* Cell Regulation Program, and Protein and Peptide Program, European Molecular Biology Laboratory, Heidelberg, D-69117
Germany; and § Max-Planck Institut for Physiology and Clinical Research, W.G. Kerckhoff-Institute, Department of Molecular
Cell Biology, Bad Nauheim, D-61231 Germany
MEP21 is an avian antigen specifically expressed on the surface of Myb-Ets-transformed multipotent hematopoietic precursors (MEPs) and of normal thrombocytes. Using nanoelectrospray tandem mass spectrometry, we have sequenced and subsequently cloned the MEP21 cDNA and named the gene thrombomucin as it encodes a 571-amino acid protein with an extracellular domain typical of the mucin family of proteoglycans. Thrombomucin is distantly related to CD34, the best characterized and most used human hematopoietic stem cell marker. It is also highly homologous in its transmembrane/intracellular domain to podocalyxinlike protein-1, a rabbit cell surface glycoprotein of kidney podocytes.
Single cell analysis of yolk sac cells from 3-d-old chick embryos revealed that thrombomucin is expressed on the surface of both lineage-restricted and multipotent progenitors. In the bone marrow, thrombomucin is also expressed on mono- and multipotent progenitors, showing an overlapping but distinct expression pattern from that of the receptor-type stem cell marker c-kit. These observations strengthen the notion that the Myb-Ets oncoprotein can induce the proliferation of thrombomucin-positive hematopoietic progenitors that have retained the capacity to differentiate along multiple lineages. They also suggest that thrombomucin and CD34 form a family of stem cell-specific proteins with possibly overlapping functions in early hematopoietic progenitors.
DURING embryonic development blood cells arise
first in the early yolk sac (primitive hematopoietic
cells) and later independently in the vicinity of the
dorsal aorta (definitive hematopoietic cells; for reviews
see Dzierzak and Medvinsky, 1995 The analysis of hematopoiesis has been greatly facilitated by the identification of a variety of cytokines (for review see Callard and Gearing, 1994 In previous work we found that the Myb-Ets oncoprotein-encoding acute leukemia virus E26 is able to transform primitive hematopoietic progenitors derived from
chicken embryo yolk sac. These cells resemble multipotent
hematopoietic progenitors since they can be induced to
differentiate into either erythrocytes, thrombocytes, myeloblasts, or eosinophils and we have therefore designated
them as MEPs1 (Myb-Ets-transformed Progenitors; Graf
et al., 1992 Using MEPs as a source of antigen for immunizations
we have generated a panel of monoclonal antibodies directed against the surface antigens of these progenitors
(McNagny et al., 1992 For several years, we had attempted to sequence MEP21
by conventional protein chemical techniques. However,
these attempts were unsuccessful due to the very low
amounts of protein that could be purified (silver stained
level). Here we report the use of nanoelectrospray mass
spectrometry (Wilm and Mann, 1996 Protein Purification and Sequencing
Proteins from ~1010 HD57 cells were solubilized in 50 ml of lysis buffer
(150 mM NaCl, 50 mM Tris, pH 7.5, 0.5% NP-40) plus protease inhibitors
(1 mM PMSF, 20 mM Staining by Coomassie brilliant blue R-250 did not reveal the location
of the band, therefore the gel was restained with silver nitrate according to
the protocol of Shevchenko et al. (1996) PCR-based Cloning and Library Construction
Degenerate PCR primers were designed based on the sense sequence of
the MEP21-1 (5 Construction of the HD100 cDNA phagemid library was described previously (McNagny et al., 1996 Sequence Comparisons
Sequence comparisons were performed using FASTA and TFASTA
searches of the GenBank/EMBL/DDBJ Data Library and Swissprot databases. Homologies were derived using "Pileup" alignments of sequences retrieved from the Swissprot database. The data for MEP21 sequences are
available from GenBank/EMBL/DDBJ under accession numbers Y13976, Y13977, and Y13978.
Nucleic Acid Hybridization
For Northern blot analysis, total RNA was prepared by lysis and fractionation in guanidinium/acetate/phenol/chloroform as described by Chromczynski and Sacchi (1987). Approximately 10 µg of each RNA was resolved
on a 1% agarose-formaldehyde gel and blotted onto nylon membranes
(GeneScreen; Dupont-NEN, Boston, MA) as described by Sambrook et
al. (1989) All hybridization probes were labeled with [ Immunohistologic Analyses
Whole mount in situ antibody stains and paraffin sections were performed
as described by Lopéz and Carrasco (1992) Animals and Cell Culture
Embryos and chicks were produced from fertilized eggs obtained from
White Leghorn chickens maintained by Lohmann (Cuxhaven, Germany).
Protocols for the isolation, virus infection, and generation of primary
transformed cells from embryonic day two, three, and four blastoderms
were as described previously (Graf et al., 1992 The origins of the cell lines used as sources of RNA have been described
previously: HD3 erythroblasts (Beug et al., 1982a All cells were grown in blastoderm medium (Graf et al., 1992 Colony Assays
Plasma clot colony assays were performed as described by Cormier and
Dieterlen-Lievre (1988) Immunofluorescence and Cell Sorting
For single color, indirect immunofluorescence analysis, 106 cells were
stained with MEP21, MEP26, EOS47 (McNagny et al., 1992 Cytological Stainings
Peroxidase staining was performed by a procedure that allows the detection of peroxidase activity (an exclusive marker of chicken eosinophil
granules; Brune and Spitznagel, 1973 MEP21 Is a 150-kD Protein Expressed
by Thrombocytes
To determine whether MEP21 antigen is coexpressed by
thrombocytes with known thrombocyte markers, and
whether in these cells it exhibits a similar molecular weight
as in MEPs (150 kD; McNagny et al., 1992
Cloning of the cDNA-encoding MEP21 Protein Reveals
a Mucinlike Structure
To clone MEP21-encoding cDNA, MEP21 protein was detergent solubilized from the MEP cell line HD57 and purified by a combination of immunoaffinity chromatography
and preparative gel electrophoresis. The protein amount
was estimated to be ~0.5 pmol based on subsequent mass
spectrometric analysis. Peptides were micropurified into a
nanoelectrospray needle and the sequences of seven tryptic peptides of MEP21 were determined as explained in
Mann and Wilm (1995)
Type 1 clones, found 39 times, contain a 5 A second type of clone, found 10 times, encodes an additional 18 amino acids in the cytoplasmic domain and includes the sequenced peptide MEP21-3 (Fig. 3, A and C).
This peptide was found at relatively low amounts in the
preparation used for sequence analysis, suggesting that the
protein encoded by this cDNA is less abundantly expressed than type 1. Half of the type 2 clones terminated
immediately after the first 62 nucleotides of the 3 Type 3 clones, found three times, are identical to type 1 and 2 clones but contain a novel cytoplasmic and 3 A comparative protein sequence analysis with those
present in the Swissprot and TFASTA data bases revealed
no significant similarity between the extracellular domain
of MEP21 and any previous entries. As shown in Fig. 3 B,
the cytoplasmic and transmembrane domain of type 1 clones, however, showed 96% similarity and 86% identity
to the cytoplasmic domain encoded by the rabbit podocalyxinlike protein (PCLP)-1 gene (Kershaw et al., 1995 Endogenous and Exogenous Expression of MEP21/
Thrombomucin Confirms the Identity of Cloned cDNAs
To verify that the cDNAs cloned indeed correspond to the
mRNAs expressed in MEP21-positive cells, Northern
blots were performed. As shown in Fig. 4 A, RNA from a
variety of chicken hematopoietic cell lines revealed a major transcript of 6 kb in the antigen-positive MEP cell lines
HD100 and HD57 but not in several antigen-negative cell
lines belonging to the B and T lymphoid as well as to the myelomonocytic, eosinophilic, and erythroid lineages. To
demonstrate that the cloned cDNAs encode a protein of
the expected size and immunoreactivity, a type 1 cDNA
was subcloned into the pSFCV viral expression vector and
transfected into the MEP21-negative avian erythroblast
cell line, HD3. As shown in Fig. 4 B, ectopically expressed
MEP21 antigen is indistinguishable from endogenously expressed MEP antigen.
Thrombomucin Is Expressed by Kidney Podocytes and
Vascular Endothelia
The homology detected between thrombomucin and PCLP-1/
podocalyxin prompted us to examine the reactivities of the
MEP21 antibody in nonhematopoietic tissues. For this
purpose we prepared fixed tissue sections from 5-d-old
chicks and examined them by immunohistochemistry. As
illustrated in Fig. 5, the antigen was found to be expressed on the lumenal face of vascular endothelia in a variety of
tissues including kidney, lung, intestine, spleen, thymus,
and brain. In the kidney, by far the most prominent expression was observed on the podocytes (cells that form
the filtration apparatus in Bowman's capsule). The antigen
is not expressed by cells comprising the thick- and thin-walled proximal and distal tubules, nor is it expressed by
cells of the collecting ducts. In the lung, thrombomucin is
expressed at high levels by capillary networks surrounding the air sacs and by major vessels, but not by bronchial-associated epithelia. In intestine, MEP21 antibody demarked the luminal face of capillaries in the lamina propria of the villi. Isolated vessels in the smooth muscle layer
were also stained, while brush borders of epithelial cells
and intra-epithelial lymphocytes scored negative. In the
spleen (and thymus, not shown) blood vessels were positive as were occasional cells in the red pulp of the spleen,
probably corresponding to thrombocytes.
Thrombomucin Is Expressed on the Surface of
Extra- and Intraembryonic Hematopoietic Cells
To study the expression of thrombomucin during ontogeny,
whole mounts were prepared from 4-d-old embryos and
analyzed by immunoperoxidase staining with MEP21 antibodies. Thrombomucin expression was detected on virtually
all embryonic and extraembryonic blood vessels (Fig. 6 A).
Sections of such embryos also showed strong antigen expression by cells of a variety of tissues including the developing neural tube, aorta, glomerulus, liver, heart, and coelomic cavity (Fig. 6 B). In each of these tissues, the antigen
appeared to be restricted to cells comprising the lumenal
surfaces of tissues or boundary elements between tissues
such as in the liver capsule, aorta, mesonephros, coelomic
cavity, and the central canal of the neural tube. As can be
seen from the inset, we also observed expression of thrombomucin by isolated cells in the ventral wall of the dorsal aorta,
in the precise region where the earliest intraembryonic hematopoietic cells develop (Cormier and Dieterlen-Lievre, 1988
MEP21/thrombomucin Is Expressed on Hematopoietic
Precursors in Early Avian Embryos
To determine whether hematopoietic progenitors express
the protein, cell suspensions were prepared from the yolk
sac of 3-d-old chick embryos and analyzed by FACS® for
the expression of thrombomucin as well as for a variety of other cell surface antigens. While a large percentage of the
cells from both stages were stained with MEP21 and JS4
antibodies (this antibody detects a late erythroid-specific
marker (Schmidt et al., 1986
MEP21-only cells also yielded mixed erythroid/thrombocytic/myelomonocytic colonies. However, the low frequency of these precursors compared to the monopotent
precursors (<1 mixed colony in 100) made it difficult to
rule out the possibility that they actually represented artifacts that arose due to an overlapping of monopotent colonies. To clarify this issue, a total of 2,112 MEP21-positive cells were sorted as single cells into 96-well plates, cultured in blastoderm medium, and evaluated without staining after 6 d. Of the cells seeded, 22% formed colonies that
could be classified relatively easily because of their small
size and the distinct morphology of nucleated erythrocytes, thrombocytes, macrophages, and eosinophils. As illustrated in Fig. 8 A, the frequencies of the various colony
types observed were comparable to those determined in
the plasma clot assay (Fig. 7). Four of the cells (that is ~1
in 500 cells seeded), yielded mixed erythrocyte, thrombocyte, and macrophage colonies, proving that thrombomucin is indeed expressed on the surface of multipotent
progenitors. These colonies were significantly larger than
the JS4hi/MEP21lo-derived ones, consisting of >100 cells
(Fig. 8 B).
Thrombomucin Is Expressed by Mono- and
Multipotent Progenitors in the Bone Marrow with a
Distribution Distinct from that of c-kit
To determine whether thrombomucin is also expressed by
hematopoietic progenitors later in ontogeny, bone marrow
from 1-wk-old chicks was stained with MEP21 antibodies
and analyzed by FACS®. Approximately 7% of the cells
were stained and their sorting resulted in an enriched population of both thrombocytes and larger cells with a
"blast"-like morphology (McNagny et al., 1992
These four subpopulations were sorted (the purity of
each fraction was >95%) and seeded in plasma clot cultures in three different media: (a) SCF plus anemic serum
(containing erythropoietin), plus cMGF; (b) SCF plus anemic serum; (c) SCF plus cMGF. 6 d later the cultures were
harvested and stained with DiffQuik for morphological analysis. As shown in Fig. 9 B, double-positive cells (R2)
were found to be highly enriched in colony forming units
(CFU) (one CFU per two cells plated), followed by c-kit-
only cells (R1, one CFU per five cells plated) and MEP21-only cells (R3, one CFU per 10 cells plated). Unsorted
cells yielded an average of one CFU per 17 cells plated,
while double-negative cells gave essentially no colonies
(data not shown) and were not characterized further. Little variations were observed between the different culture conditions except that the formation of myeloid colonies
was found to be dependent on an activity present in the
anemic serum.
Phenotypic analysis of the resulting colonies (Fig. 9 B)
revealed that a combination of MEP21 and c-kit staining
allows the separation of committed monopotent from multipotent precursors. Thus, macrophage and/or granulocyte
precursors were present in the c-kit-only fraction, but absent from all fractions expressing thrombomucin. Conversely, thrombocytic precursors were present exclusively
in the thrombomucin-only fraction, but not in the c-kit-
only fraction. Thrombomucin was also expressed by erythroid precursors but, in contrast to the expression by
thrombocytic cells, its expression decreases as a function
of erythroid maturation. Thus, thrombomucin-only cells
gave rise to mixed thrombocytic/erythroid colonies and
early erythroid colonies, including burst forming units,
whereas MEP21 low or negative fractions yielded predominantly late erythroid colonies (5-20 cells per colony).
Approximately 1 in 200 double-positive cells gave rise to
colonies containing blasts. To determine whether these
colonies correspond to multipotent progenitors, a total of
960 cells from each fraction shown in Fig. 9 A were sorted
singly into 96-well plates, cultured for 10 d in the presence
of SCF, anemic serum, and cMGF, and the wells scored
microscopically for the presence of large, blastlike colonies. As the colonies derived from bone marrow were
>50-fold larger than those from embryos they could only
be classified after cytological staining. Three colonies containing >5,000 immature-looking cells developed from the
MEP21/c-kit double-positive fraction (Fig. 10 A), while no
such colonies were detected in the other fractions. To analyze the differentiation potential of these blast colonies,
they were replated in plasma clot under the same culture
conditions, incubated for an additional 5 d, and stained
with DiffQuik. Myeloid, erythroid, and thrombocytic colonies were obtained with one of the replated blast colonies (Fig. 10 B), while the other two yielded myeloid and
thrombocytic cells (Fig. 10 C).
In this paper we describe the cloning of thrombomucin, an
antigen identified by the mAb MEP21 directed against the
surface of E26 leukemia virus-transformed multipotent
hematopoietic progenitors and of normal thrombocytes
(Fig. 11 A). We now report that it is also expressed on the
surface of both mono- and multipotent progenitors from
the bone marrow (Fig. 11 B). These findings support the
notion that the Myb-Ets oncoprotein of the E26 avian leukemia virus induces the proliferation of hematopoietic
cells that resemble normal multipotent progenitors both
functionally and phenotypically.
Here we used a recently developed method, nanoelectrospray mass spectrometry, for the sequencing of MEP21
at silver-stained levels. Despite the low amount, all amino
acids were sequenced correctly as evidenced by the agreement with the sequence deduced from the cDNA. Interestingly, the expression of a splicing variant could also be
directly verified at the protein level.
The most striking feature of the thrombomucin sequence is its Ser-Thr-Pro-rich extracellular domain that is
typical of cell membrane-associated mucins, and which is
known to be subject to extensive O-linked glycosylation,
sialylation, and sulfation (for review see Hilkens et al., 1992 Thrombomucin also bears similarities to CD34, another
highly glycosylated mucinlike cell surface protein (Simmons et al., 1992 During the development of chickens, thrombomucin
seems to be one of the earliest markers of hematopoietic
cells. Thus, it is expressed in the precirculation yolk sac and
on intraembryonic hematopoietic cells before c-kit is expressed. In mice, precirculation yolk sac cells express c-kit,
but, as in chickens, these cells are unresponsive to SCF
(Ogawa et al., 1993 We were initially surprised to find that normal and
transformed multipotent progenitors share an antigen
with mature thrombocytes. However, it is now clear that
the thrombocyte integrin The distribution of thrombomucin on embryonic progenitors is, in general, similar to that of definitive bone
marrow progenitors except that embryonic but not adult
myeloid progenitors express the antigen. This might reflect the fact that embryonic myeloid progenitors differentiate more rapidly (in about 3 d versus 10 d for the bone
marrow) and have a much more limited division potential than definitive ones (McNagny, K.M., and T. Graf, unpublished observations). Thus, multipotent progenitors might
not have sufficient time to clear thrombomucin from their
surface as they differentiate along the myeloid lineage.
This notion is supported by the observation that yolk sac-
derived myeloblasts transformed by the E26 virus express
no MEP21/thrombomucin.
What could the function of thrombomucin be? Mucins
have been shown to play both positive and negative roles
in adhesion processes. For example, specific oligosaccharide side groups of CD34 can serve as ligands for selectins
(for review see Kansas, 1996; Zon, 1995
; Cumano et
al., 1996
; Dieterlen-Lievre et al., 1996
). After the transient
production of blood cells in the spleen and fetal liver (mammals), hematopoietic progenitors are produced exclusively in the bone marrow, where their proliferation and maturation is regulated by an intricate set of microenvironmental
cues elaborated by stromal cells (Quesenberry, 1992
).
) and of specific cell
surface antigens (for reviews see Spangrude et al., 1991
;
Uchida et al., 1993
) that allow the isolation and expansion
of monopotent and multipotent precursors. In spite of
their considerable interest, antigens known to be expressed
on the surface of hematopoietic stem cells are still relatively few. They comprise tyrosine kinase receptors such
as c-kit (for review see Bernstein et al., 1991
) and flk-2 (Matthews et al., 1991
), mucins such as CD34 (Simmons et
al., 1992
), glycosylphosphatidylinositol-linked molecules
of unknown function such as Sca-1 and Thy-1 (Uchida et
al., 1993
; Miles et al., 1997
), and the AA4.1 antigen, a specific marker of yolk sac and fetal liver hematopoietic progenitors (Jordan et al., 1990
). None of these markers are
absolutely specific for hematopoietic stem cells and they
must be used in combination with lineage-specific markers
to separate monopotent from multipotent progenitors
(Uchida et al., 1993
).
).
). One of these antibodies, named
MEP21, was shown to react specifically with an antigen
present on MEPs but absent on transformed B and T lymphoid, erythroid, myelomonocytic, and eosinophilic cell
lines. Surprisingly the antigen was also found to be expressed on thrombocytes obtained after differentiation induction (through v-Myb inactivation) of MEPs transformed by a temperature mutant of E26 virus (Frampton et al., 1995
). Likewise, the MEP21 antigen could be detected on normal chicken thrombocytes, but not on lymphocytes, erythrocytes, eosinophils, neutrophil granulocytes, or macrophages (Graf et al., 1992
; McNagny et al., 1992
).
; Wilm et al., 1996
) to
sequence the MEP21 protein and clone MEP21-encoding cDNAs and a detailed analysis of the expression of the antigen during ontogeny. The data show that MEP21 is a
novel mucinlike protein distantly related to CD34, which
is expressed on the surface of mono- and multipotent progenitors of both primitive and definitive origin.
Materials and Methods
-amino-N-caproic acid, 1 mg/ml leupeptin, and 2.5 U/ml trasylol) on ice for 30 min. Nuclei were removed by centrifugation at
15,000 g for 30 min at 4°C, and the supernatant was incubated overnight at
4°C with 200 µl of MEP21 antibody-coupled Sepharose beads (2.5 mg of
antibody coupled per ml of CNBr-activated Sepharose resin; Pharmacia
Diagnostics AB, Uppsala, Sweden). Beads were washed 10 times with 2 ml of lysis buffer containing protease inhibitors, and once with PBS plus
PMSF, and bound proteins were eluted in 0.1% trifluoroacetic acid plus
PMSF. Eluted fractions were equilibrated to neutral pH by addition of
Tris buffer, lyophylized, resuspended in sample buffer, and then resolved
on a 10% SDS-PAGE gel.
. The silver-stained band was excised and digested in gel with trypsin, as described by Wilm et al. (1996)
.
Half of the peptide mixture obtained after extraction of the gel piece was
micropurified on a capillary containing 50 nl of POROS R2 material (PerSeptive Biosystems, Cambridge, MA). After washing with 5 µl of 5% formic acid, the peptides were step eluted with 1 µl of 50% MeOH in 5% formic acid, into a nanoelectrospray needle. This needle was transferred to
an APIII mass spectrometer (Perkin-Elmer Corp., Toronto, Canada) and
sprayed for 30 min. During this time, peptide ions apparent from the mass
spectrum were selected and isolated in turn, and then fragmented in the
collision chamber of the mass spectrometer. The experiment was repeated
with the remaining portion of the digest mixture that had been esterified
in 2 M HCl in MeOH. Amino acid sequences were determined by comparing spectra of esterified and nonesterified peptides, using AppleScriptTM (Apple, Cupertino, CA) based scripts developed in our group.
-AAYGARGCITTYTTYGARGTITTY-3
) and MEP21-2 (5
-GAYCCIGCIGCIGTITTYGARGAR-3
) peptides. Poly(A)+ RNA
was prepared from HD100 total RNA (see below) using oligo(dT)-cellulose (Sambrook et al., 1989
), and then converted to single-stranded cDNA
using a
ZAP-cDNA synthesis kit (Stratagene, La Jolla, CA) according to
the manufacturer's instructions. PCR of single-stranded cDNA was performed using the T7 phage arm primer (Pharmacia Diagnostics AB) and
either the MEP21-1- or MEP21-2-based primers. Taq polymerase (Pharmacia Diagnostics AB) and buffer conditions were those recommended by the manufacturer. Amplification was performed by 30 cycles of 30-s denaturation at 95°C, 30-s template annealing at 53°C, and 90-s elongation at
72°C, using an "Intelligent Heating Block" (Biometra Inc., Göttingen,
Germany). PCR products were cloned into plasmids using a TA cloning
kit (Invitrogen, San Diego, CA), transformed into Escherichia coli strain
Xl-1 blue and two positive clones were characterized by restriction map
and sequence analysis.
). The amplified HD100 library was screened
three times for MEP21 positive clones. For each screening, one million recombinant phage were plated on XL-1 MRF
bacterial host strain and
these were screened by hybridization with a 32P-labeled MEP21 PCR fragment probe (Sambrook et al., 1989
; and see below). In total, 52 recombinants were identified, plaque purified by two further rescreens, and plasmids were produced by in vivo excision using the protocols recommended
by the
ZAP-cDNA synthesis kit manufacturer (Stratagene). Inserts were
sequenced by the EMBL DNA sequencing service using an automated sequencer (A.L.F. DNA sequencer; Pharmacia Diagnostics AB).
. Hybridization of radiolabeled probes and removal of unbound
probe was performed in NaHPO4/SDS buffer as described by Church and
Gilbert (1991).
-32P]dCTP by random
hexamer priming as described by Feinberg and Vogelstein (1983)
. The following cDNA fragments were used as probes: a 1.8-kb PCR fragment of
MEP21 (see above), a 0.23-kb PstI/HindIII fragment specific for MEP21
alternatively spliced and a glyceraldehyde-3-phosphate dehydrogenase-
specific probe (Dugaiczyk, 1983
).
.
; McNagny and Graf, 1997
).
Bone marrow was flushed from the tibias and femurs of 5-d-old chickens
using PBS and a 5-ml syringe with a 21-gauge needle. Viable leukocytes
were isolated by centrifugation at room temperature on "lymphocyte separation media" (Eurobio, Les Ulis, France) for 20 min at 1,000 g. Cells
were isolated from the interface, washed three times, stained, and then
sorted as described.
); HD44 erythroblasts
(Metz and Graf, 1991
); HD11 macrophages (Beug et al., 1979
); HD13
granulocytes (Golay et al., 1988
; Kulessa et al., 1995
); HD57 MEPs (Metz
and Graf, 1991
); HD57 M1 myeloblasts (Graf et al., 1992
); HD50 1A1
eosinophils (Kulessa et al., 1995
); MSB-1 T cells (Akiyama and Kato,
1974
); RP-12 B cells (Siegfried and Olson, 1972
); and HD100 MEP/eosinophilic cells (McNagny et al., 1996
).
; McNagny et al., 1992
; McNagny and Graf, 1997
) composed of DME, supplemented with 10% fetal calf serum, 2.5% chicken serum, 0.15% NaHCO3,
56 µg/ml of conalbumin, 80 mM 2-mercaptoethanol, 0.9 µg/ml insulin, and
the standard complement of antibiotics at 37°C in 5% CO2. Medium for
HD50M and HD11 cells was supplemented with ~10 U/ml of crude
chicken myelomonocytic growth factor (cMGF; Leutz et al., 1989
). Where
indicated, colony assays were performed in the presence of 5% anemic serum as a source of erythropoietin (Radke et al., 1982
) and 4 ng/ml chicken
SCF (Hayman et al., 1993
).
. Briefly, sorted cells were resuspended in 1.2 ml
of blastoderm medium plus 120 µl of citrated bovine plasma (GIBCO
BRL, Gaithersburg, MD) plus 10 µl thrombin (100 IU/ml, Sigma) and
600-µl aliquots from this mixture were seeded in duplicate into a 24-well
tissue culture plate (Nunc, Roskilde, Denmark). Cultures were incubated
for 3-6 d at 37°C, harvested, and air dried onto microscope slides according to the methods described previously for collagen cultures (Lanotte,
1984
). Slides were stained for hemoglobin using diaminobenzidine (see below), counterstained with DiffQuik (May-Gruenwald-Giemsa-like stain; Baxter, Düdingen, Switzerland), and colony types were assessed by morphology. In some experiments, cultures were supplemented with anemic
serum, cMGF or stem cell factor (SCF). All colonies obtained from the
bone marrow and yolk sac were scored when they contained a minimum
of five cells per colony. For liquid culture colony assays, single cells were
sorted into individual wells of a 96-well plate containing 150 µl of media.
To determine the composition of large, bone marrow-derived blast colonies, these were cultured 10 d in liquid culture, replated in plasma clots and incubated an additional 5 d before DiffQuik staining and morphological analysis.
), MEP17
(anti-VLA-2; McNagny et al., 1992
; Bradshaw et al., 1995
), MYL51/2 (Kornfeld et al., 1983
), JS4 (Schmidt et al., 1986
),
IIb
3 integrin (anti-gpIIb/IIIa;
Lacoste-Eleaume et al., 1994
) or anti-c-kit (Vainio et al., 1996
) mAbs, or
with normal mouse serum followed by goat anti-mouse FITC-coupled antibodies (Dianova, Hamburg, Germany) as described previously (Graf et
al., 1992
). For two-color analysis, cells were stained as above, and then
with biotinylated anti-c-kit antibodies and streptavidin-phycoerythrin
(Dianova). Alternatively, cells were stained with the appropriate mAb
followed by goat anti-mouse antibodies coupled to phycoerythrin, and
FITC-labeled mAbs to JS4. All flow cytometric analyses were performed
using a FACScan® (Becton and Dickinson Co., Mountain View, CA). Cell
sorting was performed using FACStar® Plus and FACS® Vantage (Becton
and Dickinson Co.) cytometers. Single cell sorts were performed using the
CytocloneTM (Becton and Dickinson Co.) cell cloning attachment and
wells were checked microscopically for the presence of single cells.
) in cells suspended in culture medium (Graf et al., 1992
). Acid and neutral benzidine stains for hemoglobin
detection were performed as described previously (Beug et al., 1982b
).
Results
), peripheral
blood leukocytes were prepared and analyzed. They were
first stained with a combination of the mAbs MEP21 and 11C3, directed against the integrin
IIb
3, which is a hallmark of thrombocytes (Barclay et al., 1993
; Lacoste-Eleaume et al., 1994
). As can be seen from the FACS®
plot in Fig. 1 A, >90% of the cells that stained with the
11C3 antibody also reacted strongly with MEP21. The remaining 10% of the cells expressed low levels of MEP21
and no MEP21-positive cells could be detected that did
not also stain with 11C3. Next, the leukocytes were stained
with MEP21 antibody and sorted by FACS®. The plot in
Fig. 1 B shows that ~51% of the cells stained positive with
MEP21 antibodies while 49% were negative. Analysis of
the sorted fractions by cytocentrifugation and staining
with DiffQuik (Fig. 1 C) revealed that the MEP21-positive
fraction consisted almost entirely of thrombocytes (98%
of 309 cells counted), while the negative fraction contained
no thrombocytes at all (335 cells counted). Finally, both
fractions were subjected to Western blotting using the
MEP21 antibody. As shown in Fig. 1 D, the MEP21-positive but not the MEP21-negative fraction expressed a protein identical in size to that seen in lysates from two different MEP cell lines. In addition, erythrocytes as well as
myeloid and eosinophil cell lines, were negative in this
assay.
Fig. 1.
Expression of MEP21 protein by peripheral blood
thrombocytes. (A and B) Immunofluorescence analysis of peripheral blood leukocytes from 5-wk-old chicks. (A) Cells were
stained with a mouse mAb to IIb
3 integrin followed by a phycoerythrin-conjugated anti-mouse antibody and an FITC-coupled
MEP21 antibody. (B) Another aliquot of the above cells was
stained with MEP21 antibody followed by a phycoerythrin-conjugated anti-mouse antibody. MEP21+ and MEP21
fractions
(gates R1 and R2, respectively) were sorted by flow cytometry
and gave populations of >98% purity. (C) DiffQuik stained cells
from R1 and R2 fractions. (D) Western blot analysis of MEP21 expression of hematopoietic cell lines and sorted peripheral
blood cells. Designations of cells are indicated on the top of each lane.
[View Larger Versions of these Images (25 + 63 + 56K GIF file)]
and Wilm et al. (1996)
(Fig. 2).
Several of the peptides overlapped, leading to a total of 44 unique amino acid residues (underlined in Fig. 3). Database searches of these sequences revealed no significant homology. The sequences of peptides MEP21-1 and
MEP21-2 were used to prepare degenerate oligonucleotides to PCR amplify and to clone MEP21-specific cDNAs
from an HD100
ZAPII bacteriophage library (McNagny
et al., 1996
). One such PCR product was sequenced and found to contain the MEP21-2 peptide sequence, and this
was subsequently used to isolate 54-phage cDNA clones.
These clones fell into three categories according to differences in their coding sequence (Fig. 3 A).
Fig. 2.
Sequencing of MEP21 by nanoelectrospray mass spectrometry. (A) Mass spectrum of the unseperated peptide mixture
obtained after in-gel tryptic digestion of the protein band. Tryptic
peptide ions of MEP21 are marked by the letter T and their
charge state (number of protons attached). Peaks of trypsin autolysis products are designated by asterisks. (B) Peptide ion T4
(A) was isolated and fragmented in the collision chamber of the
mass spectrometer, leading to the spectrum shown. Fragmentation of tryptic peptides predominantly produces nested sets of
fragments containing the peptide COOH terminus (Y1, Y
2
, etc.
[Roepstorff and Fohlmann, 1984]), which allow assignment of the
sequence by their mass differences. (C) Tandem mass spectrum
of the same peptide as in B after esterification of the whole peptide mixture. Esterification results in 14-D mass shifts for the ions
containing COOH terminus plus an additional shift of 14 D for
each Asp and Glu residue as indicated by filled circles. Comparison of the tandem mass spectra of native and esterified peptide, B
and C, allowed unambiguous assignment of the peptide sequence.
Note that the isobaric amino acids Leu and Ile could not be distinguished and are designated by the letter L. The following peptide sequences were determined: T1, [AS] NEAFFEVFCSGR;
T2, [AS] NEAFFEVFCSGRR; T3, WAVHVLVHR; T4, VLDPAAVFEELK; T5, VLDPAAVFEELKEK; T6, VLDPAAVFEELKEKR; T7, ALLFLNR.
[View Larger Version of this Image (28K GIF file)]
Fig. 3.
Structure and coding capacity of MEP21 cDNA
clones. (A) Schematic representation of MEP21 type 1, 2, and 3 cDNA clones. Solid lines, 5- and 3
-untranslated
regions; stippled lines, alternative 3
-untranslated region;
and boxes, coding regions.
PAS, polyadenylation signal;
SP, signal peptide; TM, transmembrane region; S-T-P,
serine/threonine/proline rich
domain; C-C, potential disulfide-bonded domain. (B)
Amino acid sequence alignment of MEP21 (derived from
type 1 clone nucleotide sequence) and PCLP-1. Shaded
boxes, identity; dots, gaps; asterisks, potential phosphorylation sites (see text); MEP21-1,2,4, peptides sequenced by
mass spectroscopy; and TM,
the putative transmembrane domain. (C) COOH-terminal
amino acid alignments between MEP21 type 1, 2, and
3 clones. Vertical lines, sequence identity; and dots,
gaps. These sequence data
are available from GenBank/
EMBL/DDBJ under accession numbers Y13976, Y13977,
and Y13978.
[View Larger Version of this Image (45K GIF file)]
untranslated
region of 52 bp, followed by an open-reading frame of
1,713 bp encoding 571 amino acids, which includes three
out of four unique sequence stretches found by mass spectrometric sequencing. The 3
end consists of an untranslated region of 1,347 bp (Fig. 3 A). In the extracellular domain, the predicted protein contains a putative amino
terminal signal peptide and a 342-amino acid serine-, threonine-, and proline-rich domain with five potential N-glycosylation and numerous O-glycosylation sites. This is followed by a stretch of 108 amino acids containing four
cysteines that could form two globular domains, by a hydrophobic sequence of 24 amino acids that encodes a putative transmembrane region, and by a cytoplasmic region
containing a consensus protein kinase C and CKII phosphorylation site each. The observed difference between
the size of the predicted protein (54 kD) and the apparent molecular weight of MEP21 antigen (150 kD) suggests
that MEP21 is highly glycosylated. Together these features
suggest that the mep21 gene encodes a member of the mucin family of glycoproteins and we therefore now call the
MEP21 protein "thrombomucin".
untranslated region of type 1 clones in an A-rich region, suggesting that they originated because of internal priming of
A-rich sequences.
untranslated region beginning at the point where the deletion in type 1 clones begins (Fig. 3, A and C). Unlike the
other clones, type 3 clones contain a consensus polyadenylation signal.
).
Kyte-Doolittle hydrophobicity plots also revealed strong
similarity in this domain as well as a general similarity in
the extracellular domain (data not shown). The PCLP-1
gene is thought to encode the major sialoglycoprotein of
kidney podocytes and of vascular endothelia (Kershaw et
al., 1995
).
Fig. 4.
Endogenous and
exogenous expression of
MEP21 in hematopoietic
cell lines. (A) Northern blot
analysis of various hematopoietic cell lines using an
MEP21 probe: erythroid
lines HD3 and HD37; MEP
cell lines HD57 and HD100;
myeloblast line HD57M; macrophage line HD11; promyelocyte line HD13; eosinophil
line 1A1; B cell line RP-12;
T cell lines MSB1 and NPB4.
(B) Western blot analysis of
ectopically expressed MEP21
protein. Cells of the HD3
erythroid cell line were transfected with an expression
vector containing MEP21
cDNA in the sense or antisense orientation plus the
neo gene and selected for G418 resistance. Cl3 and Cl4, sense;
and Cl2, antisense transfected cells.
[View Larger Version of this Image (42K GIF file)]
Fig. 5.
Immunohistologic analysis of MEP21-reactive cells in 5-d-old chicken tissues. Arrows indicate podocytes in kidney; arteries, and capillaries in lung; capillaries in gut villi; and penicillary arteries and thrombocytes in spleen.
[View Larger Version of this Image (134K GIF file)]
).
Fig. 6.
Embryonic expression of thrombomucin. 4-d-old chick embryos were analyzed for MEP21 expression by either
whole-mount in situ staining (A) or staining of sections (B). Lower panels are high power magnification of upper panels. a,
dorsal aorta; n, neural tube; g, glomerulus;
d, duodenum; l, liver; v, heart ventricle; at,
heart atrium. Arrow, intra-aortic hematopoietic cells.
[View Larger Version of this Image (110K GIF file)]
) they were negative for most
other hematopoietic markers, including c-kit (a progenitor-specific receptor tyrosine kinase; for review see Callard and Gearing, 1994
), MEP26 (an MEP antigen that is
also expressed on thrombocytes and early erythroid cells; Graf et al., 1992
; McNagny et al., 1992
), the integrin
IIb
3, EOS47 (an eosinophil-specific transferrin; McNagny et al.,
1992
, 1996
), and MYL51/2 (a myeloid-specific antigen;
Kornfeld et al., 1983
). MEP21 and JS4 double staining revealed two distinct supbpopulations of MEP21-expressing
cells: MEP21hi only, and JS4hi/MEP21lo double-positive
cells (Fig. 7 A). To determine the colony-forming potential
of cells from the two subpopulations (R1 and R2), they were FACS® sorted (>95% purity) and seeded into
plasma clot cultures. The resulting colonies were then prepared 5 d later for DiffQuik staining and cytological evaluation. As shown in Fig. 7 B, the JS4hi/MEP21lo population
gave rise exclusively to late erythroid colonies (4-10 erythrocytes per colony), whereas all other hematopoietic precursors (early erythroid, thrombocytic, mixed erythroid/
thrombocytic, myelomonocytic, and eosinophilic) were
enriched in the MEP21hi-only fraction. Double-negative
cells yielded no colonies, suggesting that essentially all colony-forming units from these yolk sac preparations express thrombomucin.
Fig. 7.
FACS® analysis, sorting, and colony assays of day three
yolk sac cells. (A) A pool of embryonic day three yolk sac cells
were stained with MEP21 antibody followed by phycoerythrin-coupled anti-mouse antibody and an FITC-coupled antibody to
the erythroid marker JS4 and analyzed by FACS®. (B) Cells from
fractions R1 and R2 were sorted and 500 cells each plated in
plasma clot for 6 d before analysis of colonies formed. Columns
and error bars indicate the average colony numbers obtained per
culture (with standard error) of three cultures. LE, late erythroid
colonies; EE, early erythroid colonies; T/E, mixed thrombocyte
erythroid colonies; T, thrombocytic colonies; M, myelomonocytic
colonies; and Eo, eosinophilic colonies.
[View Larger Versions of these Images (42 + 27K GIF file)]
Fig. 8.
Thrombomucin expression in multipotent progenitors
of day three yolk sac. A total of 2,112 MEP21-positive cells (Fig.
7 A, R1) were sorted, seeded singly into 96-well plates, cultured
for 6 d and colonies were classified by microscopic inspection.
(A) Pie diagram illustrating the relative frequencies of the different colony types obtained: T, 177 thrombocytic; E, 128 erythroid; M, 119 myeloid; Eos, 3 eosinophilic; T/E, 23 thrombocytic/erythroid; M/T, 3 myelomonocytic/thrombocytic; and M/T/E, 4 myelomonocytic/thrombocytic/erythroid. (B) Light micrographs of
two separate colonies containing macrophages, erythrocytes, and
thrombocytes.
[View Larger Versions of these Images (70 + 108K GIF file)]
; data not
shown). As hematopoietic precursors from mammalian
bone marrow express c-kit (Ogawa et al., 1993
) and respond to kit ligand (stem cell factor or SCF) we performed
a two-color immunofluorescence analysis of chick bone
marrow using MEP21 and c-kit antibodies (no antibodies
to avian CD34 are available). As shown in Fig. 9 A, this
analysis revealed four distinct subpopulations: 13.1% c-kit
single-positive cells (R1); 5.8% c-kit/MEP21 double-positive cells (R2); 3.3% MEP21 single-positive cells (with a
small proportion of c-kitlo cells; R3); and 87.8% double-negative cells (R4).
Fig. 9.
FACS® analysis, sorting, and colony assays of bone
marrow cells. (A) Two-color immunofluorescence analysis of 5-d
chick bone marrow. Cells were stained with MEP21 antibody followed by FITC-coupled anti-mouse antibody, biotinylated anti-
c-kit antibodies and phycoerythrin-coupled streptavidin and analyzed by FACS®. (B) Cells from fractions R1, R2, and R3 were
sorted, 500 cells per plate seeded in plasma clots supplemented
with either SCF plus anemic serum (black), SCF plus cMGF
(red), or all three factors (white), and then cultured for 6 d before
analysis. Columns and error bars indicate the average and standard deviation of two separate experiments. Colony designations
are as in Fig. 6; BFU-E, burst forming unit erythroid, the earliest
detectable erythroid precursors.
[View Larger Version of this Image (29K GIF file)]
Fig. 10.
Micrographs of primary and secondary colonies obtained from bone marrow-derived progenitors. (A) Primary
blast-type colony, 10 d after seeding of MEP21-positive cells
(fraction R2) in medium containing SCF, anemic serum, and
erythropoietin. (B and C) Secondary plasma clot colonies obtained after seeding two different blast colonies into plasma clot
for 5 d and staining with DiffQuik. M, macrophages; E, erythrocytes; T, thrombocytes.
[View Larger Version of this Image (76K GIF file)]
Discussion
Fig. 11.
Differentiation potential and expression of thrombomucin by hematopoietic cells. (A) E26-transformed hematopoietic cells and (B) normal bone marrow cells. MEP, Myb-Ets-
transformed progenitors; MYE, myelobasts; THR, thrombocytes;
ERY, erythrocytes; EOS, eosinophils; GRA, granulocytes; MAC,
macrophages. The broken arrow lines in A indicate that differentiation is normally blocked at the MEP stage, but is inducible (see
text). The solid arrow lines in B denote the existence of multi-
and monopotent progenitors as inferred from colony assays. Cell
types expressing exclusively thrombomucin are indicated by the
pink shaded areas surrounded by the solid lines; those expressing
exclusively c-kit by the blue shaded area surrounded by the stippled line. Cell types expressing both antigens are indicated by the
gray shaded areas.
[View Larger Version of this Image (44K GIF file)]
).
In agreement with this notion, thrombomucin has a calculated mol wt of 54 kD and an apparent mol wt of 150 kD.
However, unlike many other mucins such as episialin, this
domain does not contain discernable sequence repeats
(Hilkens et al., 1992
; Gendler and Spicer, 1995
). The identification of differentially spliced transcripts, including one
that essentially lacks the cytoplasmic domain, suggests that
the protein has several functions. That these variant
cDNA forms are indeed translated into protein is suggested by the isolation of a peptide specific for type 2 cDNA as well as the detection of two proteins with slightly
different apparent molecular weights (Fig. 1). The cytoplasmic and transmembrane domains of thrombomucin
exhibit ~90% sequence similarity to the protein encoded
by the rabbit PCLP-1 cDNA (Kershaw et al., 1995
).
PCLP-1, in turn, probably corresponds to the kidney sialoglycoprotein, podocalyxin, an antigen defined by mAb,
which is expressed on the surface of rat and human
podocytes (Kerjaschki et al., 1984
; Kershaw et al., 1995
).
Despite the lack of sequence homology between thrombomucin and PCLP-1, in the extracellular domain both proteins
contain a Ser-Thr-Pro-rich domain. In addition, their almost-identical calculated and apparent molecular weights (Kershaw et al., 1995
), and the presence of thrombomucin
on podocytes as well as on a subset of vascular endothelial
cells (Horvat et al., 1986
) raises the possibility that these
proteins represent homologs from different species. It will
be interesting to determine whether PCLP-1/podocalyxin
is expressed on hematopoietic cells.
). This includes an intracellular domain of
about 75 amino acids containing consensus protein kinase
C and CKII phosphorylation sites and the generation of
both full-size and cytoplasmically truncated forms of the
molecule (Nakamura et al., 1993
). In addition, we have
shown that like CD34 (Fina et al., 1990
), thrombomucin is
expressed on embryonic and adult hematopoietic progenitors and vascular endothelial cells. However (at least in
mammals, and in contrast to thrombomucin), CD34 is expressed on committed granulocyte and macrophage precursors and has not been reported to be a marker of platelets (Olweus et al., 1996
). This, along with the lack of any
clear sequence identity between murine CD34 and the
murine thrombomucin homolog (McNagny, K.M., unpublished observations), argues that the two proteins are distinct. It is therefore possible that the very mild hematopoietic defect observed in CD34 ablated mice (Cheng et al.,
1996
) could be due to compensation by other mucins expressed by early progenitors such as thrombomucin.
; Bernex et al., 1996
). This suggests that
in both species c-kit is dispensible for progenitor cell development. It is possible that the small numbers of thrombomucin-positive multipotent progenitors observed in 3-d-old yolk sac are derived from definitive progenitors since
at this stage the first definitive blood cells develop and the
circulation is established (Dieterlen-Lievre et al., 1996
). In
adult bone marrow, c-kit and thrombomucin can be detected on the surface of multipotent hematopoietic progenitors as well as on erythroid precursors, but thrombomucin is present in addition on thrombocyte precursors
and c-kit on myeloid precursors (Fig. 11 B). Thus, the
combination of these two markers provides a simple
method for separation of different types of precursors.
IIb
3 is also expressed on both
MEPs (Frampton et al., 1995
) and normal mouse GEMM
cells (Tronik-Le Roux et al., 1995). Likewise, the thrombopoietin receptor is coexpressed by committed mammalian thrombocytes and multi-lineage precursors (Ku et al.,
1996
). Thrombomucin may therefore be one of a family of
molecules shared between thrombocytes and multipotent
progenitors.
) while ectopic expression of
episialin in adherent cell lines has shown that mucins can
act as anti-adhesion molecules (Hilkens et al., 1992
; Gendler and Spicer, 1995
). An anti-adhesive function has also
been discussed for thrombomucin's potential homolog,
podocalyxin. Podocalyxin maintains the glomerular filtration slits of podocytes in an open configuration via its
strong negative charge (Seiler et al., 1975
). By analogy, it
is possible that thrombomucin prevents thrombocytes
from inappropriately adhering to vessels walls. Another possibility is that, similarly to CD34, thrombomucin may
prevent the differentiation of hematopoietic cells. These
possibilities are now being explored by forced expression
of thrombomucin in myeloid and erythroid cell lines and
by ablating its expression in mice using a mouse thrombomucin homolog that we have recently isolated.
Received for publication 24 April 1997 and in revised form 26 June 1997.
Please address all correspondence to Thomas Graf, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany. Tel.: 49-62-21-387-411. Fax: 49-62-21-387-516. e-mail: Graf{at}EMBL-Heidelberg.deWe thank G. Döderlein for excellent technical support, A. Atzberger for cell sorting analysis, and T. Gibson and P. Bork for assistance in sequence analysis. We also thank T. Ilg, and L. Robb of the Walter and Eliza Hall Institute; C. von Kalle of the University of Freiburg; M. Greaves of the Chester Beatty Labs; and members of the Graf lab for discussions and comments to this manuscript. Anti-c-kit antibodies were the gift of B. Imhof and O. Vainio. We thank A. Shevchenko for expert sample preparation in mass spectrometry analysis.
cMGF, chicken myelomonocytic growth factor; MEP, Myb-Ets-transformed progenitors; PCLP, podocalyxinlike protein; SCF, stem cell factor.
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