International Immunology, Vol. 2, No. 6
© 1990 Oxford University Press 0953 8178/89 $3.00
Molecular cloning of cDNA encoding MS2 antigen, a novel cell surface antigen strongly expressed in murine monocytic lineage Seiji Yoshida, Mihoko Setoguchi, Yasunori Higuchi, Shin'ichiro Akizuki, and Shunsuke Yamamoto Department of Pathology, Medical College of Oita, Oita 879-56, Japan
Abstract cDNA clones which strongly hybridized with a 3.1 kb mRNA from mouse macrophages and macrophage cell lines and weakly with mRNA from P815 but not from a variety of other cell lines and tissues were isolated from cDNA libraries constructed using mRNA from murine macrophage cell lines and peritoneal macrophages. Treatment of a macrophage cell line with macrophage stimulators significantly enhanced transcription of the mRNA. Sequencing analysis of these clones demonstrated that the cDNA consisted of 3036 bp insert containing a 2478 bp open reading frame followed by a 538 bp 3' untranslated region. The amlno acid sequence, deduced from the nucleotide sequence of the cDNA, predicted a protein containing a signal peptide, an extracellular region, a transmembrane domain, and a cytoplasmlc tail. The extracellular region had five putative W-glycosylation sites and a cystelne-rlch domain, whereas the cytoplasmlc region consisted of a proline-rlch amlno acid sequence significantly similar to CD2. SDS - PAGE and NEPHGE SDS-PAGE analysis of the Immunoprecipitated membrane of the macrophage cell lines prepared by using rabbit anti-MS2 peptide antibody raised against a synthetic peptide preparation relative to a hydrophilic region of the MS2 amlno acid sequence confirmed that MS2 protein is a cross-linked protein having approximate molecular sizes of 89 kd and p/ 6.5-7.0. These results show that MS2 protein Is a novel cell surface antigen expressed mainly in monocytic lineages.
Identification and characterization of cell surface antigens facilitate analysis of the origin, differentiation, heterogeneity and function of cells. In the myelomonocytic lineage, a number of antigens have been discovered although those specific for the lineage are still limited (1). They include receptors for hormones, cell surface ligands, immunoglobulins and complement components, and enzymes (1). Most of the studies have been performed largely with panels of monoclonal antibodies that react with murine and human antigens. Molecular genetic techniques, however, are now recognized as being important because the overall structures of cell surface macromolecules can only be studied by these techniques. Structures deduced from nucleotide sequences also provide information about the function of the molecule and isolating the homologues of different species. We have constructed a cDNA library of a macrophage cell line by using a cDNA subtraction technique that is useful for
Correspondence to: Shunsuke Yamamoto, as above Transmitting editor: T Honjo
enrichment of cell type specific cDNA clones (2). Using one of these probes, we characterized the cDNA encoding the murine homologue of human myelomonocytic cell differentiation antgen, CD14 (3). This study describes isolation of another cDNA that is expressed in macrophages and macrophage cell lines and has a sequence encoding a novel transmembrane protein containing a cysteine rich region in the extracellular region and a proline rich cytoplasmic tail significantly similar to that of CD2 (4-8). The MS2-5 cDNA clone (450 bp) was separated from a cDNA library derived from cDNA obtained by subtractive hybridization between cDNA from a macrophage cell line, HINS-B3, and mRNA from a non-macrophage cell line, early HINS line (3). Preliminary experiments using Northern blot analysis showed that this plasmid detected 3.1 and 4.6 kb mRNA from the HINS-B3 cell line. The 3.1 kb species was the smallest and most abundant
Received 29 January 1990, accepted 14 March 1990
Downloaded from http://intimm.oxfordjournals.org/ at University of Michigan on July 3, 2015
Key words: macrophage differentiation antigen, cDNA sequencing, immunoprecipitation
586 A novel membrane antigen, MS2, in monocytic lineage to
1
2
3
H
5
6
7
8
9 10 11 12 13
(a)
(b) 1 2
3
1 5
1 2
3
5
6
7
7.464.402.371.35-
(d)
(C)
12
1 5
1 2
3
of the transcripts, suggesting that the species was a mature transcript of the MS2-5 associated gene. To obtain cDNA clones containing longer inserts, we next screened a Xgt11 cDNA library constructed by using mRNA from mouse peritoneal macrophages (Clontech, Palo Alto, CA, USA) and selected several cDNA clones using an MS2-5 insert, and separated several clones as shown in Fig. 3(a). Using the longest clone, MS2-468, spanning 2.85 kb, Northern blot hybridization using RNA from various cells was performed. The plasmid strongly hybridized with mRNA from mouse resident peritoneal macrophages and several mouse macrophage cell lines in Northern hybridization, although its expression varied among the macrophage cell lines (Fig. 1) and weakly hybridized with mRNA from the mastocytoma cell line, P815. In contrast, other tissues and cell lines showed no expression of the gene. We next examined whether expression of the MS2 gene is regulated by stimulators. As shown in Fig. 2,12-O-tetradecanoyfphorbd-13 acetate (TPA) (5 x 10~7 M) had a Diphasic effect on MS2 gene expression; the expression slightly increased after 15 min, declined after 45 min, and then again increased with time. Lipopolysaccharide (LPS) caused a significant enhancement 3 - 2 4 h after the stimulation. Crude lymphokines [a culture supernatant of spleen cells from a BALB/c mouse stimulated with Con A-Sepharose (Pharmacia-LKB, Uppsala, Sweden) (100 /ig/ml)] induced a late effect. Restmulation with LPS 8 - 72 h after initial stimulation with mouse recombinant interferon 7 (IFN7) induced time-dependent increases in MS2 expression. These results show that expression of the MS2 gene can be upregulated by macrophage stimulators. Several cDNA clones shown in Fig. 3(a) were sequenced in order to construct overall nucleotide sequences of MS2 cDNA (Fig. 3b). They spanned 3036 bp. The first codon, ATG at nucleotide position 18, was embedded in a CCATCATG sequence highly homologous to the consensus for a translation initiation site (CCACCATG) (14). An open reading frame spanning 2478 bp was followed by an in-frame stop codon, TAA at position 2496, and a 538 bp 3' untranslated region containing a
Fig. 2. Transcription of the MS2 gene by treatment of HINS-B3 cells with macrophage stimulators Total RNA (10 ^g) extracted at various times from these cells following stimulation with TPA (Sigma Chemical Co., St Louis, MO) (5 x 10~ 7 M), LPS (List Biological Lab. Inc CA) (1 /tg/ml), lymphokines (10%), and LPS (1 /tg/ml) after stimulation with IFN7 (kindly supplied by Shionogi Co., Ltd, Osaka) (100 U/ml) were electrophoresed in 1.5% formaldehyde gel, blotted on a nylon membrane and hybridized with a ^P-labelled MS2-468 cDNA probe, (a) TPA, lane 1, 0 mm, lane 2, 15 mm; lane 3, 45 min; lane 4, 2 h, lane 5, 8 h; lane 6, 48 h. (b) LPS, lane 1, Omin; lane 2, 30 mm; lane 3, 90 min; lane 4, 3 h; lane 5, 6 h; lane 6, 12 h; lane 7, 24 h; lane 8, 48 h. (c) Lymphokines, lane 1, 0 min; lane 2, 30 min; lane 3, 90 min; lane 4, 3 h; lane 5, 6 h, lane 6, 12 h; lane 7, 24 h (d) LPS stimulation for 3 h after stimulation with IFN7 for the indicated times, lane 1, no stimulation; lane 2, 0 mm, lane 3, 8 h; lane 4, 24 h; lane 5, 48 h; lane 6, 72 h. The lower parts show the same Wots rehybndized with a cDNA probe for /3-actin to demonstrate approximately equal amounts of RNA in each line.
polyadenylation signal (AATAAA). A search through the EMBL/GenBank database revealed no significant homology of the cDNA to known sequences. The cDNA encodes an 826 amino acid polypeptide which contains hydrophobic stretches capable of forming a signal sequence and a transmembrane sequence (Figs 3b and 4). The signal peptjdase normally cleaves at the carboxyl side of residues with small neutral side chains such as glycine, alanine, and senne (15). We therefore assigned the residues between the alanine and valine as a potential cleavage site for a signal peptide, although the boundary had an exceptional structure because a proline residue was found at position - 2 (15). Thus the mature core protein is composed of 812 amino acid residues and has a molecular mass of 88312.88 dalton. A hydropathicity plot (16) predicts a putative transmembrane domain that contains only hydrophobic and neutral amino acid residues (Figs 3b and 4). The domain shows a hydrophobic stretch of 25 amino acids, which is flanked on its carboxyl side by two basic ammo acids (Arg-Lys). Basic sequences on the cytoplasmic side are also found in other transmembrane proteins (17,18) and would warrant an orientation of MS2 protein such that the N-terminus is situated
Downloaded from http://intimm.oxfordjournals.org/ at University of Michigan on July 3, 2015
Rg. 1. Expression of MS2 mRNA in macropbages and macrophage cell lines. Total RNA (30 /ig) was electrophoresed in 1.5% formaldehyde gel, blotted on a nylon membrane, and hybridized with a ^P-labelled MS2-468 cDNA probe. Lanes contained RNA from the following sources 1. HINS-B3; 2, CANS-20 (mouse macrophage cell line) (9-13); 3, RAW264; 4, J774; 5, P388D1; 6, P3/NS1-Ag-1 (NS1); 7, P815, 8, EL4, 9, L929, 10, mouse resident peritoneal macrophages; 11, mouse liver; 12, mouse kidney; 13, mouse brain.
A novel membrane antigen, MS2, in monocytic lineage 587
(a)
B PB Py|FV
5' Okb
B
PvPv
B H
s
1.0
P
,. 3.0
2.0
•HS2-54
•HS2-12
•rS-2-468
MS2-8-1
OTOCTCCAaATCCCATCATOCTTaOCCTCTOOCTOCTCAOCaTCTTATaaACACCAOCA 59 -1 1ES 32 261
OTAOCCCCTOaACCTCCl 110CCCCATffTOAAA(^aTATOAAOTQOTTTGOCCTcdawiccfAOCTGCTiTC«CCCT(^aSteAGCCCfTGC«CTM Y A P a P P L P H Y J Q Y B V Y V P B B L A A B B B B B A L P S CACTOQ
© 1990 Oxford University Press 0953 8178/89 $3.00
Molecular cloning of cDNA encoding MS2 antigen, a novel cell surface antigen strongly expressed in murine monocytic lineage Seiji Yoshida, Mihoko Setoguchi, Yasunori Higuchi, Shin'ichiro Akizuki, and Shunsuke Yamamoto Department of Pathology, Medical College of Oita, Oita 879-56, Japan
Abstract cDNA clones which strongly hybridized with a 3.1 kb mRNA from mouse macrophages and macrophage cell lines and weakly with mRNA from P815 but not from a variety of other cell lines and tissues were isolated from cDNA libraries constructed using mRNA from murine macrophage cell lines and peritoneal macrophages. Treatment of a macrophage cell line with macrophage stimulators significantly enhanced transcription of the mRNA. Sequencing analysis of these clones demonstrated that the cDNA consisted of 3036 bp insert containing a 2478 bp open reading frame followed by a 538 bp 3' untranslated region. The amlno acid sequence, deduced from the nucleotide sequence of the cDNA, predicted a protein containing a signal peptide, an extracellular region, a transmembrane domain, and a cytoplasmlc tail. The extracellular region had five putative W-glycosylation sites and a cystelne-rlch domain, whereas the cytoplasmlc region consisted of a proline-rlch amlno acid sequence significantly similar to CD2. SDS - PAGE and NEPHGE SDS-PAGE analysis of the Immunoprecipitated membrane of the macrophage cell lines prepared by using rabbit anti-MS2 peptide antibody raised against a synthetic peptide preparation relative to a hydrophilic region of the MS2 amlno acid sequence confirmed that MS2 protein is a cross-linked protein having approximate molecular sizes of 89 kd and p/ 6.5-7.0. These results show that MS2 protein Is a novel cell surface antigen expressed mainly in monocytic lineages.
Identification and characterization of cell surface antigens facilitate analysis of the origin, differentiation, heterogeneity and function of cells. In the myelomonocytic lineage, a number of antigens have been discovered although those specific for the lineage are still limited (1). They include receptors for hormones, cell surface ligands, immunoglobulins and complement components, and enzymes (1). Most of the studies have been performed largely with panels of monoclonal antibodies that react with murine and human antigens. Molecular genetic techniques, however, are now recognized as being important because the overall structures of cell surface macromolecules can only be studied by these techniques. Structures deduced from nucleotide sequences also provide information about the function of the molecule and isolating the homologues of different species. We have constructed a cDNA library of a macrophage cell line by using a cDNA subtraction technique that is useful for
Correspondence to: Shunsuke Yamamoto, as above Transmitting editor: T Honjo
enrichment of cell type specific cDNA clones (2). Using one of these probes, we characterized the cDNA encoding the murine homologue of human myelomonocytic cell differentiation antgen, CD14 (3). This study describes isolation of another cDNA that is expressed in macrophages and macrophage cell lines and has a sequence encoding a novel transmembrane protein containing a cysteine rich region in the extracellular region and a proline rich cytoplasmic tail significantly similar to that of CD2 (4-8). The MS2-5 cDNA clone (450 bp) was separated from a cDNA library derived from cDNA obtained by subtractive hybridization between cDNA from a macrophage cell line, HINS-B3, and mRNA from a non-macrophage cell line, early HINS line (3). Preliminary experiments using Northern blot analysis showed that this plasmid detected 3.1 and 4.6 kb mRNA from the HINS-B3 cell line. The 3.1 kb species was the smallest and most abundant
Received 29 January 1990, accepted 14 March 1990
Downloaded from http://intimm.oxfordjournals.org/ at University of Michigan on July 3, 2015
Key words: macrophage differentiation antigen, cDNA sequencing, immunoprecipitation
586 A novel membrane antigen, MS2, in monocytic lineage to
1
2
3
H
5
6
7
8
9 10 11 12 13
(a)
(b) 1 2
3
1 5
1 2
3
5
6
7
7.464.402.371.35-
(d)
(C)
12
1 5
1 2
3
of the transcripts, suggesting that the species was a mature transcript of the MS2-5 associated gene. To obtain cDNA clones containing longer inserts, we next screened a Xgt11 cDNA library constructed by using mRNA from mouse peritoneal macrophages (Clontech, Palo Alto, CA, USA) and selected several cDNA clones using an MS2-5 insert, and separated several clones as shown in Fig. 3(a). Using the longest clone, MS2-468, spanning 2.85 kb, Northern blot hybridization using RNA from various cells was performed. The plasmid strongly hybridized with mRNA from mouse resident peritoneal macrophages and several mouse macrophage cell lines in Northern hybridization, although its expression varied among the macrophage cell lines (Fig. 1) and weakly hybridized with mRNA from the mastocytoma cell line, P815. In contrast, other tissues and cell lines showed no expression of the gene. We next examined whether expression of the MS2 gene is regulated by stimulators. As shown in Fig. 2,12-O-tetradecanoyfphorbd-13 acetate (TPA) (5 x 10~7 M) had a Diphasic effect on MS2 gene expression; the expression slightly increased after 15 min, declined after 45 min, and then again increased with time. Lipopolysaccharide (LPS) caused a significant enhancement 3 - 2 4 h after the stimulation. Crude lymphokines [a culture supernatant of spleen cells from a BALB/c mouse stimulated with Con A-Sepharose (Pharmacia-LKB, Uppsala, Sweden) (100 /ig/ml)] induced a late effect. Restmulation with LPS 8 - 72 h after initial stimulation with mouse recombinant interferon 7 (IFN7) induced time-dependent increases in MS2 expression. These results show that expression of the MS2 gene can be upregulated by macrophage stimulators. Several cDNA clones shown in Fig. 3(a) were sequenced in order to construct overall nucleotide sequences of MS2 cDNA (Fig. 3b). They spanned 3036 bp. The first codon, ATG at nucleotide position 18, was embedded in a CCATCATG sequence highly homologous to the consensus for a translation initiation site (CCACCATG) (14). An open reading frame spanning 2478 bp was followed by an in-frame stop codon, TAA at position 2496, and a 538 bp 3' untranslated region containing a
Fig. 2. Transcription of the MS2 gene by treatment of HINS-B3 cells with macrophage stimulators Total RNA (10 ^g) extracted at various times from these cells following stimulation with TPA (Sigma Chemical Co., St Louis, MO) (5 x 10~ 7 M), LPS (List Biological Lab. Inc CA) (1 /tg/ml), lymphokines (10%), and LPS (1 /tg/ml) after stimulation with IFN7 (kindly supplied by Shionogi Co., Ltd, Osaka) (100 U/ml) were electrophoresed in 1.5% formaldehyde gel, blotted on a nylon membrane and hybridized with a ^P-labelled MS2-468 cDNA probe, (a) TPA, lane 1, 0 mm, lane 2, 15 mm; lane 3, 45 min; lane 4, 2 h, lane 5, 8 h; lane 6, 48 h. (b) LPS, lane 1, Omin; lane 2, 30 mm; lane 3, 90 min; lane 4, 3 h; lane 5, 6 h; lane 6, 12 h; lane 7, 24 h; lane 8, 48 h. (c) Lymphokines, lane 1, 0 min; lane 2, 30 min; lane 3, 90 min; lane 4, 3 h; lane 5, 6 h, lane 6, 12 h; lane 7, 24 h (d) LPS stimulation for 3 h after stimulation with IFN7 for the indicated times, lane 1, no stimulation; lane 2, 0 mm, lane 3, 8 h; lane 4, 24 h; lane 5, 48 h; lane 6, 72 h. The lower parts show the same Wots rehybndized with a cDNA probe for /3-actin to demonstrate approximately equal amounts of RNA in each line.
polyadenylation signal (AATAAA). A search through the EMBL/GenBank database revealed no significant homology of the cDNA to known sequences. The cDNA encodes an 826 amino acid polypeptide which contains hydrophobic stretches capable of forming a signal sequence and a transmembrane sequence (Figs 3b and 4). The signal peptjdase normally cleaves at the carboxyl side of residues with small neutral side chains such as glycine, alanine, and senne (15). We therefore assigned the residues between the alanine and valine as a potential cleavage site for a signal peptide, although the boundary had an exceptional structure because a proline residue was found at position - 2 (15). Thus the mature core protein is composed of 812 amino acid residues and has a molecular mass of 88312.88 dalton. A hydropathicity plot (16) predicts a putative transmembrane domain that contains only hydrophobic and neutral amino acid residues (Figs 3b and 4). The domain shows a hydrophobic stretch of 25 amino acids, which is flanked on its carboxyl side by two basic ammo acids (Arg-Lys). Basic sequences on the cytoplasmic side are also found in other transmembrane proteins (17,18) and would warrant an orientation of MS2 protein such that the N-terminus is situated
Downloaded from http://intimm.oxfordjournals.org/ at University of Michigan on July 3, 2015
Rg. 1. Expression of MS2 mRNA in macropbages and macrophage cell lines. Total RNA (30 /ig) was electrophoresed in 1.5% formaldehyde gel, blotted on a nylon membrane, and hybridized with a ^P-labelled MS2-468 cDNA probe. Lanes contained RNA from the following sources 1. HINS-B3; 2, CANS-20 (mouse macrophage cell line) (9-13); 3, RAW264; 4, J774; 5, P388D1; 6, P3/NS1-Ag-1 (NS1); 7, P815, 8, EL4, 9, L929, 10, mouse resident peritoneal macrophages; 11, mouse liver; 12, mouse kidney; 13, mouse brain.
A novel membrane antigen, MS2, in monocytic lineage 587
(a)
B PB Py|FV
5' Okb
B
PvPv
B H
s
1.0
P
,. 3.0
2.0
•HS2-54
•HS2-12
•rS-2-468
MS2-8-1
OTOCTCCAaATCCCATCATOCTTaOCCTCTOOCTOCTCAOCaTCTTATaaACACCAOCA 59 -1 1ES 32 261
OTAOCCCCTOaACCTCCl 110CCCCATffTOAAA(^aTATOAAOTQOTTTGOCCTcdawiccfAOCTGCTiTC«CCCT(^aSteAGCCCfTGC«CTM Y A P a P P L P H Y J Q Y B V Y V P B B L A A B B B B B A L P S CACTOQ
