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Identification of human homologue of mouse IFN-γ induced protein from human dendritic cells
Immunology Letters 74 (2000) 221 – 224
www.elsevier.com/locate/
Identification of human homologue of mouse IFN-g induced protein from human dendritic cells Nan Li, Weiping Zhang, Xuetao Cao * Department of Immunology, Second Military Medical Uni6ersity, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China Received 27 April 2000; received in revised form 13 August 2000; accepted 13 August 2000
Keywords: Gene cloning; Dendritic cell; Interferon; Large-scale sequencing
Dendritic cells (DC) are the most potent antigen-presenting cells (APC) [1,2] possessing the unique capacity to induce primary T cell response in vivo, which depends on their functions such as Ag uptake and processing, and the capacity for maturation and migration. Upon Ag capture in periphery, immature DC migrate to the secondary lymphoid organs, and activate naive T cells. They change the Ag-uptake/processing phenotype to a distinctive Ag-presenting one [3]. To better understand the molecular mechanisms underlying the unique capacity of DC, efforts have been engaged in analyzing the repertoire of genes expressed by human DC in recent years [4–6]. In the present study, we have used large-scale screening of cDNA library to isolate novel cDNAs expressed in human peripheral blood monocyte-derived DC. Culture of DC, construction of DC cDNA library and large-scale sequencing were performed as described previously [7]. An in-house EST database was generated for human DC cDNA library, from which a cDNA clone was identified as a human homologue of mouse IFN-g induced gene MG11 [8] and designated as dendritic cell-derived IFN-g induced protein (DCIP). The full length of DCIP cDNA was obtained by RACE (Marathon cDNA Amplification Kit, Clontech). One gene-specific primer, 5%-GCT GCA GAT AAG TGA ACG AGA TGT-3% and one nested primer 5%-CAA Abbre6iations: APC, antigen presenting cells; DC, dendritic cells; DCIP, dendritic cell-derived interferon-gamma induced protein; IFNg, interferon-gamma; MLR, mixed lymphocyte reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription polymerase chain reaction. * Corresponding author. Tel.: + 86-21-25070273; fax: + 86-2165382502. E-mail address: [email protected] (X. Cao).
TAT GGT CTC ATC CCT G-3% were used in the touch-down PCR, which allowed us to extend DCIP cDNA to a final length of 2283 bp (Fig. 1). The DCIP cDNA sequence contained a methionine codon situated in a consensus Kozak sequence. There was an inframe stop codon (TGA) in the 5% upstream, therefore, the methionine codon was considered as the correct start codon. The DCIP cDNA showed a 153 bp 5% untranslated sequence, rich in G + C (109 out of 153, 71.24%), an 1881 bp ORF, and 249 bp of 3% untranslated region including a consensus polyadenylation sequences (AATAA starting at position 2188, Fig. 1). The ORF of DCIP cDNA revealed a protein of 626 amino acids with a calculated molecular weight of 72142.8 D and an isoelectric point of 7.0. Known peptide motifs that could suggest the cellular location or function of the DCIP protein were searched in PROSITE databank. The protein exhibited few structural motifs indicative of its function. However, frequently found consensus sequences such as casein kinase II phosphorylation sites, protein kinase C phosphorylation sites, and N-myristoylation sites were present. As shown in Fig. 1, an amidation site and a N-Glycosylation site (Asn Glycosylation) indicated that the DCIP protein could be a glycoprotein or a proteoglycan. As illustrated by the hydrophobicity plot (data not shown), the protein showed no obvious hydrophobicity, and no signal peptide or transmembrane regions were indicated, suggesting DCIP was a intracellular protein. Significant homolog found in the relevant databases was MG11, a mouse IFN-g induced protein [8]. It showed 72% identity (82% similarity) to the predicted DCIP protein (Fig. 2). It contained 594 aa and was cloned from a IFN-g induced mouse macrophage sub-
0165-2478/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 0 ) 0 0 2 7 6 - 5
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N. Li et al. / Immunology Letters 74 (2000) 221–224
traction library. In addition, the DCIP protein revealed high similarities with some proteins from distantly related organisms. For example, it shared 33% identity and 51% similarity with a hypothetical 66.5 KD protein of Caenorhabditis elegans, a rhabditid nematode used as a model studying nervous system development and cell apoptosis (data not shown). Distant relationship was also found with hypothetical proteins of thermophilic Eubacteria Thermotoga maritima, hyper-thermophilic archaebacterium Pyrococcus horikoshii and hyperthermophilic bacterium Aquifex aeolicus. This suggests that DCIP is of close analogy to protein sequences between
prokaryotes and eukaryotes, which reflects the conservation of gene products necessary for cell function. Although true relationships between prokaryotic and eukaryotic proteins would be difficult to establish, the high homology shared among these proteins could suggest that DCIP may be a member of a novel and large family of highly conserved proteins. Further experiments such as genome hybridization will facilitate to establish the relationship between DCIP and proteins of other organisms. The mRNA expression pattern of DCIP was analyzed by RT-PCR in DC and various cell lines(Fig.
Fig. 1. Nucleotide sequence of the DCIP cDNA and deduced amino acid sequence. Potential amidation site (X,G,[R,K],[R,K]) was shadowed, N-glycosylation site (N,P,S/T,P) was boxed and N-myristoylation (G,E/D/R/K/H/P/F/Y/W,X2,S/T/A/G/C/N,P) site was underlined. This sequence has been deposited in the GenBank/EMBL database with the accession number AF228421.
N. Li et al. / Immunology Letters 74 (2000) 221–224
223
Fig. 2. Multiple alignment of the predicted amino acids of DCIP (SBBI88) and MG11 and a homolog from C. elegans (Q09374). Identities and positives are shown. The alignment was performed with the GCG package.
3A). The RT-PCR analysis of the DCIP mRNA expression was performed using total cellular RNA extracted by Trizol reagent (Gibco BRL) and first strand cDNA synthesis was primed using an Oligo(dT)15 primer. Synthesis of cDNA was checked by PCR using b-actin primers. The amplification of a 1535bp DCIP fragment with the primers 5%-GCT GCA GAT AAG TGA ACG AGA TGT-3% (forward) and 5%-TCG AAG GAG AAA GGC TTT AA-3% (backward) was performed for 35 cycles (15 s at 95°C, 30 s at, and 45 s at 72°C). As shown in Fig. 3A, monocyte-derived DC cultured with
GM-CSF/IL-4 expressed detectable DCIP, which confirmed its derivation. Expression was detected at different levels in cells such as THP-1 (monocytic leukamia), K562 (myelogenous leukemia), HL60 (promyelocytic leukemia), Daudi and Raji (B cell lines), MCF-7 (breast adenocarcinoma), PC-3 (prostate adenocarcinoma), LoVo (colon adenocarcinoma), SMMC 7721 (hepatoma) and A172 (glioblasoma). No mRNA expression was detected in Molt-4, Jurkat (both T cell lines) and Hela (cerix epitheloid carcinoma).
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N. Li et al. / Immunology Letters 74 (2000) 221–224
Moreover, Northern Blot analysis was performed using human adult MTN Blots (Clontech). The 1535bp RT-PCR product was labeled by random priming with a-32P-dCTP (50 mCi, 3000 Ci/mmol, Amersham) as a probe. Hybridization was performed in ExpressHybTM hybridization solution (Clontech Laboratories) under high stringency. As shown in Fig. 3B, constitutive expression of DCIP mRNA was detected in most normal tissues including heart, skeleton muscle, spleen, liver, small intestine, placenta, lung and peripheral blood leukocytes. DCIP mRNA was expressed as a strong 5.2 kb message without alternatively spliced products. Database searches also produced highly homologous partial ESTs from the placenta, heart, and lung (not shown). Very faint expression of DCIP expression was detected in colon and kidney. Brain and thymus was negative for DCIP expression. It is known that IFN-g could influence the expression of over 200 genes that can be described in terms of several major molecular programs that collectively regulate immunity[9]. In the activation of DC by infectious agents, IFN-g was induced by IL-12 secreted by DC in an autocrine manner[10]. DC-derived IFN-g together with IL-12, may be important in the upregulation of surface molecules on DC such as MHC II and CD11a,
and activation of T cells[10]. In vitro experiments with monocyte-derived DC showed that the presence of IFN-g might contribute to the maturation of DC which can drive a Th1 immune response. IL-18 produced by DC can augments IL-12-induced IFN-g production by DC also in an autocrine manner. Thus, a feedback system was comprised among DC, IFN-g, IL-12, and maybe IL-18, in which IFN-g induced proteins could play important roles. Because of the genetic/functional complexity of the response to IFN-g, the functional significance of a variety of genes stimulated or repressed by IFN-g, referred as IFN-induced genes, remains obscure by far. In the present study, a human homolog of mouse IFN-g induced protein, DCIP was identified from monocyte-derived DC. DCIP protein showed 72% identity with MG11, the mouse IFN-g induced protein. DCIP mRNA expression was ubiquitously detected in various human tissues and cell lines. The identification of DCIP from human DC indicates its potential involvement in the IFN-g-regulated DC function and will aid the comprehension of the complex interaction between cytokines and important cell populations of immune system. Further work will focus on determining the inducible expression of DCIP on other immune cells and the molecules that interacted with DCIP.
.
Acknowledgements We thank Dr Tao Wan, Dr Minghui Zhang and Dr Daoming Zhang for expert technical assistance. This work was supported by grants from National Natural Science Foundation of China (39825123 and 39730420) to X.C.
References
Fig. 3. RT-PCR and Northern blot analysis of DCIP mRNA expression. (A) RT-PCR with DCIP- and b-actin-specific primers on the indicated cells. All the cells were similarly positive for b-actin (lower panel). Identity of the DCIP PCR product was confirmed by sequencing. (B) Clontech human MTN blots was analyzed with a probe generated by RT-PCR, which contained the stop codon of DCIP ORF. A major band at 5.2 kb is seen.
[1] R.M. Steinman, Annu. Rev. Immunol. 9 (1991) 271 – 296. [2] J. Banchereau, R.M. Steinman, Nature 392 (1998) 245–252. [3] G. Girolomoni, P. Ricciardi-Castagnoli, Immunol. Today 18 (1997) 102 – 104. [4] C.G. Mueller, S. Ho, C. Massacrier, S. Lebecque, Y.J. Liu, Eur. J. Immunol. 27 (1997) 3130 – 3134. [5] B. de Saint-Vis, J. Vincent, S. Vandenabeele, B. Vanbervliet, J.J. Pin, S. Ait-Yahia, S. Patel, M.G. Mattei, J. Banchereau, S. Zurawski, J. Davoust, C. Caux, S. Lebecque, Immunity 9 (1998) 325 – 336. [6] T. Shimada, M. Matsumoto, Y. Tatsumi, A. Kanamaru, S. Akira, FEBS Lett. 425 (1998) 490 – 494. [7] X. Cao, W. Zhang, T. Wan, L. He, T. Chen, Z. Yuan, S. Ma, Y. Yu, G. Chen, J. Immunol., 2000. [8] W.P. Lafuse, D. Brown, L. Castle, B.S. Zwilling, J. Leukoc. Biol. 57 (1995) 477 – 483. [9] U. Boehm, T. Klamp, M. Groot, J.C. Howard, Annu. Rev. Immunol. 15 (1997) 749 – 795. [10] T. Ohteki, T. Fukao, K. Suzue, C. Maki, M. Ito, M. Nakamura, S. Koyasu, J. Exp. Med. 189 (1999) 1981 – 1986.
www.elsevier.com/locate/
Identification of human homologue of mouse IFN-g induced protein from human dendritic cells Nan Li, Weiping Zhang, Xuetao Cao * Department of Immunology, Second Military Medical Uni6ersity, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China Received 27 April 2000; received in revised form 13 August 2000; accepted 13 August 2000
Keywords: Gene cloning; Dendritic cell; Interferon; Large-scale sequencing
Dendritic cells (DC) are the most potent antigen-presenting cells (APC) [1,2] possessing the unique capacity to induce primary T cell response in vivo, which depends on their functions such as Ag uptake and processing, and the capacity for maturation and migration. Upon Ag capture in periphery, immature DC migrate to the secondary lymphoid organs, and activate naive T cells. They change the Ag-uptake/processing phenotype to a distinctive Ag-presenting one [3]. To better understand the molecular mechanisms underlying the unique capacity of DC, efforts have been engaged in analyzing the repertoire of genes expressed by human DC in recent years [4–6]. In the present study, we have used large-scale screening of cDNA library to isolate novel cDNAs expressed in human peripheral blood monocyte-derived DC. Culture of DC, construction of DC cDNA library and large-scale sequencing were performed as described previously [7]. An in-house EST database was generated for human DC cDNA library, from which a cDNA clone was identified as a human homologue of mouse IFN-g induced gene MG11 [8] and designated as dendritic cell-derived IFN-g induced protein (DCIP). The full length of DCIP cDNA was obtained by RACE (Marathon cDNA Amplification Kit, Clontech). One gene-specific primer, 5%-GCT GCA GAT AAG TGA ACG AGA TGT-3% and one nested primer 5%-CAA Abbre6iations: APC, antigen presenting cells; DC, dendritic cells; DCIP, dendritic cell-derived interferon-gamma induced protein; IFNg, interferon-gamma; MLR, mixed lymphocyte reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription polymerase chain reaction. * Corresponding author. Tel.: + 86-21-25070273; fax: + 86-2165382502. E-mail address: [email protected] (X. Cao).
TAT GGT CTC ATC CCT G-3% were used in the touch-down PCR, which allowed us to extend DCIP cDNA to a final length of 2283 bp (Fig. 1). The DCIP cDNA sequence contained a methionine codon situated in a consensus Kozak sequence. There was an inframe stop codon (TGA) in the 5% upstream, therefore, the methionine codon was considered as the correct start codon. The DCIP cDNA showed a 153 bp 5% untranslated sequence, rich in G + C (109 out of 153, 71.24%), an 1881 bp ORF, and 249 bp of 3% untranslated region including a consensus polyadenylation sequences (AATAA starting at position 2188, Fig. 1). The ORF of DCIP cDNA revealed a protein of 626 amino acids with a calculated molecular weight of 72142.8 D and an isoelectric point of 7.0. Known peptide motifs that could suggest the cellular location or function of the DCIP protein were searched in PROSITE databank. The protein exhibited few structural motifs indicative of its function. However, frequently found consensus sequences such as casein kinase II phosphorylation sites, protein kinase C phosphorylation sites, and N-myristoylation sites were present. As shown in Fig. 1, an amidation site and a N-Glycosylation site (Asn Glycosylation) indicated that the DCIP protein could be a glycoprotein or a proteoglycan. As illustrated by the hydrophobicity plot (data not shown), the protein showed no obvious hydrophobicity, and no signal peptide or transmembrane regions were indicated, suggesting DCIP was a intracellular protein. Significant homolog found in the relevant databases was MG11, a mouse IFN-g induced protein [8]. It showed 72% identity (82% similarity) to the predicted DCIP protein (Fig. 2). It contained 594 aa and was cloned from a IFN-g induced mouse macrophage sub-
0165-2478/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 0 ) 0 0 2 7 6 - 5
222
N. Li et al. / Immunology Letters 74 (2000) 221–224
traction library. In addition, the DCIP protein revealed high similarities with some proteins from distantly related organisms. For example, it shared 33% identity and 51% similarity with a hypothetical 66.5 KD protein of Caenorhabditis elegans, a rhabditid nematode used as a model studying nervous system development and cell apoptosis (data not shown). Distant relationship was also found with hypothetical proteins of thermophilic Eubacteria Thermotoga maritima, hyper-thermophilic archaebacterium Pyrococcus horikoshii and hyperthermophilic bacterium Aquifex aeolicus. This suggests that DCIP is of close analogy to protein sequences between
prokaryotes and eukaryotes, which reflects the conservation of gene products necessary for cell function. Although true relationships between prokaryotic and eukaryotic proteins would be difficult to establish, the high homology shared among these proteins could suggest that DCIP may be a member of a novel and large family of highly conserved proteins. Further experiments such as genome hybridization will facilitate to establish the relationship between DCIP and proteins of other organisms. The mRNA expression pattern of DCIP was analyzed by RT-PCR in DC and various cell lines(Fig.
Fig. 1. Nucleotide sequence of the DCIP cDNA and deduced amino acid sequence. Potential amidation site (X,G,[R,K],[R,K]) was shadowed, N-glycosylation site (N,P,S/T,P) was boxed and N-myristoylation (G,E/D/R/K/H/P/F/Y/W,X2,S/T/A/G/C/N,P) site was underlined. This sequence has been deposited in the GenBank/EMBL database with the accession number AF228421.
N. Li et al. / Immunology Letters 74 (2000) 221–224
223
Fig. 2. Multiple alignment of the predicted amino acids of DCIP (SBBI88) and MG11 and a homolog from C. elegans (Q09374). Identities and positives are shown. The alignment was performed with the GCG package.
3A). The RT-PCR analysis of the DCIP mRNA expression was performed using total cellular RNA extracted by Trizol reagent (Gibco BRL) and first strand cDNA synthesis was primed using an Oligo(dT)15 primer. Synthesis of cDNA was checked by PCR using b-actin primers. The amplification of a 1535bp DCIP fragment with the primers 5%-GCT GCA GAT AAG TGA ACG AGA TGT-3% (forward) and 5%-TCG AAG GAG AAA GGC TTT AA-3% (backward) was performed for 35 cycles (15 s at 95°C, 30 s at, and 45 s at 72°C). As shown in Fig. 3A, monocyte-derived DC cultured with
GM-CSF/IL-4 expressed detectable DCIP, which confirmed its derivation. Expression was detected at different levels in cells such as THP-1 (monocytic leukamia), K562 (myelogenous leukemia), HL60 (promyelocytic leukemia), Daudi and Raji (B cell lines), MCF-7 (breast adenocarcinoma), PC-3 (prostate adenocarcinoma), LoVo (colon adenocarcinoma), SMMC 7721 (hepatoma) and A172 (glioblasoma). No mRNA expression was detected in Molt-4, Jurkat (both T cell lines) and Hela (cerix epitheloid carcinoma).
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N. Li et al. / Immunology Letters 74 (2000) 221–224
Moreover, Northern Blot analysis was performed using human adult MTN Blots (Clontech). The 1535bp RT-PCR product was labeled by random priming with a-32P-dCTP (50 mCi, 3000 Ci/mmol, Amersham) as a probe. Hybridization was performed in ExpressHybTM hybridization solution (Clontech Laboratories) under high stringency. As shown in Fig. 3B, constitutive expression of DCIP mRNA was detected in most normal tissues including heart, skeleton muscle, spleen, liver, small intestine, placenta, lung and peripheral blood leukocytes. DCIP mRNA was expressed as a strong 5.2 kb message without alternatively spliced products. Database searches also produced highly homologous partial ESTs from the placenta, heart, and lung (not shown). Very faint expression of DCIP expression was detected in colon and kidney. Brain and thymus was negative for DCIP expression. It is known that IFN-g could influence the expression of over 200 genes that can be described in terms of several major molecular programs that collectively regulate immunity[9]. In the activation of DC by infectious agents, IFN-g was induced by IL-12 secreted by DC in an autocrine manner[10]. DC-derived IFN-g together with IL-12, may be important in the upregulation of surface molecules on DC such as MHC II and CD11a,
and activation of T cells[10]. In vitro experiments with monocyte-derived DC showed that the presence of IFN-g might contribute to the maturation of DC which can drive a Th1 immune response. IL-18 produced by DC can augments IL-12-induced IFN-g production by DC also in an autocrine manner. Thus, a feedback system was comprised among DC, IFN-g, IL-12, and maybe IL-18, in which IFN-g induced proteins could play important roles. Because of the genetic/functional complexity of the response to IFN-g, the functional significance of a variety of genes stimulated or repressed by IFN-g, referred as IFN-induced genes, remains obscure by far. In the present study, a human homolog of mouse IFN-g induced protein, DCIP was identified from monocyte-derived DC. DCIP protein showed 72% identity with MG11, the mouse IFN-g induced protein. DCIP mRNA expression was ubiquitously detected in various human tissues and cell lines. The identification of DCIP from human DC indicates its potential involvement in the IFN-g-regulated DC function and will aid the comprehension of the complex interaction between cytokines and important cell populations of immune system. Further work will focus on determining the inducible expression of DCIP on other immune cells and the molecules that interacted with DCIP.
.
Acknowledgements We thank Dr Tao Wan, Dr Minghui Zhang and Dr Daoming Zhang for expert technical assistance. This work was supported by grants from National Natural Science Foundation of China (39825123 and 39730420) to X.C.
References
Fig. 3. RT-PCR and Northern blot analysis of DCIP mRNA expression. (A) RT-PCR with DCIP- and b-actin-specific primers on the indicated cells. All the cells were similarly positive for b-actin (lower panel). Identity of the DCIP PCR product was confirmed by sequencing. (B) Clontech human MTN blots was analyzed with a probe generated by RT-PCR, which contained the stop codon of DCIP ORF. A major band at 5.2 kb is seen.
[1] R.M. Steinman, Annu. Rev. Immunol. 9 (1991) 271 – 296. [2] J. Banchereau, R.M. Steinman, Nature 392 (1998) 245–252. [3] G. Girolomoni, P. Ricciardi-Castagnoli, Immunol. Today 18 (1997) 102 – 104. [4] C.G. Mueller, S. Ho, C. Massacrier, S. Lebecque, Y.J. Liu, Eur. J. Immunol. 27 (1997) 3130 – 3134. [5] B. de Saint-Vis, J. Vincent, S. Vandenabeele, B. Vanbervliet, J.J. Pin, S. Ait-Yahia, S. Patel, M.G. Mattei, J. Banchereau, S. Zurawski, J. Davoust, C. Caux, S. Lebecque, Immunity 9 (1998) 325 – 336. [6] T. Shimada, M. Matsumoto, Y. Tatsumi, A. Kanamaru, S. Akira, FEBS Lett. 425 (1998) 490 – 494. [7] X. Cao, W. Zhang, T. Wan, L. He, T. Chen, Z. Yuan, S. Ma, Y. Yu, G. Chen, J. Immunol., 2000. [8] W.P. Lafuse, D. Brown, L. Castle, B.S. Zwilling, J. Leukoc. Biol. 57 (1995) 477 – 483. [9] U. Boehm, T. Klamp, M. Groot, J.C. Howard, Annu. Rev. Immunol. 15 (1997) 749 – 795. [10] T. Ohteki, T. Fukao, K. Suzue, C. Maki, M. Ito, M. Nakamura, S. Koyasu, J. Exp. Med. 189 (1999) 1981 – 1986.