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Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2940-2950
Molecular Identification and Functional Characterization of a Novel
Protein That Mediates the Attachment of Erythroblasts to
Macrophages
By
Manjit Hanspal,
Yva Smockova, and
Quang Uong
From the Department of Biomedical Research, St Elizabeth's Medical
Center, Tufts University School of Medicine, Boston, MA.
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ABSTRACT |
We have previously identified a novel protein that mediates the
attachment of erythroblasts to macrophages in vitro. This attachment
promotes terminal maturation and enucleation of erythroblasts (Hanspal
and Hanspal, Blood 84:3494, 1994). This protein is referred to
here as Emp for erythroblast macrophage
protein. Two immunologically related isoforms of Emp with
apparent molecular weights of 33 kD and 36 kD were detected in
macrophage membranes. The complete amino acid sequence of the larger
isoform of Emp was deduced from the nucleotide sequence of a
full-length 2.0-kb cDNA that was isolated from a human macrophage cDNA
library using affinity-purified anti-Emp antibodies. Of the 2,005 bp,
1,185 bp encode for 395 amino acids representing 43 kD (the sodium
dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]
molecular mass is 36 kD). Northern blot analysis of human macrophage
poly(A) RNA detected a message for Emp of 2.1 kb. The deduced amino
acid sequence contains a putative transmembrane domain near the
N-terminus. To investigate the structure/function relationships of Emp,
recombinant fusion proteins of full-length and truncated Emp were
produced in bacteria, COS-7, and HeLa cells. Cell binding assays showed
that the N-terminus is exposed on the cell surface. The recombinant Emp
functions as a cell attachment molecule when expressed in heterologous
cells. Furthermore, we showed that the demise of erythroblasts in the absence of Emp-mediated erythroblast-macrophage association is accompanied by apoptosis. We postulate that Emp-mediated contact between erythroblasts and macrophages promotes terminal maturation of
erythroid cells by suppressing apoptosis.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE ASSOCIATION OF erythroblasts with
macrophages plays a central role in the human bone marrow, where
erythropoiesis occurs in distinct anatomic units called erythroblastic
islands. These islands consist of a central macrophage surrounded by a
ring of developing erythroblasts.1-4 Although
erythroblastic islands have been well characterized morphologically,
virtually nothing is known about the structural and functional basis of
cellular contacts within erythroblastic islands. We have previously
shown that physical contact between erythroblasts and macrophages
promotes the terminal maturation of erythroblasts, leading to their
enucleation in vitro. Importantly, we identified a novel protein now
termed Emp (erythroblast macrophage
protein) that mediates the association of erythroblasts with
macrophages.5
The molecular basis of erythroblast-macrophage interactions is poorly
understood. In addition to the Emp-mediated association noted above,
very late activation antigen-4 (VLA-4; or  4 1
integrin)-vascular cell adhesion molecule-1 (VCAM-1) has
been reported to mediate the contact of erythroblasts with
macrophages.6 This was evidenced by the finding that
central macrophages in erythroblastic islands isolated from anemic
mouse spleens express VCAM-1, whereas the surrounding erythroblasts
express 41 integrin. Moreover, monoclonal antibodies against 4
integrin and VCAM-1 disrupted erythroblastic islands, suggesting that
these molecules play an important role in erythroblast-macrophage
interactions. Whereas VCAM-1 is expressed by central macrophages of
erythroblastic islands but not by other macrophages, such as those of
peritoneal origin,6 VLA-4 is distributed on a variety of
hematopoietic cells, including stem cells, lymphocytes, erythroblasts,
monocytes, and immature granulocytes.7-9 Thus, if VLA-4 and
VCAM-1 were the only molecules involved in erythroblast-macrophage
interactions, several types of hematopoietic cell islands could be
formed. However, in vivo, only erythroid cells are found in association
with bone marrow macrophages. This observation suggests that other
molecules, such as Emp, may also be required for the formation of
erythroblastic islands in vivo.
In contrast to the limited understanding of erythroblast-macrophage
cell interaction, there is considerable data on the adhesion of
hematopoietic progenitor cells to bone marrow stromal cells. Such
adhesive interactions promote the localization of hematopoietic progenitor cells to the bone marrow and support their proliferation and
differentiation.10,11 In the bone marrow, the cell-cell and
cell-extracellular matrix (ECM) interactions involve multiple adhesive
molecules. Cell-cell interactions among hematopoietic progenitors in
the bone marrow mainly involve cell adhesion molecules of the Ig
superfamily. However, recently, a member of the cadherin family of
cell-cell adhesion receptors was also shown to be involved in the
hematopoietic system. E-cadherin, which is mainly expressed by cells of
the epithelial origin, was detected in human erythroblasts and was
shown to be involved in the differentiation of erythroid cells.12 The interactions of hematopoietic progenitor cells with ECM, on the other hand, are largely mediated by integrins, whose
expression is developmentally regulated.13
We have previously shown that the Emp-mediated association of
macrophages with erythroblasts promotes terminal maturation of
erythroblasts followed by their enucleation.5 We report here the complete amino acid sequence of the larger isoform of Emp
translated from the nucleotide sequence of human macrophage cDNA. The
apparent molecular weight, cellular localization, and binding
specificities distinguish Emp from other known adhesive molecules
present in hematopoietic tissues. Also, we show that Emp-mediated
attachment of erythroblasts to macrophages prevents apoptosis of
erythroblasts. Based on this observation, we propose that, in the
absence of this attachment, apoptosis contributes to the inability of
erythroid cells to fully mature. The availability of the primary
structure of Emp will permit further characterization of its
physiological functions in both erythroid and nonerythroid cells.
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MATERIALS AND METHODS |
Production of anti-Emp antibodies.
Polyclonal antibodies were raised against gel-purified Emp isolated
from human macrophage membranes. The antibodies were affinity-purified on nitrocellulose membrane containing immobilized Emp, as described by
Olmsted,14 and were tested against macrophage membranes
separated by sodium dodecyl sulfate (SDS) gels containing 18%
polyacrylamide by Western blotting. The immunoreactive bands were
detected by an enhanced chemiluminescence (ECL) system (Amersham,
Arlington Heights, IL).
Cloning of Emp cDNA.
For immunoscreening, a cDNA library constructed from the human
monocytic cell line, U937, was used. U937 cells express high levels of
Emp as tested by Western blotting using affinity-purified anti-Emp
antibodies (data not shown). A U937 cDNA library made in the expression
vector  gt11 (Clonetech, Palo Alto, CA) was used to
infect overnight cultures of Escherichia coli strain Y1090 at a
density of approximately 5 × 104 plaque-forming
units/180-mm diameter plate. The infected cells, after appropriate
dilutions, were then placed on 1.5% LB agar plates with the top 0.7%
agar and incubated for 3.5 hours at 42°C. Protein expression was
induced by overlaying the plate with a nitrocellulose membrane that had
been saturated with 10 mmol/L isopropyl -D-thiogalactoside (IPTG).
Incubation was continued for another 3.5 hours at 37°C.
Nitrocellulose membranes were blocked with 5% nonfat dried milk in
phosphate-buffered saline (PBS) containing 3 mmol/L sodium azide for 1 hour. Incubation with affinity purified anti-Emp IgG prepared from
rabbit antiserum was performed for 1 hour. This was followed by
incubation with [125I] protein A. Positive plaques were
visualized by autoradiography. Positive recombinant phage plaques were
then purified to 100% homogeneity by three cycles of rescreening, and
the cDNA inserts were subcloned into pGEM7Z (Promega, Madison,
WI) for further analysis.
A 1.6-kb positively reacting partial cDNA clone was obtained by
immunoscreening. This clone covered almost the entire coding region,
with the exception of the very amino terminus of Emp. The amino
terminus was completed by 5 RACE (rapid amplification of cDNA
ends) using the Marathon cDNA amplification method (Clonetech). First-strand synthesis of 1.0 µg of U937 Poly(A) RNA was
performed using a modified lock-docking oligo (dT) primer that consists of two degenerate nucleotide positions at the 3
end.15 After second-strand synthesis, the cDNA pool was
blunt-end ligated to the cDNA adaptors. The first polymerase chain
reaction (PCR) was performed with a 27-mer sense primer (AP1) specific
for the adaptor and an Emp-specific antisense primer
(5-TCCCAGCTGGTG-TAGTCGGTAGTT-3 nucleotides 744 to 721;
see Fig 3). The second round of PCR was performed with a nested 23-mer
sense primer (AP2) and an Emp-specific antisense primer
(5-GAAGGC-CAGCATGCCCATGG-3 nucleotides 623 to 604; see
Fig 3). The conditions for both PCRs were 95°C for 30 seconds,
60°C for 30 seconds, and 68°C for 5 minutes. The nucleotide sequences of the collinear primers, AP1 and AP2, are described in the
RACE protocol (Clonetech). The amplified product was subcloned in a TA
cloning vector system (Invitrogen, San Diego, CA) and sequenced by the dideoxynucleotide chain-termination
method.16 Both strands of the cDNA clones were sequenced.
Generation of peptide-specific antibodies.
A peptide (p1) corresponding to residues 15-26 (YETLNKRFRAAQ) of human
Emp was synthesized. Antibodies to the peptide were raised in rabbits,
and the antiserum was tested for immunospecificity for Emp by Western
blots of macrophage membranes. Peptide-specific antibodies were
obtained by affinity purification of antiserum on cyanogen bromide
immobilized peptide. Antibodies were eluted with 0.1 mol/L glycine, pH
2.5, immediately neutralized with Tris, and dialyzed against PBS.
Northern blot analysis.
Poly(A) RNA was isolated by using the FastTrack kit from Invitrogen.
Five micrograms of poly(A) RNA was electrophoresed on a 1% agarose gel
containing 6.6% formaldehyde. RNA was then transferred to
nitrocellulose and hybridized with a full-length
[32P]-labeled Emp cDNA probe overnight at 42°C in
50% formaldehyde, 4× SSC, 0.5 mg/mL salmon testes DNA
(denatured), 1× Denhardt's solution, 0.25 mg/mL yeast t-RNA,
10% dextran sulphate, and 1% SDS. This was followed by washes in
0.1× SSC, 0.1% SDS at room temperature for 5 minutes, at
52°C for 20 minutes, and finally in 0.1× SSC, 0.5% SDS at
60°C for 30 minutes. The filter was air-dried briefly and subjected
to autoradiography using Kodak x-ray films (Eastman Kodak, Rochester,
NY).
Construction and expression of GST-Emp fusion protein.
Human recombinant Emp was expressed in bacteria. Two constructs
(designated GST-Emp-1 and GST-Emp-2) were prepared in the pGEX-2T
vector (Pharmacia, Uppsala, Sweden). The GST-Emp-1
construct contained the entire coding region of Emp that was amplified
by the PCR using the following primers containing EcoRI
adaptors: (sense) 5-AAGAATTCCGGTGGAGTCGGCGGCTCAG-3,
nucleotides 20 to 38; and (antisense) 5-CCGAATTCCAGAGTA
TTTATCGTTGCTCA-3, nucleotides 1210 to 1229. The sense primer was
designed upstream of the initiator methionine. Thus, the GST-Emp-1
construct encodes full-length Emp with an additional eight amino acids
upstream of the initiator methionine. The GST-Emp-2 construct contained
cDNA insert encoding sequence between amino acids 98 and 395. The
insert was amplified by PCR using the following sense primer containing
EcoRI adaptor: 5-AAGAAT TCATAGCAGCGACCAGCCCGCG-3
nucleotides 336 to 356, together with the same antisense primer used
for the GST-Emp-1 construct. After digestion with EcoRI, the
PCR-amplified products were ligated into the pGEX-2T vector. All
constructs were sequenced from both directions to ascertain sequence
fidelity and proper orientation. Transformants containing Emp cDNA
inserts were grown in the presence of IPTG to induce expression of the
fusion proteins. Expression of fusion proteins in transformants was
determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of
total cell lysates, followed by the cell attachment assay. A parallel
control was included that contained only the GST protein.
The cell attachment assay.
The attachment of erythroblasts to Emp was studied by the cell
attachment assay as described previously.5 Briefly,
bacterial cell lysates expressing full-length or truncated Emp were
separated by SDS-PAGE and transferred to a nitrocellulose membrane
using the standard transfer procedure.17 The nitrocellulose
membrane was blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour at 22°C and incubated with [125I]-labeled
erythroblasts for 2 hours at 4°C. The membrane was washed 4 to 5 times with PBS, air-dried, and exposed to Kodak x-ray film for
autoradiography.
Expression of Emp in mammalian cells.
A eukaryotic expression vector pMT3, containing a hemagglutinin (HA)
tag, was used. The vector pMT3 and monoclonal antibodies against the HA
tag were generously provided by Dr K. Andrabi (Harvard Medical School, Boston, MA). Two constructs of human Emp cDNA were
made: one containing nucleotides 21 to 1229 (designated Emp-1) encoding
full-length Emp (residues 1-395), whereas the other containing nucleotides 336 to 1229 (designated Emp-2) encoding truncated Emp from
residues 114 to 395. The inserts were PCR amplified using the following
primers containing Nsi I adaptors: (sense for Emp-1) 5-TGCATGCAT CGGTGGAGT CGGCGGCT CA GT-3; (sense for Emp-2)
5-TGCATGCATCCCATAG CAG CGAC CAGCCCGC-3; and (antisense
for both Emp-1 and Emp-2) 5-GGCATGCATCAGAG TAT
TTATCGTTGCTCA-3. After digestion with Nsi I, the
PCR-amplified products were ligated into the unique Pst1 site of pMT3.
cDNAs were inserted to produce an in-frame fusion with the HA tag at
the C-terminus, thus expressing a fusion protein containing the HA tag
and some vector sequence, thereby adding 35 amino acids (3.9 kD) to the
molecular mass of Emp. All constructs were sequenced from both
directions to ascertain sequence fidelity and proper orientation. The
plasmids were purified on Qiagen columns (Qiagen Inc, Chatsworth, CA)
and transiently transfected into COS-7 cells by diethyl aminoethyl
(DEAE)-dextran method.18
Three days after the transfection, COS-7 cells were harvested,
metabolically labeled with [35S] methionine, and
immunoprecipitated with monoclonal antibodies against the HA tag. For
immunoprecipitation, metabolically labeled cells were treated with
diisopropyl fluorophosphate (Sigma Chemical Co, St Louis, MO) and lysed
in 0.5 mL of immunoprecipitation buffer (10 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA, 1% NP-40, 0.5% sodium deoxycholate, 2 mg/mL BSA, 0.2 mmol/L TLCK, 0.2 mmol/L TPCK, and 2 mmol/L phenylmethyl
sulfonyl fluoride [PMSF]). The samples were centrifuged
at 800g for 5 minutes to remove nuclei. The supernatants were
diluted 10-fold with the immunoprecipitation buffer. Ten microliters of
monoclonal antibody against the HA tag was added to each and the
samples were incubated overnight at 4°C with gentle shaking.
Thereafter, 100 µL of protein A-Sepharose (Pharmacia; 50 mg/mL of
buffer A [10 mmol/L Tris-HCl, pH 7.5, 130 mmol/L NaCl, 5 mmol/L EDTA,
and 1% NP-40]) was added and the samples incubated for another 4 hours at 4°C. The protein A-Sepharose beads were then collected and
washed three times with buffer A. The final pellet was resuspended in
70 µL SDS sample buffer and boiled for 2 minutes. Beads were removed
by centrifugation and the supernatant was directly loaded on
SDS-polyacrylamide gels according to the buffer system of
Laemmli.19
To determine the cellular orientation of the expressed Emp cDNA,
transfected COS-7 cells were surface labeled with [125I]
using the glucose oxidase-lactoperoxidase method,20
followed by immunoprecipitation with monoclonal antibodies against the HA tag as described above.
Stable transfections.
To study the attachment of erythroblasts to Emp cDNA-transfected cells,
a stably transfected HeLa cell line was generated. HeLa cells were
cotransfected with Emp cDNA constructs (either Emp-1 or Emp-2) and
pHook vector (Invitrogen) (containing the neomycin resistance gene
under the direction of a cytomegalovirus [CMV]
promoter) by DEAE-dextran method. Three days after transfection, cells
were harvested and replated in the selective medium containing 200 µg/mL of G418. Resistant cells were further propagated in selective
medium to establish a cell line. Human erythroblasts cultured by the
two-phase liquid culture system5 were mixed with
transfected or nontransfected HeLa cells and grown in complete culture
medium in the absence of G418 for 3 to 4 days. On day 3 or 4, floating
cells were removed and the adherent cells were washed two times with
PBS. Cells adhering on the petri dishes were fixed and stained with
Wright-Giemsa without detaching from the dishes and examined by
bright-field microscopy.
In vitro maturation of erythroid progenitors.
Erythroblasts were generated by in vitro culture of peripheral
blood-derived mononuclear cells using a two-phase liquid culture system
as described previously.5 This system supports the
proliferation and maturation of largely erythroid progenitors. At the
end of the culture, approximately 90% of the cells are late
erythroblasts and approximately 10% of the remaining cells are
macrophages. For experiments involving macrophage-depleted cultures,
macrophages were removed using monoclonal antibodies MO2 against
macrophage surface antigen as described earlier.5 Briefly,
macrophages were removed on day 4 or 5 of the second phase of culture,
when clusters of proerythroblasts and macrophages started to appear. The mixture of cells at a concentration of about 107/mL was
incubated with 1:20 rat antihuman MO2 antibodies at 4°C for 45 minutes. After three washings in cold Iscove's modified Dulbecco's
medium (IMDM), the cells were resuspended in IMDM
containing 1:40 fluorescein isothiocyanate (FITC)-conjugated rabbit
antirat IgG and incubated for a further 45 minutes. The cells were then washed, resuspended in IMDM, and incubated with sheep anti-FITC antibody attached to magnetic particles for 30 minutes at 4°C. MO2-positive cells were removed by magnetic separation using a Bio-Mag
separator (Advanced Magnetic Inc, Cambridge, MA). The control cultures,
ie, macrophage-containing cultures, were treated identically but were
not exposed to MO2 monoclonal antibodies. Cell viability, as tested by
trypan blue exclusion staining, was found to be greater than 95% in
both the control and macrophage-depleted cultures before and after the
magnetic separation procedure. The cultures were continued for 7 to 8 days after the removal of macrophages (a total of 12 days in the second
phase of culture).
In situ detection of apoptosis.
Erythroblasts cultured in the presence and absence of macrophages were
analyzed for DNA fragmentation, characteristic of apoptosis, using the
TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling)
reaction. The latter was performed using the peroxidase-conjugated in
situ cell death kit (Boehringer Mannheim, Indianapolis,
IN) according to the manufacturer's instructions.
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RESULTS |
Detection of two immunologically related Emp isoforms in macrophage
membranes.
Anti-Emp antibodies detected two isoforms of Emp in the macrophage
membranes (Fig 1, lane 1). These isoforms
migrate on SDS gels with apparent molecular weights of 33 kD and 36 kD
and are referred to as Emp-33 and Emp-36, respectively. The cell
attachment assay involving incubation of radiolabeled erythroblasts
with immobilized macrophage membrane proteins showed that both isoforms bind to radiolabeled erythroblasts (Fig 1, lane 2). Occasionally, we
have observed a lower band of approximately 26 kD that also appears to
bind to radiolabeled erythroblasts. Because this lower band is not
always detected, it is likely that it represents a degradation product
of Emp. Previously,5 the two isoforms were not easily
discernible because of the diffuse appearance of protein bands on low
polyacrylamide concentration gels. Western blot analysis using anti-Emp
antibodies detected Emp-33 and Emp-36 from erythroblast membranes as
well (data not shown).
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| Fig 1.
Detection of Emp isoforms in macrophage membranes.
Macrophage membrane proteins were separated by 18% SDS-PAGE and
transferred to a nitrocellulose (NC) membrane. The NC membrane was
probed with anti-Emp antibodies (lane 1) and immunoreactive bands were
detected with an enhanced chemiluminescence system, or the NC membrane
was incubated with radioiodinated erythroblasts (lane 2) and processed
for autoradiography. Molecular weight standards are shown on the
left.
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Cloning and sequencing of the Emp cDNA.
As a first step to elucidate the mechanism by which the Emp-mediated
attachment of erythroblasts to macrophages promotes erythroid cell
maturation, studies were initiated to determine the primary structure
of Emp. A human macrophage (U937) library constructed in bacteriophage
expression vector gt11 was used for expression cloning. The library
was screened with affinity-purified anti-Emp antibodies using standard
techniques. A 1.6-kb cDNA clone was obtained by immunoscreening. This
clone contained almost the entire coding region of Emp with the
exception of its very amino terminus. The amino terminal sequence was
obtained by 5 RACE, which yielded a 0.7-kb overlapping cDNA
fragment. Both cDNA clones were subcloned and sequenced
(Fig 2).
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| Fig 2.
Organization of human Emp cDNA. (A) The restriction map
was obtained from two overlapping clones isolated from a human U937
gt11 cDNA library. The central block (nucleotides 45-1229)
represents the coding region. Both strands were sequenced, and arrows
indicate the sequencing strategy. (B) Hydrophilicity plot of the
deduced amino acid sequence of Emp. Positive values indicate
hydrophilicity, whereas negative values indicate hydrophobicity. AA,
amino acids. (C) Immunochemical identification of the Emp sequence.
Macrophage membrane proteins were separated by 18% SDS-PAGE,
transferred to NC membrane, and probed with affinity purified anti-p1
antibodies (lane 1) or affinity-purified antibodies against native Emp
(lane 2). Anti-p1 was raised against the synthetic peptide
corresponding to residues 15-26. The position of the molecular weight
standards (same as those used in Fig 1) is shown on the left.
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Together, these two clones gave a sequence of 2,005 bps that
represented the complete sequence containing a translation initiation codon at nucleotide 45 and a stop codon at nucleotide 1229 (Fig 3). An open reading frame of 1,185 bp
predicted a protein containing 395 amino acids with a calculated
molecular mass of 43 kD (although the protein expressed in E
coli and COS-7 cells migrates with an apparent molecular weight of
~36 kD; see below) and an isoelectric point of 9.86. The stop codon
is followed by an untranslated region of 776 bp with a consensus
polyadenylation sequence (AATAAA) 10 bp before the poly A tail. The
hydrophilicity plot shows a hydrophobic segment near the N-terminus
between amino acids 56 and 80 (Fig 2). Although the hydrophobicity
index is relatively low, this segment is followed immediately by highly
charged residues, consistent with the stop transfer signals found
following most membrane spanning domains.21
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| Fig 3.
Nucleotide and deduced amino acid sequence of human Emp
cDNA. The putative membrane spanning domain is underlined, the putative
PTB binding motif is dotted underlined, and the polyadenylation
consensus sequence (AATAAA) is doubly underlined.
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The predicted amino acid sequence of Emp contains a consensus sequence,
N-D-K-Y at the C-terminus (Fig 3), which, if tyrosine phosphorylated,
could serve as a binding site for phosphotyrosine binding (PTB)
domains. The PTB domains are structurally different from SH2 domains
and specifically recruit tyrosine-phosphorylated proteins into
signaling complexes by binding to phosphotyrosine within a sequence
motif N-X-X-Y (where X represents any amino acid).22,23 The
recognition sequence of the PTB domain is specified by residues
N-terminal of the tyrosine and especially the  3 and the 5
residues, which correspond respectively to an asparagine and to a
hydrophobic residue.22,24-27 Emp also contains tyrosine residues that could participate in other cell signaling events. For
example, tyrosine 127 (which is part of a YXN sequence), if phosphorylated, could possibly be recognized directly by the SH2 domain
of GRB2.28,29 Similarly, tyrosine 312 of Emp could
potentially bind to the SH2 domain of phospholipase C .30
Database searches did not show any protein that is highly homologous
over the entire coding sequence of Emp. However, short segments of Emp
were found to have homology with the  chain of sheep T-cell surface
glycoprotein CD3. The segment of Emp between amino acids 236 and 264 is
41% identical, and the segment between amino acids 67 and 96 is 33%
identical with the chain of T-cell receptor. The CD3/T-cell
receptor complex has been shown to prevent apoptosis of immature T
cells in thymic cultures.31
Peptide-specific antibody detects native protein in macrophage
membranes.
To confirm the authenticity of cloned Emp cDNA, we have raised
antibodies against a selected domain of recombinant Emp. Based on the
deduced amino acid sequence, a peptide (p1) corresponding to residues
15-26 was synthesized. Rabbits were immunized with the peptide p1, and
antibodies were purified on a p1 affinity matrix. In immunoblots of
macrophage membranes, affinity-purified anti-p1 antibodies detected a
single 36-kD band that comigrates with the larger isoform of Emp (Fig
2C). These results show that the full-length recombinant Emp
corresponds to the native 36-kD Emp isoform from macrophages. Also,
this result suggests that the smaller isoform of Emp lacks the peptide
p1.
Emp is widely distributed in nonerythroid cells.
To determine whether Emp is expressed in various cell types and
tissues, Northern blot analysis was performed. Analysis of poly(A) RNA
from U937 cells, the human monocytic cell line used for cDNA cloning,
showed a message size of 2.1 kb for Emp, which is in agreement with the
2.0-kb nucleotide sequence of the cDNA clones. After induction with
12-O-tetradecanoylphorbol-13-acetate (TPA), which results in their
differentiation into adherent macrophage-like cells,32,33
the U937 cells showed a 50% decrease in the message for Emp
(Fig 4A). A transcript of the same size was
also observed using poly(A) RNA isolated from human heart, brain,
placenta, lung, liver, skeletal muscle, kidney, and pancreas (Fig 4B).
To address the concern regarding possible contamination of tissues with
peripheral blood, Northern blot analysis was repeated with poly(A) RNA
isolated from various cell lines. As shown in Fig 4C, Emp transcripts
were also present in K562, COS-7, HeLa, and Jurkat cell lines. These
results show that Emp transcripts are widely distributed in
nonerythroid cells and tissues.
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| Fig 4.
Northern blot analysis. (A) Five micrograms of Poly(A)
RNA from uninduced (1) and induced (2) U937 cells. (B) Northern blot
containing 20 µg of total RNA from human tissues was obtained from
Clonetech. (C) Five micrograms of Poly(A) RNA from different cell
lines. Each blot was hybridized with a full-length
[32P]-labeled Emp cDNA probe. Blot A was stripped and
reprobed with human -actin cDNA. The size of the markers in
kilobases is shown.
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Emp expressed in E coli behaves as an erythroblast binding
protein.
To determine if recombinant Emp binds to erythroblasts, GST-fusion
protein was expressed in bacteria. Two constructs were prepared in the
pGEX-2T vector: one containing the entire coding sequence (GST-Emp-1)
and the other containing cDNA sequence from which 97 amino acids from
the N-terminus were deleted (GST-Emp-2). These constructs were
expressed as GST fusion proteins in E coli. Expression of
fusion proteins in the transformants was determined by SDS-PAGE of
total cell lysates, followed by the cell attachment assay. As shown in
Fig 5A, the GST-Emp-1 construct produced a 65-kD product (indicated by an asterisk in lane 1) that contains full-length Emp (36 kD). The GST-Emp-2 construct was expressed as a
58-kD fusion protein (indicated by an asterisk in lane 2) containing
GST and a truncated Emp (29 kD). Our cell attachment assay, which
involves incubation of immobilized recombinant proteins with
radiolabeled erythroblasts, showed a strong binding of erythroblasts to
the 65-kD (full-length) product and only a weak binding to the 58-kD
(truncated) product. Quantification of cell binding, as determined by
scanning the autoradiogram, showed a 20-fold greater attachment of
erythroblasts to the 65-kD product than to the 58-kD product under
identical conditions. These results show that the recombinant Emp
behaves identically to native protein in the cell attachment assay and
that the cell binding domain of Emp is contained within the 97 amino
acid region at the N-terminus. Affinity-purified anti-Emp antibodies
detected both 65-kD and 58-kD products by Western blotting (Fig 5A).
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| Fig 5.
Expression of recombinant Emp. (A) Full-length (Emp-1)
and truncated (Emp-2) human Emp were expressed as glutathione
S-transferase (GST) fusion proteins in bacteria. Cell lysates of
bacteria transformed with GST-Emp-1 and -2 constructs (lanes 1 and 2, respectively) were separated by SDS-PAGE and stained directly with
Coomassie blue or transferred to nitrocellulose, probed with
radiolabeled erythroblasts followed by autoradiography, or probed
with affinity-purified anti-Emp antibody and immunoreactive bands were
detected by ECL. (B) Full-length (Emp-1) and truncated (Emp-2) human
Emp were expressed in COS-7 cells as fusion proteins
containing HA tag at the C-terminus. Transfected and
nontransfected COS-7 cells were either metabolically labeled with
[35S]methionine or surface labeled with
[125I], followed by immunoprecipitation with anti-HA tag
antibodies. Autoradiographs of the immunoprecipitates are shown. The
position of the molecular weight standards is indicated on the left.
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The N-terminus of Emp is extracellular.
To confirm the topology of Emp in the plasma membrane, an
epitope-tagged protein was produced in mammalian cells. Two constructs of human Emp cDNA were made in a eukaryotic expression vector pMT3
containing an HA tag: one containing the entire coding sequence (Emp-1)
and another containing truncated Emp (113 amino acids deleted from the
N-terminus) (Emp-2). The cDNA fragments were inserted in frame to
produce a fusion with the HA tag at the C-terminus. These two
constructs were transiently transfected into COS-7 cells by the
DEAE-dextran method. Three days after transfection, COS-7 cells were
harvested, metabolically labeled with [35S] methionine,
and immunoprecipitated with monoclonal antibodies against the HA tag. A
41-kD protein was immunoprecipitated from COS-7 cells transfected with
the Emp-1 construct, and a 32-kD protein was immunoprecipitated from
COS-7 cells transfected with the Emp-2 construct (Fig 5B). As described
in Materials and Methods, recombinant Emp is expressed as a fusion
protein containing the HA tag and some vector sequence, thereby adding
35 amino acids (3.9 kD) to the molecular mass of Emp. Thus, the
SDS-PAGE molecular mass of full-length recombinant Emp expressed in
COS-7 cells is 37.1 kD and that of the truncated protein is 28.1 kD.
Next, COS-7 cells transfected with the Emp constructs were surface
labeled with [125I], followed by immunoprecipitation with
monoclonal antibodies against the HA tag. As shown in Fig 5B, a
radiolabeled product of 41 kD was immunoprecipitated from COS-7 cells
transfected with the Emp-1 plasmid containing the entire coding region
of Emp cDNA. However, immunoprecipitations under identical conditions
from COS-7 cells transfected with the Emp-2 plasmid lacking 113 amino acids from the N-terminus did not precipitate a radiolabeled 32-kD product. These results suggest that the N-terminus, which is encoded only by the Emp-1 construct, must be exposed on the cell surface because it was radiolabeled by surface iodinating agents, whereas the
C-terminus carrying the HA tag must be intracellular and hence inaccessible to the surface labeling reagents. This
observation is further supported by the fact that the recombinant
GST-Emp-1 fusion protein showed a strong attachment with erythroblasts
(Fig 5A).
Recombinant Emp expressed in HeLa cells mediates attachment of
erythroblasts.
Because transient transfections allow only a small number of cells to
acquire DNA, it was difficult to assess the attachment of erythroblasts
to Emp-transfected COS-7 cells. Therefore, a stably transfected cell
line was generated using HeLa cells. We have previously shown that
erythroblasts do not bind to nontransfected HeLa cells.5
The latter were cotransfected with Emp cDNA constructs (either Emp-1 or
Emp-2) and pHook vector (Invitrogen). pHook vector contains CMV
promoter for high-level transcription and a neomycin resistance gene
for the selection of stable cell lines. To demonstrate that the
transfected HeLa cells support the attachment of erythroblasts in
culture, human erythroblasts were isolated by the two-phase liquid
cultures of peripheral blood mononuclear cells as described previously.5 Erythroblasts at the
proerythroblast/basophilic normoblast stage harvested on day 7 or 8 of
the second phase were mixed with transfected or nontransfected HeLa
cells, and cultures were continued in petri dishes for 3 to 4 days at
37°C in a 5% CO2 incubator. On day 3 or 4, floating
cells were removed. The adherent cells were washed, fixed, stained with
Wright-Giemsa without detaching from the dishes, and examined by
bright-field microscopy. As shown in
Fig
6A through F, erythroblasts specifically attached to HeLa cells that
had been transfected with the Emp-1 plasmid containing the entire
coding sequence of the Emp cDNA insert in the sense orientation. No
attachment was observed with HeLa cells transfected with the plasmid
containing a truncated Emp cDNA or to nontransfected HeLa cells (Fig 6G
and H). Hence, the transfection of full-length cDNA of Emp can produce
erythroblast binding characteristics in HeLa cells. These results
indicate that the cDNA encodes an authentic Emp. Furthermore, these
results provide evidence that the Emp cDNA encodes a novel cell
adhesion molecule and that the N-terminal of Emp is extracellular and
is involved in cell:cell contact.
View larger version (123K):
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| Fig 6.
Attachment of erythroblasts to transfected and
nontransfected HeLa cells. Early erythroblasts were cultured with HeLa
cells transfected with the full-length (Emp-1) plasmid (A through F),
with the truncated (Emp-2) plasmid (H), or with nontransfected HeLa
cells (G) for 3 to 4 days. On day 3 or 4, floating cells were removed
and the adherent cells were washed two times to remove any trapped
nonadherent cells. Cells adhering on the petri dishes were fixed and
stained with Wright-Giemsa without detaching from the dishes and
examined by bright-field microscopy. Original magnification: (A through
F) × 100; (G and H) × 40.
|
|
View larger version (92K):
[in this window]
[in a new window]
| Fig 7.
In situ analysis for DNA
fragmentation. (Top panel) Wright-Giemsa-stained cytocentrifuged
preparations of cells obtained on day 12 of the second phase of (a)
macrophage-containing and (b) macrophage-depleted cultures. An
erythroblastic island consisting of a central macrophage (M) surrounded
by a ring of late erythroblasts (E) is shown in (a). (Middle and bottom
panels) Erythroblasts harvested on day 12 of the second phase of each
culture were examined by the TUNEL method, (c) macrophage-containing
culture, (d) macrophage-depleted culture, (e) macrophage-containing
culture in the presence of anti-Emp peptide (p1) antibodies, and (f)
macrophage-depleted culture supplemented with macrophage conditioned
medium. Apoptotic cells are identified by more dense staining.
|
|
Emp-mediated association inhibits apoptosis of erythroblasts.
We have previously shown that the association of erythroblasts with
macrophages mediated by Emp promotes erythroid maturation leading to
their enucleation. In the absence of this association, erythroid cells
mature to the late erythroblast stage but they do not enucleate and
ultimately die. To determine whether the demise of erythroblasts in the
absence of Emp-mediated erythroblast-macrophage association is due to
apoptosis, erythroblasts cultured in the presence and absence of
macrophages were analyzed for DNA fragmentation in situ using the TUNEL
reaction.
Erythroblasts isolated on day 12 of the second phase of
macrophage-containing as well as macrophage-depleted cultures are typically at the late stage of erythroid maturation as assessed by
Wright-Giemsa staining of cytocentrifuged cell preparations (Fig 7a and
b). However, if the cultures are continued for another 3 to 4 days,
erythroblasts in macrophage-containing cultures undergo enucleation,
whereas those in macrophage-depleted cultures die. For the following
studies, we examined erythroblasts on day 12 of the second phase.
Erythroblasts were subjected to TUNEL assay for in situ detection of
apoptosis. As shown in Fig 7d, erythroblasts cultured in absence of
macrophages stained intensely by the TUNEL method that detects in situ
endonucleolytic cleavage characteristic of apoptosis.
Quantification of the TUNEL reaction showed that 66% erythroblasts in
macrophage-depleted cultures versus 10% in macrophage-containing
cultures were apoptotic (Table 1). To
determine if the magnetic separation procedure for macrophage removal
induced any apoptosis of erythroblasts, TUNEL assay was performed on
erythroblasts harvested from cultures that underwent mock macrophage
removal. No apoptosis was observed in these erythroblasts (data not
shown).
The results noted above suggest that macrophages inhibit apoptosis of
erythroblasts. To determine whether the effect of macrophages is
through Emp-mediated contact with erythroblasts, this contact was
specifically interfered by adding affinity-purified antibodies against
the peptide, p1, in the extracellular domain of Emp to the
macrophage-containing cultures. Antibodies (20 µg/mL) were added on
day 4 of the second phase and cultures were continued up to day 12. Erythroblasts from control and antibody-containing cultures were
subjected to TUNEL assay. As seen in Fig 7e and Table 1, 59% to 60%
erythroblasts cultured in the presence of anti-Emp antibody were
apoptotic, suggesting that Emp-mediated cell:cell contact inhibits
apoptosis of erythroblasts. Similar results were obtained when the
monovalent Fab fragment of anti-p1 IgG was used (data not shown).
Apoptosis was also observed in erythroblasts when macrophage-depleted
cultures were reconstituted with macrophage conditioned medium (Fig
7f): 10% macrophage conditioned medium was added to cultures on day 4 of the second phase after macrophage removal and the cultures were
continued up to day 12. These results further document the role of
erythroblast-macrophage contact in inhibiting apoptosis.
| |
DISCUSSION |
In this study, we have characterized Emp, a novel protein present on
the surface of erythroblasts and macrophages. Emp mediates erythroblast-erythroblast and erythroblast-macrophage interactions in
vitro, suggesting a role of Emp in homophilic interactions that involve
binding to homotypic and heterotypic cells.5 Two isoforms
of Emp with apparent molecular weights of 33 kD and 36 kD were detected
in macrophage membranes. Using cell attachment assay, we have shown
that both isoforms of Emp bind to radiolabeled erythroblasts. This
report describes the complete amino acid sequence of the larger isoform
of human macrophage Emp, which will facilitate assignment of structural
and functional domains within the protein.
The complete amino acid sequence of Emp was deduced from the nucleotide
sequence of a full-length cDNA that was isolated by screening a human
macrophage cDNA library using affinity-purified anti-Emp antibodies. To
demonstrate that the predicted sequence encodes an authentic Emp,
antibodies were raised against a peptide p1, corresponding to residues
15-26 in the amino terminus of recombinant Emp. Affinity-purified
anti-p1 antibodies detected the larger isoform of Emp in macrophage
membranes, suggesting that recombinant Emp corresponds to the 36-kD
isoform native to macrophage membranes.
Northern blot analysis showed the presence of a single 2.0-kb species
of Emp mRNA in U937 cells, a human macrophage cell line (Fig 4). Upon
induction with phorbol ester TPA, the U937 cells differentiate into
adherent macrophage-like cells. Our observation that the Emp mRNA is
downregulated twofold upon induction of U937 cells with TPA (Fig 4A) is
consistent with the fact that adherent macrophages do not participate
in the formation of erythroblastic islands. Moreover, the presence of
Emp transcripts in various human tissues and cell lines suggests that
Emp or its homologues are widely distributed and may be involved in
other cell:cell interactions.
The expression of full-length recombinant Emp in heterologous cells and
erythroblast attachment assays provide conclusive evidence that Emp is
a cell attachment molecule. A 20-fold reduction in the erythroblast
attachment to the truncated Emp lacking 97 amino acids from the
N-terminus shows that the cell binding domain of Emp is located at the
N-terminus within 97 residues. Recombinant Emp was also expressed in
COS-7 cells as a fusion protein with an HA tag at the C-terminus.
Surface labeling of transfected cells provided further evidence that
the N-terminus is exposed on the cell surface. Furthermore, full-length
and truncated (113 amino acids from the N-terminus deleted) Emp cDNA
fragments were stably transfected into HeLa cells. Incubation of
transfected cells with human erythroblasts confirmed that the
N-terminal extracellular domain is involved in cell:cell contact. In
summary, the deduced primary structure of Emp consists of a relatively
small amino terminal extracellular domain involved in cell-cell
contact, a single transmembrane domain, and a large cytoplasmic domain.
The N-terminal extracellular domain is the likely site for homophilic binding interactions between homotypic and heterotypic cells. The
C-terminal cytoplasmic domain of Emp contains several tyrosine residues
that, when phosphorylated, could be recognized by well-characterized protein recognition modules such as PTB or SH2
domains.22-30 Through these interactions, Emp may be
recruited into signaling complexes and may provide mechanism to connect
with signal transduction pathways.
The results presented here provide the convincing evidence that Emp is
an integral membrane protein with the N-terminus on the extracellular
side and the C-terminus on the cytoplasmic side. But, because it lacks
an amino-terminal cleavable signal sequence, the mechanism by which the
amino terminus of Emp is translocated across the endoplasmic reticulum
is less clear. Nevertheless, it is believed that the membrane insertion
of proteins without a cleavable signal sequence is assisted by internal
hydrophobic sequences that are inserted in the membrane by the same
mechanism that operates for cleavable signal sequences, except that no
postinsertional proteolysis occurs.21,34-36 We believe that
a similar mechanism is applicable for Emp's insertion in the membrane.
Most adhesion proteins, for example, cadherins, integrins, and Ig
superfamily, consist of a relatively large extracellular domain, a
single membrane spanning domain, and a short cytoplasmic domain.37 In contrast, Emp appears to contain a relatively
small extracellular domain at the N-terminus and a large cytoplasmic domain at the C-terminus. Thus, Emp may belong to a subfamily of novel
adhesion proteins displaying large cytoplasmic domains or Emp may be a
component of a large complex of which all the components are required
for proper functioning.
Two other macrophage receptors that have been proposed to be involved
in adhesion to erythroid cells include erythroblast receptor,
EbR,38 and a lectin-like sheep erythrocyte receptor, SER.39,40 EbR mediates reversible divalent cation-dependent binding of hematopoietic cells to murine fetal liver macrophages. In
contrast, Emp, which is present on the surface of both macrophages and
erythroblasts, does not require divalent cations to mediate cellular
contact.5 SER, a 185-kD plasma membrane glycoprotein, mediates binding of unopsonized sheep erythrocytes via recognition of
sialylated glycoconjugates and may interact with sialylated ligands on
bone marrow cells. Using monoclonal antibodies, Crocker et
al41 have shown that SER is diffusely localized at the
contact zones between macrophages and erythroblasts within
erythroblastic islands. The apparent molecular weight of Emp
distinguishes it from SER. These observations indicate that Emp is a
novel molecule that is involved in the attachment of macrophages to
erythroid cells.
In the absence of Emp-mediated interaction of erythroblasts with
macrophages, erythroid cells mature to the late erythroblast stage but
fail to enucleate and undergo apoptosis. This observation is of
particular interest because portions of Emp are homologous to the chain of the sheep T-cell surface glycoprotein CD3 and because
antibodies to CD3/T-cell receptor complex have been shown to induce
cell death in immature T cells by apoptosis. It is also of relevance to
note that the interaction of follicular dendritic cell-B-cell clusters
mediated by VLA-4-VCAM-1 interactions inhibits apoptosis of germinal
center B cells.42 Similarly, the central macrophage in
erythroblastic islands may inhibit apoptosis of erythroblasts through
the VLA-4-VCAM-1 interaction or through interactions involving Emp.
The data presented here show that the disruption of
erythroblast-macrophage contact by anti-Emp antibodies causes
apoptosis, thus suggesting that one of the functions of the
Emp-mediated cell-cell contact is to prevent apoptosis of
erythroblasts. Numerous erythroid-expressed genes have been linked to
regulation of apoptosis. For example, erythropoietin regulates survival
of erythroid progenitors by preventing apoptosis.43 Recently, GATA-1, an erythroid transcription factor, has been shown to
support the viability of erythroid precursors by suppressing apoptosis.44
In summary, the molecular cloning of Emp describes a novel protein
involved in erythroblast-macrophage interaction. This interaction promotes the terminal maturation of erythroid cells leading to their
enucleation by suppressing apoptosis. To elucidate the mechanism by
which Emp mediates erythroblast-macrophage contact and promotes terminal maturation and enucleation of erythroid cells, it will be
essential to identify the interactions of Emp with other cellular components, including cytoskeletal and signaling proteins. These interactions are likely to show a new mechanism of cellular adhesion that may mediate cell:cell contacts in an hematopoietic
microenvironment.
| |
FOOTNOTES |
Submitted April 15, 1998;
accepted June 11, 1998.
Supported in part by a grant from the American Cancer Society to M.H.
and a grant from the National Institutes of Health (HL 27215) to the
late Dr Jiri Palek.
Presented in part in abstract form at the 38th Annual Meeting of the
American Society of Hematology, Orlando, FL, 1996.
Genbank accession no. AFO84928.
Address reprint requests to Manjit Hanspal, PhD, Department of
Biomedical Research, St Elizabeth's Medical Center, 736 Cambridge St,
Boston, MA 02135; e-mail: mbh{at}tiac.net.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Athar Chishti for his advice and encouragement
during the course of these studies, Jennifer Wu for the preparation of
the peptide p1 and anti-p1 antibodies, Dr Eugenia Cifuentes for
preparing the Fab fragment of anti-p1, and Donna-Marie Mironchuk for
the artwork.
| |
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Abstract
Introduction
Abstract