From the Human malignant cells are targeted by homologous
complement C3b if they express M161Ag, a 43-kDa protein with
C3-activating property. cDNA of M161Ag cloned from human leukemia
cell lines predicted M161Ag as a novel secretory protein comprised of
428 amino acids including 5 amino acids encoded by TGA codons
(Matsumoto M., Takeda, J., Inoue, N., Hara, T., Hatanaka, M.,
Takahashi, K., Nagasawa, S., Akedo, H., and Seya, T. (1997) Nat.
Med. 3, 1266-1270), although the origin of this gene was
obscure. Here we clarified this point through genomic and biochemical
analysis: 1) 5'-UT and genomic sequences represented the prokaryote
promoter and ribosomal binding site; 2) the TGA codons in M161Ag
cDNA were translated not into selenocysteines but into tryptophans;
3) M161Ag anchored onto the membrane secondary to its N-terminal
palmitoylation like prokaryote lipoproteins; 4) genomic and cDNA
clones of M161Ag were highly homologous to Mycoplasma
fermentans gene encoding P48, a monocytic
differentiation/activation factor, recently released in the data base,
although the resultant proteins were different in the amino acid
sequences. Additionally, purified soluble M161Ag efficiently provoked
IL-1 Selective tumor destruction has long been desired for tumor
immunity. Recently we discovered a membrane-associated novel gene product expressed on some malignant human cells/cell lines, but not on
normal cells, in close conjunction with apoptotic stimuli such as Fas
or x-irradiation. This protein, with a molecular mass of 43 kDa and
named M161Ag (1-3), activates homologous complement (C).1 The C-opsonized tumor
cells are rapidly cleared, presumably through the expressed M161Ag.
Its cloned cDNA, however, suggested that M161Ag was a secretory
protein and suprisingly contained 5 amino acids encoded by TGA codons
(3). A possible C-activation characteristic of M161Ag is that once
secreted from the tumor cells, it activates homologous C via the
alternative pathway on the cell membrane, thereby allowing for
homologous C3 targeting (3). Thus, M161Ag appeared to have unique
structural, functional and expression profiles. Yet, information about
the relevant amino acids encoded by TGA codons, genomic organization,
the type of protein anchoring onto membranes, and the regulatory
mechanism of protein expression remain to be settled.
We previously thought that the M161Ag gene was of human origin because
M161Ag was expressed on malignant human cells from patients. However, a
similar DNA sequence of non-human origin, named P48, was released after
the submission of the amino acid sequence of M161Ag (3). The P48
protein was first described as a novel human cytokine inducing the
production of IL-1 Here we demonstrate that M161Ag is a lipoprotein derived from M. fermentans. Furthermore, we found that M161Ag is a potent biological response modifier that provokes IL-10 and IL-12 in addition
to inflammatory cytokines in human monocytes.
Reagents, Cell Lines, and Cells--
Monoclonal antibodies
(mAbs) against M161Ag (M161 (2), MK53 (11)) were produced in our
laboratory and purified on DEAE-Sephacel or Protein G-Sepharose
(Pharmacia, Uppsala, Sweden). Restriction enzymes were purchased from
Takara (Kusatsu, Japan). ELISA kits for TNF- Department of Immunology,
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, tumor necrosis factor
, and IL-6 like P48, and further
IL-10 and IL-12 in human peripheral blood monocytes. Thus, M161Ag
originates from M. fermentans, and latently infected
M. fermentans allows human cells to produce M161Ag. The liberated protein serves as a potent modulator of innate and cellular immune responses via its complement-activating and cytokine-producing activities.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, TNF-
, and IL-6 in human monocytes and then
corrected into a product of Mycoplasma fermentans, a
parasitic prokaryote (5-8). Irrespective of the high homology between
these two genes, there are significant differences in the primary
structures as well as functional profiles of these gene products.
Meanwhile, several papers have been published suggesting that leukemia
(9) and AIDS (10) are frequently associated with parasitic M. fermentans and its gene products.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, IL-1
and IL-6 were
purchased from Perseptive Biosystems (Framingham, MA), and those for
IL-10 and IL-12 were from Endogen (Woburn, MA).
Southern Blotting Analysis--
Human genomic DNA was isolated
from peripheral blood leukocytes, primary cultures of human
fibroblasts, various leukemia cell lines, and spleen using an Iso Quick
nucleic acid extraction kit (ORCA Research Inc., Bothell, WA) or QIAamp
tissue kit (Qiagen Inc., Chatsworth, CA) according to the
manufacturers' directions. For each digest, 10 µg of DNA was
digested with EcoRV, KpnI, BamHI, or
HaeIII and then separated by electrophoresis on a 0.7%
agarose gel. The DNA was transferred onto Hybond N+ nylon
membranes (Amersham, Buckinghamshire, UK) and immobilized using a
Stratalinker UV cross-linker (Stratagene, La Jolla, CA). The blots were
prehybridized for 30 min, hybridized for 2 h at 65 °C in rapid
hybridization buffer (Amersham) with 32P-labeled
full-length M161Ag cDNA, and washed at high stringency (65 °C,
0.2 × SSC, 0.1% SDS). The membranes were exposed to Kodak HyperfilmTM MP for 24 h at 70 °C.
Cloning of Genomic M161Ag Gene-- Genomic DNA from WI38 cells (50 µg) was digested with HaeIII at 37 °C overnight and run on a 0.7% agarose gel. An ~3-kb fragment was recovered from the gel using a QIAEX II kit (Qiagen) and ligated into EcoRV-digested pBluescript II KS+ vector (Stratagene) with T4 ligase. The ligated DNA was transformed into competent MC1061 cells by electroporation using a Gene-Pulser (Bio-Rad). The transformants consisting of 1.21 × 104 colonies were divided into 20 tubes (600 colonies/tube), cultured at 37 °C overnight, and then subjected to PCR screening for the M161Ag gene using specific primers, 5'-TTGAGTCCTATTGCTGCTATTC-3' and 5'-CACCAAATGATGCAACAACTCT-3'. Colonies (5 × 103) from one positive tube were screened for the M161Ag gene by colony hybridization using a 32P-labeled full-length M161Ag cDNA. Positive clones were subjected to sequence analysis.
DNA Sequencing and Computer Analysis-- DNAs were sequenced on both strands using a dideoxy terminator cycle sequencing kit (Applied Biosystems). Homology search was performed on NCBI, and nucleotide/protein analysis was performed with Gene Works, GENETYX, Clustal W in a Macintosh 7200.
RT-PCR and PCR-- Poly(A)+ RNA from M161Ag-positive cell lines were reverse-transcribed using a random primer with RNase H-free reverse transcriptase (Superscript, Life Technologies, Inc.). The full-length M161Ag was amplified using forward 5'-AAGGAGATTATATGAAAAAGTC-3' and reverse 5'-AAGTGTACTTCTCTAGTCAATC-3' primers with exTaq (Takara). The thermocycle conditions were 35 cycles of 94 °C for 30 s, 60 °C for 45 s, and 72 °C for 2 min for denaturation, annealing and extension, respectively. PCR products were cloned into the pCRTMII vector (Invitrogen) and sequenced. PCR reactions with Mycoplasma-specific primers were run according to the manufacturer's directions (Takara) using 1 µg DNA template. PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide.
Protein Isolation-- Purified MK53 was coupled to CNBr-activated Sepharose 4B. M161Ag was purified from P39(+) cell lysates (5 × 109) using column chromatography as described previously with slight modifications (2). After the Q-Sepharose and chromatofocusing columns, fractions containing M161Ag were pooled, and the pH was adjusted to 7.4 and applied to a MK53-Sepharose equilibrated with 10 mM Tris-HCl, 0.14 M NaCl, 0.02% Nonidet P-40, 0.5 mM PMSF, pH 7.4. The column was sequentially washed with starting buffer containing 0.5 M NaCl and then starting buffer without Nonidet P-40. M161Ag was eluted with 0.1 M triethylamine, 0.5 mM PMSF. Prior to amino acid analysis, the eluate was further purified by high performance liquid chromatography (HPLC) using a Phenyl-5PWRP column (4.6 × 75 mm, Tosoh Corp., Tokyo) and a HPLC Cosmosil 5C4-AR-300 column (4.6 × 150 mm, Nakalai tesque, Kyoto). In each step, protein elution was checked by immunoblotting. The final sample gave a single band on 12.5% SDS-PAGE and silver staining.
Secreted M161Ag was purified from conditioned media (CM) of P39(+) cells in the absence of Nonidet P-40. Five liters of CM were concentrated by 50% ammonium sulfate precipitation and dialyzed against 20 mM PBS, 0.5 mM PMSF, pH 6.0, overnight at 4 °C. The sample was applied to an S-Sepharose column equilibrated with the same buffer and eluted with 1 M NaCl in the starting buffer. M161Ag-positive fractions (checked by immunoblotting) were pooled and dialyzed against PBS, 0.5 mM PMSF, pH 7.4. The soluble M161Ag was further purified by the MK53-Sepharose as described in the purification of the membrane-bound form. The buffer was exchanged to Dulbecco's PBS by ultrafiltration (YM-10, Amicon). The purified M161Ag gave a 43-kDa singlet on SDS-PAGE/silver staining, and its concentration was 60 ng/ml as determined by an ELISA established in our laboratory.2Amino Acid Analysis of M161Ag-- The principle of amino acid analysis used in this study was based on the method of Ishida et al. (13). The purified M161Ag was hydrolyzed in 6 M HCl at 110 °C for 24 h in an evacuated sealed tube. To examine Trp content of M161Ag, the sample was also hydrolyzed in 3 M mercaptoethanesulfonic acid at 115 °C for 24 h under evacuated conditions. These hydrolysates were applied to an L8500 amino acid analyzer equipped with an L1050 fluorescence detector (Hitachi, Ltd., Japan) to quantify amino acid derivatives.
Biosynthetic Labeling and Immunoprecipitation-- P39(+) cells (5 × 106) were labeled with 300 µCi of [9,10-3H]palmitic acid in 5 ml of RPMI supplemented with 10% FCS or 10 µCi of [14C]tryptophan in 2.5 ml of Trp-depleted RPMI supplemented with 10% FCS for 24 h at 37 °C. Cells were lysed in lysis buffer (PBS, pH 7.4, 1% Nonidet P-40, 10 mM EDTA, 25 mM iodoacetamide, 2 mM PMSF) for 30 min at room temperature. Lysates were clarified by centrifugation at 10,000 × g for 15 min at 4 °C. The supernatants were precleared with protein G-Sepharose (Amersham Pharmacia Biotech). M161Ag was immunoprecipitated with a mAb against M161Ag (MK53), followed by protein G-Sepharose. Nonimmune mouse IgG was used as a control antibody. Immunoprecipitates were washed and analyzed by SDS-PAGE on 10% gel (3). The gels were fixed, soaked in AmplifyTM (Amersham) for 30 min, dried, and exposed to Fluorograph film for 10 days (3H) or 12 days (14C).
For 75Se labeling, 10 µCi of [75Se]selenite (Research Reactor Center, University of Missouri, Columbia) was added to P39(+) and P39(Determination of Cytokines--
THP-1 cells or monocytes from
individual healthy donors (1 × 106 cells/ml)
were stimulated with LPS (5 µg/ml for THP-1 cells, 10 ng/ml for
monocytes; Escherichia coli, 026:B6, Sigma) and soluble M161Ag (2.4 ng/ml, 6 ng/ml, 12 ng/ml). After 24 h of stimulation, supernatants were removed and cells were lysed by two cycles of freezing/thawing. Cytokine titers in both supernatants and cell lysates
were determined by ELISA. The IL-1 ELISA is highly specific for
mature IL-1
, and the IL-12 ELISA is highly specific for total human
IL-12 (p70 and p40). The cell lines used in this study were free of
Mycoplasma infection.
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RESULTS |
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Southern Analysis-- To clarify the origin of M161Ag, we performed Southern analysis using DNAs extracted from human PBMCs, spleen, primary cultures of fibroblasts, and various human cell lines. Hybridizing bands against M161Ag probe were observed in lanes with M161Ag-positive cell lines (3), for example WI38 DNA (Fig. 1A), but not in lanes with primary cultures of human fibroblasts (Fig. 1A), PBMC, and spleen DNAs (not shown). Fig. 1B shows a Southern blot with HaeIII digests of DNAs from a variety of M161Ag-positive and -negative cell lines. DNA bands appeared in parallel with M161Ag protein expression in the cell lines tested. The size of the HaeIII digests was variable; ~3 kb in Jurkat(+), CEM(+), and WI38, 7.7 kb in P39(+), and 7 kb in K562(+).
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Cloning of Genomic M161Ag Gene-- Because the genome of M161Ag was absent in normal organs and was not uniform in the M161Ag-positive cell lines, it is unlikely to reside definitely in the human chromosomal DNA. Thus, genomic analysis was carried out. Finally, one positive clone (CL1) was obtained, which consisted of a single exon based on the sequence on both strands. CL1 was 3141 bp including the 5' regulatory and coding regions of M161Ag, and had 99% identity with 1621 bp of M161Ag cDNA obtained from the P39(+) cDNA library (Fig. 2A). One base (C (cDNA) to T (CL1)) transition at CL1 1820 bp caused a His to Tyr conversion, and a three-nucleotide (corresponding to Ala) in-frame insertion at CL1 2261 bp resulted in generation of a putative 429-amino acid precursor protein.
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M161Ag Is a Gene Product of M. fermentans-- Two methods were used to determine whether M161Ag is related to a M. fermentans gene product. Firstly, Mycoplasma infection was confirmed by RT-PCR using primers of Mycoplasma genus-specific rRNA. There was a correlation in the results of PCR analysis between M161Ag expression and Mycoplasma infection (Fig. 3).
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M161Ag Is Modified by Palmitate-- Prokaryotic signal peptidase II cleaves precursor polypeptides upstream of a Cys residue to which a lipid moiety is then attached (17). M161Ag carried in its N terminus a four-amino acid motif (AVSC) characteristic of bacterial lipoproteins. To test the lipid modification, P39(+) cells were biosynthetically labeled with [9,10-3H]palmitic acid. As shown in Fig. 4C, palmitic acid was incorporated into M161Ag. Thus, M161Ag is a lipoprotein.
Inflammatory Cytokine-inducing Activity of M161Ag--
M161Ag
shared the identical N-terminal 114 amino acids with P48, a cytokine
(IL-1, TNF-, and IL-6) inducer. This prompted us to test whether
these cytokines were produced in monocytes and THP-1 cells in response
to M161Ag stimulation. As shown in Fig.
5A, purified soluble M161Ag
induced IL-1
production, but not secretion, in THP-1 cells. Minimal
TNF-
and IL-6 were detected in conditioned media. These cytokines
were produced in an M161Ag dose-dependent manner.
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DISCUSSION |
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We initially expected M161Ag to be a human gene product, because its cDNA had a polyadenylation signal followed by the poly(A) tail and the protein was originally detected in bone marrow cells of patients with leukemia undergoing chemotherapy and in those with aplastic anemia.3 Additionally, the 5 TGA codons in the ORF of this protein could be read into selenocysteines if it were a human protein. At that time, no protein or nucleotide sequence similar to M161Ag was found in the data base.
The findings of the present study can be summarized as follows. 1) 5'-UT of cDNA and genomic sequences predicted the presence of the prokaryote promoter and ribosomal binding site (Fig. 2). 2) A poly(A) tail-like sequence as well as an AATAAA polyadenylation signal was conserved not only in the cDNA but also in the genome (Fig. 2). 3) There was a correlation between M161Ag expression and Mycoplasma infection (Fig. 3). 4) Homology searches indicated that the P48 monocyte differentiation/activation factor gene originating from M. fermentans (accession number U70254) was highly homologous to our M161Ag cDNA and genome. 5) The TGA codons encoded Trp but not selenocysteine residues in the purified protein as in Mycoplasma proteins. Therefore, we conclude that M161Ag is a protein of Mycoplasma but not human origin. However, the size variety of genomic Southern analysis remains unexplained. Strain-to-strain difference and/or genomic integration of the M161Ag gene may account for this unusual result.
These points are reminiscent of the suggestion that mitochondria are derived from parasitic bacteria because they have their own genome and proteins. Some of the genomic structure still bear the marks of prokaryotic origin. An intriguing point is that the M161Ag mRNA partly mimics those of eukaryotes, and this may be advantageous for attainment of stable dynamics and steady-state level in the parasitic environment (18).
M161Ag is a multifunctional protein with abilities of C activation and
cytokine induction. P48 of M. fermentans induces the production of IL-1B, TNF-, and IL-6 in human monocytes and
monocytic cell lines (4-8). Because the amino acid sequences were
largely different throughout the C-terminal regions between M161Ag and P48, the IL-1
-, TNF-
-, and IL-6-inducing activity must be mapped within the N-terminal domain conserved between these two proteins. However, M161Ag, but not P48, stimulates monocytes to induce IL-10 and
IL-12 which affect the polarization and development of naive T-helper
cells. Again, M161Ag has C-activating ability which has not been
determined in P48. The stretched sequence of M161Ag including 5 Trp
residues may play a role in the latter functions.
M161Ag is a putative membrane protein with a lipid anchor since M161Ag was palmitoylated (Fig. 4C). The lipid moiety on bacterial lipoproteins strongly potentiates the humoral as well as the cellular immune responses (19). Indeed, MALP-2 (a recently isolated M. fermentans-derived 2-kDa lipopeptide, macrophage-activating lipopeptide-2), carries 1 mol of C16:0 and an additional mole of a mixture of C18:0 and C18:1 fatty acids per lipopeptide molecule (20) and acts as an inducer of NO at picomolar concentrations (20). Surprisingly, the amino acid sequence of this lipopeptide was entirely consistent with the N-terminal 14-amino acid sequences of M161Ag and P48. It is likely that soluble forms of this M. fermentans gene product confer another function on macrophages besides C-activation and cytokine production. However, the mechanisms whereby soluble M161Ag is generated from the membrane-associated forms to express its functions still remain unknown.
M. fermentans is a mycoplasma species capable of infecting humans and has been suspected of serving as a cofactor of AIDS development (21, 22). Several groups (10, 12) speculated that M. fermentans facilitates depletion of T cells or immature myelomonocytic cells, favoring the progression of functional immunodeficiency in AIDS. Yet, the products of M. fermentans responsible for immune modulation, polyclonal B or T cell activation, cytokine production, and cytocidal effect (23-26) have not been identified. A possible interpretation is that M161Ag and/or P48 is a molecule relevant to AIDS progression. C3-activating function of M161Ag is also consistent with the observation that C3 deposition is induced on CD4+ T cells of HIV-infected individuals (27, 28). Coinfection of M. fermentans with HIV may actually support progression to AIDS in latent patients via the functions of M161Ag.
These results also explain why M161Ag-positive myeloid cell lines were
obtained after most of the cells died. Like human myeloid cell lines
P39(+) and K562(+), infection with M. fermentans may cause
cell death in affected cells, and some that survive are persistently
infected with M. fermentans and are M161Ag-positive. The
parasitic growth of M. fermentans may be regulated by
signals related to cell death, since M161Ag synthesis is induced by
x-irradiation and Fas stimulation and up-regulated with TNF-
(3).
Our sequential studies showed that the M. fermentans gene product M161Ag had dual functions: complement activation and cytokine induction. Once M161Ag is expressed because of latent infection of fermentans, it converts self cells to non-self and elicits innate immune responses via activation of C3/C5 and monocytes. However, the roles of autologous C3 activation and deposition on host cells and Th1-activating cytokine production in the acquired immune responses are still poorly understood. Furthermore, parasitic infection of M. fermentans has been associated with oncogenic properties (29, 30). These issues will be further clarified using recombinant M161Ag and deletion mutants, and such studies are currently in progress in our laboratory.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. K. Toyoshima and H. Akedo (Osaka Medical Center) for support of this work and Drs. J. Takeda and N. Inoue (Osaka University), K. Takahashi (Hokkaido University), and M. Nomura and M. Hatanaka (Osaka Medical Center) for invaluable discussions. Thanks are also due to Dr. N. A. Begum for the critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported in part by grant from the Ministry of Public Welfare.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Immunology, Osaka Medical Center for Cancer and Cardiovascular
Diseases, Higashinari-ku, Osaka 537, Japan. Fax: 81-6-981-3000.
1 The abbreviations used are: C, complement; CM, conditioned media; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; PMN, polymorphonuclear cell; PMSF, phenylmethylsulfonyl fluoride; IL, interleukin; TNF, tumor necrosis factor; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; FCS, fetal calf serum; RT-PCR, reverse transcription-polymerase chain reaction; kb, kilobase(s); bp, base pair(s); HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
2 S. Kikkawa, M. Matsumoto, M. Kurita, M. Nishiguchi, and T. Seya, manuscript in preparation.
3 M. Matsumoto and T. Seya, unpublished data.
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REFERENCES |
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