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Department of Immunology, Saga Medical School, Saga, Japan; and
Research and Development Division, Yokohama Research Center, Mitsubishi Chemical Corporation, Kanagawa, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RP105 is another LRR molecule that is expressed on B lymphocytes. It was first identified by a mAb that protects spleen B cells from irradiation-induced apoptosis (7). The RP105 molecule, when cross-linked by an Ab, transmits an activation signal that leads to massive B cell proliferation as well as resistance against apoptosis. Interestingly, these activated and proliferating B cells arrest their growth and undergo apoptosis upon a signal from the AgR (8). RP105 may be involved in the regulation of B cell growth and Ag-induced death. RP105 is a type I transmembrane protein of 105 kDa with LRR on the extracellular domain (9). In the course of biochemical studies, we conducted transfection of the RP105 cDNA, but RP105 was always poorly expressed on the cell surface. The expression of RP105 by transient transfection is significantly lower than that seen with other molecules. We speculated that the LRRs of RP105 might be associated with another molecule that is required for efficient expression. In the present study, we sought such a molecule and identified it as a mouse homologue of chicken MD-1. Chicken MD-1 was first isolated as a v-myb-regulated gene from chicken myeloblasts that had been transformed with the avian myeloleukemia virus. Burk et al. (10) used the estrogen-dependent v-myb expression system to show that MD-1 expression rapidly increases after v-myb induction. It was predicted that MD-1 was a secretory molecule, because it has only one hydrophobic stretch at the amino-terminus. It does not have any homology to other molecules, and no functional study has been reported since. This report has rediscovered MD-1 as a B cell surface molecule. We also describe a role of MD-1 in cell surface expression of RP105.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The B cell lines M12.4, CH12, A20, and WEHI231 were obtained from Drs. D. Mckearn (Mayo Clinic, Rochester, MN), R. J. Hodes (National Cancer Institute, Bethesda, MD), T. Kurosaki (Kansai Medical School, Osaka, Japan), and N. Sakaguchi (Kumamoto University, Kumamoto, Japan), respectively. A human kidney epithelial cell line, 293T, was provided by Dr. T. Hirano (Osaka University, Osaka, Japan). BaRP30 was established previously by transfecting the IL-3-dependent line Ba/F3 with an expression vector encoding RP105 (9). BaRP30 was further transfected by electroporation with another expression vector coding for MD-1flag. Cells that express MD-1flag were selected by cell surface staining of the flag epitope and referred to as BaRPMD. The rat anti-mouse RP105 Ab RP/14 was established in our laboratory (7). The mouse anti-flag mAb M2 was purchased from Eastman Kodak (New Haven, CT).
Microsequencing of N-terminal aa
The purified anti-RP105 mAb (10 mg RP/14) was coupled to the N- hydroxy-succinidyl-activated HiTrap column according to the manufacturer’s instructions (Pharmacia Biotech Japan, Tokyo, Japan). Up to 2 x 1010 cells from the B cell line M12.4 were collected. Lysis buffer was added to create the cell lysate at 5 x 107 cells/ml. Lysis buffer consisted of 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 50 mM iodoacetamide, 1 mM PMSF, 10 µg/ml soybean trypsin inhibitor (Wako Pure Chemical Industries, Osaka, Japan), and 5 mM EDTA. After a 1-h incubation on ice, lysate was cleared by centrifugation and loaded onto an anti-RP105-coupled column. The column was washed with buffer containing 20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 0.02% sodium azide. Bound proteins were eluted with buffer containing 50 mM glycine/HCl (pH 2.6) and 0.1% Triton X-100. Each fraction was neutralized immediately with 1 M Tris/HCl (pH 8.0) and subjected to SDS-PAGE analysis. Peak fractions were combined, dialyzed against 0.1% Triton X-100 and 10 mM NaCl, and lyophilized. Purified proteins were resolved with SDS-PAGE, blotted onto a Immobilon P membrane (Nihon Millipore, Tokyo, Japan), and visualized with Coomassie brilliant blue R-250 (Nippon Bio-Rad Laboratories, Kanagawa, Japan). The target proteins were excised and loaded onto a protein sequencer (Applied Biosystems, Foster City, CA).
cDNA cloning
A cDNA library was prepared previously from a B cell lymphoma
(BCL1) (9). Degenerate primers were designed according to the
N-terminal aa sequence (double underlined in Fig. 2
A). Two
primers were prepared for each direction to reduce degeneracy. The
sequences of primers were as follows: forward primer 1,
GA(CT)CA(CT)GG(ACGT)TC(ACGT)GA(AG)AA(CT)GG(ACGT)TGG; forward primer
2: GA(CT)CA(CT)GG(ACGT)AG(CT)GA(AG)AA(CT)GG(ACGT)TG; reverse primer
1: TG(AG)TA(ACGT)AC(ACGT)AC(CT)TC(ACGT)AG(ACGT)CC(ACGT)CC;
and reverse primer 2:
TG(AG)TA(ACGT)AC(ACGT)AC(CT)TC(CT)AA(ACGT)CC(ACGT)CC. Purified DNA
from the BCL1 cDNA library was used as a template, and amplification
was conducted as follows: a cycle of 94°C for 2 min, 55°C for 2
min, and 72°C for 30 s followed by 29 cycles of 94°C for
45 s, 55°C for 2 min, and 72°C for 30 s. The final step
at 72°C was extended to 10 min. Amplified products were subjected to
PAGE. A fragment of expected size (71 bp) was recovered using the QIAEX
II Gel Extraction kit (Qiagen GmbH, Hilden, Germany) and was cloned
into a plasmid by T-A cloning. Cloned fragments were sequenced,
and those encoding the obtained aa sequence were used as a probe to
screen the cDNA library. Colony hybridization was performed as
described previously (9). Sequencing was conducted with an
ALFexpress DNA sequencer (Pharmacia) and a Thermo Sequenase
cycle sequencing kit (Amersham Japan, Tokyo, Japan).
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Total RNA was extracted from various tissues with Isogen (Nippon Gene, Toyama, Japan) and subjected to agarose electrophoresis (20 µg/lane). After transfer to a nylon membrane (Hybond-N+, Amersham), RNA was hybridized to a probe that had been labeled by the random priming of a cDNA clone encoding mouse MD-1. The hybridization buffer consisted of 10% dextran sulfate (Pharmacia), 1 M NaCl, 1% SDS, and 50 mM Tris/HCl (pH 7.5). Hybridization was conducted at 65°C for 20 h. Washing was conducted in 2 x SSC and 0.1% SDS at 65°C. Radioactive signals were visualized using an image-analyzer BAS2000 (Fuji Film, Tokyo, Japan). The same membrane was reprobed for glyceraldehyde-3-phosphate dehydrogenase (11).
Cell surface biotinylation and immunoprecipitation
Cell surface biotinylation and immunoprecipitation was conducted as described previously (12). Briefly, cells were washed in HBSS and adjusted to 5 x 107/ml in saline containing 100 mM HEPES (pH 8.0). Sulfosuccinimidobiotin (Pierce, Rockford, IL) was added to the cell suspension to make a final concentration at 0.5 mg/ml. After a 30-min incubation at room temperature with occasional shaking, cells were washed in HBSS and lysed in lysis buffer (see above). After a 30-min incubation on ice, lysate was centrifuged, and nuclei were removed. We used either anti-RP105-coupled HiTrap beads or anti-flag mAb (M2)-coupled Sepharose 4B (Eastman Kodak); mouse IgG1-coupled HiTrap beads were used as control beads. The beads were added to cell lysate and rotated for 2 h at 4°C. Beads were washed in lysis buffer, and bound proteins were subjected to SDS-PAGE and Western blotting. Biotinylated proteins were detected with streptavidin-peroxidase (Amersham) and SuperSignal chemiluminescent substrate (Pierce).
Transient transfection
The human kidney cell line 293T was plated onto a 24-well plate at 1.5 x 105/well on the day before transfection. The calcium phosphate/DNA coprecipitate was prepared as follows. DNA (1 µg) was diluted in 100 µl of deionized water, and 2 M of calcium chloride (14 µl) was added to DNA. An equal amount (114 µl) of 2 x HEPES-buffered saline (280 mM NaCl, 50 mM HEPES, and 1.5 mM sodium phosphate (pH 7.05)) was added slowly in a dropwise manner. After 30-min incubation at room temperature, coprecipitates were added to 293T cells. Medium was changed on the following day and then cultured for 1 to 2 days. Cells were harvested with PBS containing 1 mM EDTA and were used for further analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In an attempt to search for RP105-associated molecules, we
conducted immunoprecipitation studies. Because the extracellular domain
of RP105 has LRRs and the intracellular domain is very short, we
expected that the associated molecule would be a cell surface molecule.
Therefore, cell surface biotinylation was used for labeling proteins.
We used several B cell lymphomas that express RP105. After extracting
membrane proteins with buffer containing 1% Triton X-100, RP105 was
precipitated with an anti-RP105 Ab. Two extra signals of 
22 and
25 kDa (glycoprotein (gp)22/25) were apparent in precipitates
from all five B cell lines and normal spleen cells (Fig. 1, arrows). Interestingly, the amount of
gp22/25 was variable with each B cell line, and gp22 was dominant over
gp25 in BCL1, WEHI231, and A20. Both signals migrated faster
under nonreduced conditions, suggesting an intramolecular disulfide
bond (data not shown).
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We subsequently proceeded to purify the 22/25-kDa molecules and
determine their N-terminal aa sequence (see Materials and
Methods). A total of 24 residues were successfully determined from
the 25-kDa molecule (Fig. 2
A,
double underline). The same sequence was obtained from the 22-kDa
molecule. Because the sequence did not show any identity to previously
identified sequences, we moved on to cDNA cloning (see Materials
and Methods). A DNA fragment encoding the N-terminal aa sequence
was obtained by PCR using degenerate primers. A cDNA library was
screened using the DNA fragment as a probe. A total of 18 clones were
obtained from 2 x 105 colonies. After the size
determination and restriction mapping of cDNA inserts, we focused on a
1-kb cDNA clone. An entire sequence of the clone was determined and is
shown in Figure 2A. The longest open reading frame begins
with a codon at position 95–97 and ends with a stop codon at 578–580.
This sequence encodes 162 aa, and the experimentally determined
sequence starts at the aa-1 (double underlined in Fig. 2A).
Therefore, the hydrophobic stretch from the aa-19 to the aa-1 is a
signal sequence (underlined in Fig. 2A). Poly(A) signals
locate in a 3' untranslated region. Mature peptide consists of 143 aa
and a deduced molecular mass of 17.8 kDa. Asparagine residues at
positions 77 and 137 are canonical N-glycosylation sites
(represented by an asterisk in Fig. 2A). The databases were
searched again using the entire aa sequence, and chicken MD-1 (10), a
v-myb-regulated gene, showed significant homology (Fig. 2B). Chicken MD-1 cDNA is 1 kb and codes for 160 aa. With
5 gaps between the aa 19 and aa 20 in chicken MD-1, 64 aa are identical
(40% identity). We conclude that the cDNA encodes a mouse homologue of
chicken MD-1. Hereafter, it is referred to as mouse MD-1. Northern
hybridization was conducted to visualize tissue distribution (Fig. 3). The MD-1 transcript is 1 kb in
size and is most abundant in the spleen. It was also detectable in
other tissues such as the liver, brain, thymus, and kidney. MD-1 may
have an additional role in nonimmune systems. Most B cell lines
expressed the MD-1 transcript, but an IL-3-dependent line, Ba/F3, did
not (data not shown).
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The cDNA clone does not have the second hydrophobic stretch,
suggesting that MD-1 is a secretory molecule. We created a construct
that tagged MD-1 with the flag epitope at the C terminus (MD-1flag).
This construct was transfected into 293T cells. As expected, MD-1flag
was not expressed on 293T cells but was demonstrable in the supernatant
(Fig. 4, A1 and B).
A broad signal was apparent that was 22 to 35 kDa. However, the
flag epitope appeared on the cell surface when RP105 was coexpressed
(Fig. 4A2). The expression of MD-1flag showed a linear
correlation with that of RP105 (Fig. 4C). An irrelevant cDNA
did not confer MD-1 expression on the cell surface (data not shown).
Consequently, MD-1 is a secreted protein but can be present on the cell
surface in the presence of RP105.
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To prove that MD-1 binds to RP105, coprecipitation studies were
conducted using the BaRPMD cell line expressing RP105 and MD-1flag (see
below). After cell surface biotinylation, immunoprecipitation was
conducted with either anti-RP105 or anti-flag mAb (Fig. 5).
Precipitated proteins were probed with either avidin (for cell surface
molecules) or anti-flag Ab (for MD-1flag). The anti-RP105 mAb
precipitated RP105 and additional molecules that were 25 to 30 kDa
(lane 2). These molecules are MD-1flag, since
similar signals of 28 and 25 kDa were apparent by probing with
anti-flag mAb (lane 5). Signals in lane
2 are stronger than those in lane 5, since lanes
1 through 3 were exposed to film longer than
lanes 4 through 6 to show MD-1 signals in
lane 3. When immunoprecipitation was conducted with
anti-flag mAb, signals were weaker than with anti-RP105 mAb.
Anti-flag mAb may have difficulty recognizing the flag epitope on MD-1
when it is associated with RP105. Although weaker, MD-1 signals were
apparent in lane 3 as well as lane 6. RP105 was
also visible when probed with avidin (lane 3). In
addition, we noticed another signal just below the upper MD-1 signal in
lane 3; it was not detected by probing with anti-flag
mAb (lane 6). A similar signal was observed in
lane 2 with shorter exposure. Further study is underway to
identify this signal. Immunoprecipitation with control IgG (mouse IgG1)
had no signal (lanes 1 and 4). From these
results, we conclude that MD-1 is associated with RP105.
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With an aim to understand a role of MD-1 in RP105 expression and
function, we attempted to differentiate the RP105/MD-1 complex from the
RP105 monomer. The cell line 293T was transiently transfected
either with RP105 alone or with RP105 and MD-1 and then stained with
the anti-RP105 mAb. Three independent experiments were conducted,
and the percentages of RP105-positive cells are shown in Table I. In a control transfection in which
RP105 was expressed with an irrelevant molecule, Ly-6A/E, the mean
percentage of RP105-positive cells was less than half of the
transfection of RP105 alone. On the other hand, the transfection of
RP105 and MD-1 resulted in more than twice as many RP105-positive cells
when compared with the transfection of RP105 alone. Moreover, we
established and used stable transfectants. The BaRP30 cell line was
previously established by transfecting an expression vector encoding
RP105 into the IL-3-dependent line Ba/F3 (9). BaRP30 was further
transfected with another expression vector encoding MD-1. Therefore,
the newly established line, BaRPMD, differs from BaRP30 in MD-1
expression only. BaRP30 expresses RP105 alone, while BaRPMD expresses
RP105 and MD-1. Indeed, MD-1 is present on BaRPMD but not on BaRP30
(Fig. 6, A and C). The introduction of
MD-1 conferred approximately a sevenfold increase in the mean
fluorescence intensity of RP105 (Fig. 6B: 75.4; Fig. 6D: 519.0). The binding of anti-RP105 mAb RP/14 to
BaRP30 is as saturable as it is to BaRPMD, and a sufficient amount of
anti-RP105 Ab was used in these experiments. Therefore, the
difference in fluorescence intensity stems from the increased
expression of RP105 molecules. We have established eight other lines by
transfecting BaRP30 with MD-1flag. All of these lines showed an
increased expression of RP105 (data not shown). Taken together with the
results presented in Table I, these results lead to the conclusion that
MD-1 positively regulates the cell surface expression of RP105 (see
Discussion).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). A cDNA clone for gp22/25
was isolated and found to be a mouse homologue of chicken MD-1 (10).
The physical association of MD-1 and RP105 was confirmed by the
following results: 1) MD-1 is a secretory molecule but is tethered to
the cell surface in the presence of RP105. The expression of MD-1 shows
a linear correlation with that of RP105. 2) MD-1 was coprecipitated in
combination with RP105 (Fig. 5).
The Ba/F3 cell line does not express the MD-1 transcript (data not
shown), but RP105 was able to come out by stable transfection (Ref. 9
and Fig. 6). Therefore, MD-1 is
dispensable for RP105 expression. However, MD-1 has an effect on RP105
expression. The transient transfection of RP105 and MD-1 resulted in
about twice a many RP105-positive cells compared with the transfection
of RP105 alone (Table I). The stable transfection of MD-1 conferred
approximately a sevenfold increase in cell surface RP105 on a cell line
that expresses RP105 alone. Moreover, we also found that human MD-1
plays a similar role in the expression of human RP105 (Miura, Y., R.
Shimazu, K. Miyake, S. Akashi, H. Ogata, Y. Yamashita, Y. Narisawa, and
M. Kimoto. 1998. RP105 is associated with MD-1, and transmits an
activation signal in human B cells. Blood In press).
Considering the physical association, the RP105/MD-1 complex would be
favored over the RP105 monomer for cell surface expression. MD-1 seems
to be requisite for efficient expression of RP105. There are several
other molecules that require an additional, physically associated
subunit for proper expression on the cell surface. A heavy chain of the
class I MHC complex is associated with ß2-microglobulin
(ß2m). ß2m is similar to MD-1 in that it
does not have a transmembrane portion; both are either secreted or
attached to another molecule on the cell surface. Some class I MHC
heavy chains such as H-2Db are able to reach the cell
surface without ß2m. However, those free heavy chains are
not stable on the cell surface and during intracellular transport, most
likely due to an inappropriate conformation (13). This may also be the
case with RP105. RP105 may take a more stable conformation in the
presence of MD-1. We recently obtained an anti-RP105 mAb. The mAb
recognized the RP105/MD-1 complex but not the RP105 monomer (our
unpublished observations). Therefore, the RP105 monomer would be
different from the RP105/MD-1 complex in its conformation. MD-1 is
likely to have an important role in the proper conformation and
efficient expression of RP105.
Two distinct bands of 22 and 25 kDa were coprecipitated with RP105.
These two species behaved similarly during SDS-PAGE under either
reduced or nonreduced conditions and have the same aa sequence at the N
termini. They are probably the same protein with a distinct
posttranslational modification. Indeed, we obtained two distinct
signals of MD-1 by transfecting an MD-1 cDNA (Fig. 5). Glycosylation
would be one of the most probable causes of the size difference. Gp25
would have heavier glycosylation than gp22.
RP105 belongs to the LRR superfamily, in which some members are implicated in linking innate and adaptive immunity (14). CD14 is the monocyte/macrophage LPSR and activates these monocytes/macrophages in response to LPS. Activated macrophages secrete cytokines that then act on lymphocytes. The Toll receptor protects Drosophila from fungal infection (5). A human homologue of the Toll receptor was recently isolated (6). It is expressed on lymphocytes as well as on monocytes/macrophages and delivers an activation signal. These LRR molecules are activated in innate immunity and potentiate adaptive immunity (14). RP105 is another LRR molecule that is specifically expressed on B lymphocytes. Given that RP105 has a function that is similar to other LRR proteins, it may help B cells to communicate with and respond to innate immunity. In this regard, a ligand of RP105 could be a product of pathogens, as is the case with tomato genes cf-2 and cf-9. In seeking a ligand, MD-1 must be taken into consideration. It may regulate the ligand binding of RP105 by modulating its conformation or even by providing a ligand-binding site. Our attempts to search for a ligand have been conducted using a fusion protein that contains the extracellular domain of RP105. However, two new probes must be prepared (fusion proteins of MD-1 or RP105/MD-1). The preparation of new fusion proteins is currently underway; such proteins will be used in our continuous effort to search for a ligand of RP105 in lymphocytes, monocytes/macrophages, and pathogens.
Previously, we have proposed two subdomains in the LRRs of RP105 (15).
There is a cluster of nonhomologous repeats (from 7–10) in the middle
of the 22 LRRs. This cluster would divide the RP105 LRRs into two
subdomains, an N-terminal subdomain and a membrane-proximal subdomain.
Preliminary studies localized the MD-1-binding site at the N-terminal
LRRs (our unpublished observations). RP105 may allow another
molecule to bind to membrane-proximal LRRs. The cytoplasmic region of
RP105 consists of only 11 aa. An associated signal transducer is
expected to transmit an activation signal. Such a signaling molecule
might bind to membrane-proximal LRRs. Other LRR molecules in the immune
system have given us an idea regarding signaling in RP105. The Toll
receptor has a cytoplasmic portion that is similar to the IL-1R and
activates the NF-
B pathway in both Drosophila and humans (6, 16, 17). Also, plant resistance genes have LRRs and activate the NF-B
pathway (reviewed in 4 . This connection of LRR molecules and the
NF-B pathway seems to be true of RP105, because B cell activation
with RP105 ligation is severely affected in c-rel knockout
mice (18). Moreover, we confirmed IB
degradation in response to
RP105 cross-linking (our unpublished observations). Thus, a
signal through RP105 is likely to activate the NF-B pathway, and a
signal transducer, if any, would link RP105 with the NF-B pathway.
In conclusion, the present study determined that MD-1 is an RP105-associated molecule. Its association changes the conformation of RP105 and facilitates its cell surface expression. This finding should accelerate our search for a ligand and a signaling molecule of RP105.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kensuke Miyake, Department of Immunology, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan. E-mail address:
3 Abbreviations used in this paper: LRR, leucine-rich repeat; ß2m, ß2-microglobulin; gp, glycoprotein.
Received for publication January 16, 1998. Accepted for publication March 26, 1998.
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R. M. Gorczynski, Z. Chen, D. A. Clark, J. Hu, G. Yu, X. Li, W. Tsang, and S. Hadidi Regulation of Gene Expression of Murine MD-1 Regulates Subsequent T Cell Activation and Cytokine Production J. Immunol., August 15, 2000; 165(4): 1925 - 1932. [Abstract] [Full Text] [PDF]
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 H. Ogata, I-h. Su, K. Miyake, Y. Nagai, S. Akashi, I. Mecklenbrauker, K. Rajewsky, M. Kimoto, and A. Tarakhovsky The Toll-like Receptor Protein Rp105 Regulates Lipopolysaccharide Signaling in B Cells J. Exp. Med., July 3, 2000; 192(1): 23 - 30. [Abstract] [Full Text] [PDF]
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R. Shimazu, S. Akashi, H. Ogata, Y. Nagai, K. Fukudome, K. Miyake, and M. Kimoto MD-2, a Molecule that Confers Lipopolysaccharide Responsiveness on Toll-like Receptor 4 J. Exp. Med., June 7, 1999; 189(11): 1777 - 1782. [Abstract] [Full Text] [PDF]
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Y. Miura, R. Shimazu, K. Miyake, S. Akashi, H. Ogata, Y. Yamashita, Y. Narisawa, and M. Kimoto RP105 Is Associated With MD-1 and Transmits an Activation Signal in Human B Cells Blood, October 15, 1998; 92(8): 2815 - 2822. [Abstract] [Full Text] [PDF]
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J. da Silva Correia, K. Soldau, U. Christen, P. S. Tobias, and R. J. Ulevitch Lipopolysaccharide Is in Close Proximity to Each of the Proteins in Its Membrane Receptor Complex. TRANSFER FROM CD14 TO TLR4 AND MD-2 J. Biol. Chem., June 8, 2001; 276(24): 21129 - 21135. [Abstract] [Full Text] [PDF]
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 A. Visintin, A. Mazzoni, J. A. Spitzer, and D. M. Segal Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4 PNAS, October 9, 2001; 98(21): 12156 - 12161. [Abstract] [Full Text] [PDF]
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