better aligned structurally (as seen by superposition) than do the IL-1ra structures, so both templates were used for modeling. Modeler (30.Sali A. Potterton L. Yuan F. van Vlijmen H. Karplus M. Proteins. 1995; 23: 318-326Crossref PubMed Scopus (968) Google Scholar) was used to generate a family of 20 structures for each query sequence. All structures showed a well defined core β-trefoil, with higher variability in the outer loops; the per molecule probability density function (PDF) used by modeler varied from 1194 to 1984. The structure with the lowest overall PDF violation was visualized using a variable width ribbon based on the per residue PDF violation and showed that the highest violation was in the region of highest structural difference between IL-1ra and IL-1β. At this point the cysteine positions for the three models were revisited to ensure that no disulfide links were missed. A representative structure for each model was chosen by first ordering the models within a family by total PDF violation. After discarding structures with obvious problems, such as knots, the remaining members were then superimposed onto their mean. The structure with the lowest all atom root mean square deviation from the mean was chosen as the representative structure. Finally, the models were analyzed using Procheck (31.Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4569) Google Scholar) and it was shown that the structures are valid at 2.0-Å resolution with no major structural problems. The structure models for FIL1δ, FIL1ε, and FIL1ζ can be examined by contacting the authors. Intron placement was determined by direct cloning or amplification of genomic DNA from the various novel IL-1 loci. Primers were designed so that the PCR products overlapped one another to ensure that small introns were not overlooked. The sequence of the PCR products from genomic DNA was compared with the cDNA sequence in order to determine the exon/intron junctions. Chromosome mapping was performed using the Genebridge 4 radiation hybrid panel (32.Gyapay G. Schmitt K. Fizames C. Jones H. Vega-Czarny N. Spillett D. Muselet D. Prud'Homme J.F. Dib C. Auffray C. Morissette J. Weissenbach J. Goodfellow P.N. Hum. Mol. Genet. 1996; 5: 339-346Crossref PubMed Scopus (424) Google Scholar) (catalog number RH02.05 from Research Genetics) which consists of 93 human/hamster hybrid cell lines. Genomic DNA from the cell lines was amplified using PCR primers specific for the human version of each novel IL-1 family gene. Products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Each hybrid was scored in the following manner: 0 was assigned if there was clearly no amplification; 1 was assigned where there clearly was amplification; a score of 2 was assigned where the data was ambiguous. Scores were then submitted to the Whitehead Institute/MIT for chromosomal assignment and placement relative to known framework markers on the radiation hybrid map. Scores for each gene are as follows: FIL1δ, 100000010000000010001201001001200001101100001000000000101000010001120000000100010000100010100;FIL1ε, 101000010000100010001101121001100001101101001000001000101010021011110000001000010001100010100;FIL1ζ, 101000010000100010001201221001000001101101001000001000101010011021110000001000010001120010100;FIL1η, 122000200010000010000001001001000120001100201000000000101000001000220000001000010001100010100. Scores used for concomitant map placement of IL-1α, IL-1β, and IL-1ra were obtained from the NCBI (IL-1α, X02851;IL-1β, D20737 and AA150507; IL-1ra, H50548,R49297, and T72887). First-strand cDNAs present in CLONTECH(Palo Alto, CA) Human Multiple Tissue cDNA Panels I (catalog number K1420-1) and II (catalog number K1421-1) and the Human Immune Panel (catalog number K1426-1) were screened by PCR amplification using primers given in Table I. The primers were designed to span introns so that products arising from genomic DNA and cDNA could be distinguished. In some cases, nested primers were used in a second PCR reaction. The presence of an amplification product for each gene/tissue combination was determined by analysis on agarose gels stained with ethidium bromide.Table IPCR primer sequences used for analyzing expression of novel IL-1 family member mRNAMoleculePrimary or nestedSense/antiSequenceNo. cyclesAnneal °CFIL1δPrimarySenseGGGAGTCTACACCCTGTGGAGCTCAA3058FIL1δPrimaryAntisenseCTGCTGGAAGTAGAAGTCTGTGATGGFIL1δNestedSenseGGAGCTCAAGATGGTCCTGAGTGGGGCGCT3058FIL1δNestedAntisenseGCATTCCAGCCACCATTCTCGGGAAGCTFIL1εPrimarySenseGACACACCTCAGCAGGGGAGCATTCAGG4060FIL1εPrimaryAntisenseAACAGCATAGTTAACCCAAAGTCAGTAGFIL1ζPrimarySenseTGAGATCCTATGTCAGGCTGTGATAGG4060FIL1ζPrimaryAntisenseTGCTATGAGATTCCCAGAGTCCAGGACCFIL1ηPrimarySenseACATCATGAACCCACAACGGGAGGCAGCAC3560FIL1ηPrimaryAntisenseCTCTATCCTGGAACCAGCCACCCACAGCFIL1ηNestedSenseCCAAATCCTATGCTATTCGTGATTCTCGAC3558FIL1ηNestedAntisenseGGATTTATTCCACAGAATCTAAGTAGAAGOn some occasions, a second PCR reaction using nested primers was performed for FIL1δ and FIL1ε. Cycle numbers and annealing temperatures are also given. Open table in a new tab On some occasions, a second PCR reaction using nested primers was performed for FIL1δ and FIL1ε. Cycle numbers and annealing temperatures are also given. Alternatively, individual cell types from human peripheral blood were isolated from multiple donors, and stimulations were performed as described (33.Kubin M. Kamoun M. Trinchieri G. J. Exp. Med. 1994; 180: 211-222Crossref PubMed Scopus (349) Google Scholar, 34.Kubin M. Chow J.M. Trinchieri G. Blood. 1994; 83: 1847-1855Crossref PubMed Google Scholar, 41.Kubin M.Z. Parshley D.L. Din W. Waugh J.Y. Davis-Smith T. Smith C.A. Macduff B.M. Armitage R.J. Chin W. Cassiano L. Borges L. Petersen M. Trinchieri G. Goodwin R.G. Eur. J. Immunol. 1999; 29: 3466-3477Crossref PubMed Scopus (83) Google Scholar). In brief, NK cells were incubated with IL-12 (R & D Biosystems; 1 ng/ml) for either 2 or 4 h. T cells were unstimulated or stimulated with anti-CD3 (OKT-3 antibody, immobilized on plastic at 5 μg/ml) or with the combination of anti-CD3 and anti-CD28 (the anti-CD28 antibody was CK248, used in soluble form as a 1:500 dilution of ascites fluid), for 4 h. Monocytes were unstimulated, or stimulated with LPS (Sigma, 1 μg/ml) for 2 or 3 h. B cells were unstimulated, or stimulated with a combination of 005% SAC + 500 ng/ml CD40L trimer (Immunex) + 5 ng/ml IL-4 (Immunex) for 3.5 or 4 h. Dendritic cells were stimulated with LPS as for monocytes for 2 or 4 h. After isolation of RNA and synthesis of first-strand cDNA, PCR amplifications and gel analysis were performed as described above. Novel IL-1 family members, as well as control IL-1β and IL-18 molecules, were generated by transfection of expression vector constructs into COS cells using DEAE-dextran (35.Cosman D. Cerretti D.P. Larsen A. Park L. March C. Dower S. Gillis S. Urdal D. Nature. 1984; 312: 768-771Crossref PubMed Scopus (220) Google Scholar). Expression vectors used were pDC409 (36.Giri J.G. Ahdieh M. Eisenman J. Shanebeck K. Grabstein K. Kumaki S. Namen A. Park L.S. Cosman D. Anderson D. EMBO J. 1994; 13: 2822-2830Crossref PubMed Scopus (979) Google Scholar) for FIL1ε and FIL1ζ, or pDC412, a close relative, for FIL1δ and FIL1η. The unmodified open reading frames were used for FIL1δ, ε, and η. For FIL1ζ, the sequence beginning with Lys27 (KNLN. . . . . .) was fused downstream of the human immunoglobulin κ light chain signal peptide. IL-1β, with an ATG codon added to the N terminus of the mature form (beginning with Ala117), was expressed in pDC409. Human IL-18 was expressed as the mature form fused to the IL-7 signal peptide in the expression vector pDC206 (37.Kozlosky C.J. Maraskovsky E. McGrew J.T. VandenBos T. Teepe M. Lyman S.D. Srinivasan S. Fletcher F.A. Gayle III, R.B. Cerretti D.P. Beckmann M.P. Oncogene. 1995; 10: 299-306PubMed Google Scholar). For radiolabeling, 48 h after transfection cells were starved of cysteine and methionine for 60 min, then labeled with 70 μCi/ml of a [35S]cysteine/methionine mixture (Amersham; >1000 Ci/mmol) for 4–6 h. It is perhaps of interest that FIL1δ, FIL1ε, and FIL1η appear to be secreted from the COS cells despite the absence of either signal peptide or prodomain. C-terminal FLAG-tagged FIL1δ and -ε were partially purified from the conditioned medium using the tags, and their N termini sequenced. The N-terminal amino acid of the secreted FIL1ε was methionine 1; it had been modified by N-terminal acetylation. The N-terminal amino acid of the secreted FIL1δ is valine 2. Thus, there does not appear to have been cleavage of an unrecognized signal peptide or prodomain in either molecule. There are a number of proteins which, when transfected into COS cells, do not later appear in the medium, so this is not a general phenomenon attributable to leaky cells. However, the intracellular version of IL-1ra (icIL-1ra, a kind gift of William Arend, University of Colorado) also appears in the medium following transfection of COS cells. The significance of these findings is currently unknown. The novel IL-1 family members, present as35S-labeled proteins in conditioned medium from transfected COS cells, were tested for binding to Fc fusion proteins of the IL-1 receptor superfamily members (see Footnote 2 for general methods) 2Born, T. L., Morrison, L. A., Esteban, D. J., VandenBos, T., Thebeau, L. G., Chen, N., Spriggs, M. K., Sims, J. E., and Buller, M. L. (2000) J. Immunol., in press. IL-1R type I, IL-1R AcP, IL-1Rrp1, IL-1Rrp2, IL-1R AcPL, and T1/ST2 as follows: 0.5–1.0 ml of conditioned medium was pre-cleared for 2 h at 4 °C with 50 μl of protein G-Sepharose (Amersham Pharmacia Biotech; 50% solution in phosphate-buffered saline) containing 1% Triton X-100, 0.02% NaN3, and protease inhibitors (Roche Molecular Biochemicals catalog number 1 836 145). After a brief spin (3 min, 1000 rpm), the supernatant was transferred to a fresh tube and incubated overnight at 4 °C with 1 μg of receptor/Fc fusion protein plus another 50 μl of protein G-Sepharose. The mixture was centrifuged briefly, and the supernatant mixed with 0.5 ml of phosphate-buffered saline containing 5% glucose and protease inhibitors and spun again. The pellet was washed four times with 1 ml of a solution containing 0.4 m NaCl, 0.05% SDS, 1% Nonidet P-40, and protease inhibitors, and resuspended in 25 μl of 2 × reducing sample buffer (Zaxis, catalog number 220-2110106). Samples were run on 4–20% Tris glycine gels (Novex) and autoradiographed. The four previously known members of the IL-1 family (IL-1α, IL-1β, IL-1ra, and IL-18), while possessing a low overall fractional amino acid identity, share certain common amino acid sequence motifs, the most obvious of which can be summarized as F(X 10–12)FXS(AVS)XX(PE)XX(FY)(LI)(CAS)(TC) where X is any amino acid, and parentheses indicate that one of the included amino acids is present at that position. There are similarities in intron placement within the family as well. Relying on the sequence similarity, we searched public EST data bases and found sequences corresponding to three novel IL-1 family members, described below as FIL1δ, FIL1ε, and FIL1ζ. A fourth novel family member, described below as FIL1η, was originally revealed in a published patent application. Examination of the sequence (called IL-1δ by the inventors) in the patent application suggested that it was derived from an aberrantly spliced mRNA. We searched for and found an alternative form of mRNA that contains the conserved family sequence motif in the extreme 3′ exon. A brief description of the cloning and characteristics of each of the family members is given below. The sequences, and a comparison with the previously known IL-1 superfamily members, are given in Fig.1. A search of GenBankTM revealed a murine EST, accession number W08205, that resembled the known IL-1s but was not identical to any. The IMAGE clone corresponding to the EST was sequenced and found to contain the entire open reading frame of an IL-1-like molecule. Unlike the known family members, this novel polypeptide (called FIL1δ) appeared to contain neither a signal peptide nor a prodomain at the N terminus. A human FIL1δ cDNA was then isolated from a human placenta cDNA library, using mouse FIL1δ as a probe. The human sequence predicted an open reading frame similar to that of mouse FIL1δ. Multiple FIL1δ cDNA clones from both species were subsequently isolated, and all had the same predicted open reading frame, with no evidence for isoforms containing either signal peptide or prodomain. Interestingly, among the cDNA clones from both species were found several different 5′-untranslated region sequences (data not shown). These different 5′ sequences appear to derive from separate exons, in that they can be found (separately) in genomic sequence upstream of the FIL1δ coding region, and have potential splice donor sites at their 3′ ends. Presumably theFIL1δ gene is transcribed from at least two promoters. A later search of GenBankTM revealed a murine EST, accession number AA030324, that resembled a second novel IL-1 family member. Sequencing of the IMAGE clone corresponding to the EST showed an open reading frame that appeared to encode the C-terminal portion of an IL-1 molecule, but which could not be extended in the 5′ direction. The mouse sequence was used to probe a human genomic library, and a positive clone was identified and the insert sequenced. The sequence revealed a 212-base pair region with homology to the 3′-most exon of mouse FIL1ε. There was a potential splice acceptor site at the 5′ end of this region, and a stop codon at the 3′ end of the 70-amino acid open reading frame. More human genomic DNA to the 5′ side of this open reading frame was then isolated and sequenced, revealing three additional putative exons with sequence similarity to the mouse EST and to other IL-1 family members. On the assumption that the four putative exons were spliced to form a single coding region, PCR primers were designed and used successfully to amplify the predicted product from several different human RNA sources. As for FIL1δ, the predicted FIL1ε reading frame contains neither a signal peptide nor a prodomain. A third EST, accession number AI014548, was found in GenBankTM that appeared to encode an IL-1-like molecule. However, further sequencing revealed that the corresponding (human) IMAGE clone contained a stop codon upstream of the open reading frame but no initiating methionine. Screening of two other cDNA libraries resulted in isolation of a second, distinct aberrant clone, as well as a clone that contained an open reading frame that did begin with a methionine and that extended for 192 amino acids. This last clone was named FIL1ζ. Sequence comparison with other family members suggests that FIL1ζ has a prodomain of some 15–30 amino acids. Analysis of genomic DNA demonstrated that an intron lies between the nucleotides encoding the 23rd and 24th amino acids of the 192 amino acid open reading frame form (see Figs. 1 and4). The stop codon-containing sequences found in the aberrant cDNA clones lie within this intron, and appear to be incorporated into mRNA by cryptic splicing events. Since we had found three different cDNA isoforms for FIL1ζ, only one of which appeared to contain a functional open reading frame, it was important to determine the relative levels of the different transcripts. This was done by designing PCR primers that would amplify and distinguish the three isoforms, and using them to examine expression in a panel of first-strand cDNAs. The (presumably functional) FIL1ζ transcript was found in lymph node, thymus, bone marrow stroma, lung, testis, and placenta (Table II). We could not detect the form of mRNA represented by the EST in any tissue, whereas that represented by the other form of "aberrant" mRNA was present in bone marrow stroma (from which we had originally isolated it), lung, and placenta but not in the other tissues (not shown). The mRNA encoding that form appeared to be much less abundant than the functional FIL1ζ mRNA.Table IIExpression data for novel IL-1 family membersSourceFIL1δFIL1εFIL1ζFIL1ηHuman tissue Spleen−a−− Lymph nodeaaa− Thymusaaa− Tonsilaa−a Bone marrow−ba,ba Fetal liver−−−− Leukocyte−a−− Heart−−−a Braina−− Placentaa,b−aa Lung(a)−aa Liver−−−− Skeleton musclea−−− Kidney−−−− Pancreas−−−− Prostatea−−− Testisa−aa Ovary−−−− Small intestine−−−− Colon−−−a Fetal brainb NK cellsb Parathyroid tumorc Colon tumorc PoolcHuman cell lines Mo-Tb HUT-102b Rajib THP-1dd U937d,e IMTLHdd HL60dd HPT-4bb T84bMouse tissue Spleend,ed,e Kidne
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