The Eosinophil-Specific Cell Surface Antigen

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The Eosinophil-Specific Cell Surface Antigen, EOS47, Is a Chicken
Homologue of the Oncofetal Antigen Melanotransferrin
By Kelly M. McNagny, Fabio Rossi, Graham Smith, and Thomas Graf
The EOS47 antigen is a 100-kD cell sutface glycoprotein selectively expressed by avian retrovirus-transformed eosinophils and their precursors. We have purified theEOS47 protein t o homogeneity andused peptide sequence information
t o clone EOS47-encoding cDNAs. The open reading frames
from these cDNAs predict a 738 amino acid protein with
homology t o human melanotransferrin, a membrane-bound,
transferrin-like protein that isexpressed at high levels by a
subset of melanomas, tumor cell lines, fetal intestine, and
liver, but not by most normal adulttissues. The predicted
protein sequence of EOS47 displays a 61% sequence identity
with melanotransferrin and conservation of all 28 cysteine
residues, indicating a similar tertiary structure. The finding
that EOS47 lacks several of the iron-coordinating amino
acids present in all transferrins suggests that it may be impaired in its abilityt o bind iron. In nonhematopoietic tissues,
EOS47 is expressed at high levels by epithelial brushborders
of small intestine and kidney and at lower levels by cells
lining the sinusoids of the liver. Within hematopoietic tissues,EOS47 is restricted t o a subpopulation of cells (1%
t o 5%) in bone marrow and early spleen and fluorescenceactivated cell sorting of EOS47+ cells leads t o a dramatic
(>JO-fold) enrichment of peroxidase+ eosinophils. In contrast, peripheral blood eosinophils are EOS47-, suggesting
that theantigen is expressed by newly formedeosinophils
and that expression ceases shortly before these cells emigrate from the bone marrow into theperipheral blood. Our
results showthat
melanotransferrin is a stage-specific
marker of eosinophils and should be useful for theirisolation
and further characterization.
0 1996 by The American Society of Hematology.
E
transformed progenitors, myeloblasts, and eosinophils.” One
antibody, named EOS47, specifically reacts with peroxidase’
eosinophils (an enzyme that in avian hematopoietic cells is
expressed exclusively by eo~inophilsl~”~)
and their precursors, butnotby transformed cells of other hematopoietic
lineage^.^',^^ We report here the cloning and sequencing of
several EOS47 encoding cDNAs. The predicted protein sequence shows a high degree of homology with human melanotransfemn, a transferrin-like molecule expressed selectively by melanomas and other nonhematopoietic tumor cell
lines. Analysis of antigen expression by normal hematopoietic cells suggests that melanotransferrin may become a valuable lineage and maturation stage-specific marker of eosinophils.
OSINOPHILS ARE ONE OF three distinct subpopulations of polymorphonuclear granulocytes produced in
vertebrate bone
Although they comprise a minor
subpopulation of the peripheral blood leukocytes (approximately 2% to 5%), they are rapidly recruited to sites of
hypersensitivity (particularly in bronchial asthma) and to
sites of parasitic (helminths) infections. The primary immunologic role of eosinophils appears to be the destruction
of multicellular parasites by degranulation and release of a
number of toxic, granule proteins including major basic protein, eosinophil cationic protein, eosinophil derived neurotoxin, and eosinophil per~xidase.~
In chronic bronchial
asthma, eosinophils play a major role in inflammation and
pathologic respiratory cell damage by the release of this
same complement of granular protein^.^.^ Several cytokines
are known to promote the production of eosinophils from
bone marrow hematopoietic precursors. Interleukin-3 (IL-3)
and granulocyte-macrophage colony-stimulating factor
(GM-CSF) enhance the production of eosinophils and precursors for several other hematopoietic lineages, whereas
IL-5 exclusively promotes the production and terminal differentiation of eosinophil^.^^^ Colony-forming assays in the
presence of mixtures of these cytokines have suggested that
eosinophil precursors represent a heterogeneous pool of cells
that vary intheir cytokine requiremenk6 However, relatively
little is known about the early stages of eosinophil differentiation, largely due to the lack of lineage-specific surface
markers that would allow the isolation and characterization
of normal precursors and due to the limited number of differentiation-inducible eosinophilic cell lines.’”’
We reported previously the ability of the myb-ets-containing avian retrovirus E26 to transform cells with the properties of multipotent hematopoietic precursors (referred to
as MEPs for Myb-Ijts ~ r o g e n i t o r ~ ’These
~ ~ ~ ~transformants
).
spontaneously differentiate into erythrocytes and thrombocytes and can be efficiently induced to differentiate into
either myeloblasts or eosinophils by treatment with phorbol
esters or by over-expression of kinase type oncogenes.’*J3
Using this in vitro differentiation system, we have generated
a panel of monoclonal antibodies (MoAbs) specific for E26Bfood,Vol 87, No 4 (February 15). 1996: pp 1343-1352
MATERIALS AND METHODS
Animals, primary tissues, and cells. All primary tissues and cells
werederivedfromcommerciallyproducedValoeggs
and birds
which were maintained at the EMBL animal care facility.
Cell lines and culture conditions. Theoriginsofthecelllines
used as sources of RNA have been described previously:HD3 erythroblasts”; HD44 erythroblasts”; HDl1 macrophages(described
HD13
HD57
earlier as LSCC-MCMAl)”;
multipotent cells”; HD57M myelobla~tsl~;
HD5OM-GATA-l + eosinophils”; MSB-I T cellsz3;and RP-l2 B cell^.'^ To generate the
From the Differentiation Program, European Molecular Biology
Laboratory, Heidelberg, Germany.
Submitted July 27, 1995; accepted September 27, 1995.
K.M.M was supported by International Human Frontier Science
Program Organization fellowship No. LT-434/92 and by NRSA Fellowship No. F32 HM736from the National Heart, Lung and Blood
Institute, National Institutes of Health.
Address reprint requests to Thomas GraJ; PhD, Differentiation
Program, European Molecular Biology Laboratory, D-691I7 Heidelberg, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0004-4971/96/8704-00$3.00/0
1343
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1344
HDlOO cell line, 2-day-old chicken blastoderms were infected with
the E26 virus and plated in semisolid medium as described previously.13 Transformed colonies were pooled and serially passaged
for 6 months to select for continuously proliferating cell lines.
Screening subclones of these cell lines by surface immunofluorescence led to the identification of one clone that constitutively expresses the EOS47
All cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal calf serum, 2.5% chicken
serum, 0.15% NaHC03, 56 pg/mL of conalbumin, 80 mmol/L 2mercaptoethanol, 0.9 pg/mL insulin, and the standard complement
of antibiotics at 37°C in 5% CO,. Medium for HDSOM and HDl1
cells was supplemented with chicken myelomonocytic growth fact ~ r . ~ ~ . ~ ~
Protein puri’cation and sequencing. Proteins from approximately 1010 HDl00 cells were solubilized in 50 mL of lysis buffer (150 mmol/L NaCI, 50 mmol/L Tris [pH 7.51, 0.5% NP-40)
plus protease inhibitors (1 mmol/L phenylmethylsulfonylfluoride
[PMSF], 20 rnmoVL e-amino-n-caproic acid, 1 mg/mL leupeptin,
and 2.5 U/mL trasylol) on ice for 30 minutes. Nuclei were removed
by centrifugation at 15,OOOg for 30 minutes at 4”C, and the supernatant was incubated overnight at 4°C with200 pL of EOS47 antibodycoupled Sepharose beads (4 mg of antibody coupled per milliliter
of CNBr-activated Sepharose resin; Pharmacia, Uppsala, Sweden).
Beads were washed 10 times with 2 mL of lysis buffer containing
protease inhibitors and once with phosphate-buffered saline (PBS)
plus PMSF, and bound proteins were eluted in 0.1% trifluoroacetic
acid plus PMSF. Eluted fractions were equilibrated to neutral pH
by the addition of Tris buffer, lyophilised, resuspended in sample
buffer, and resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Resolved proteins were
blotted onto Immobilon-P filters (Millipore, Bedford, MA) using
CAPS buffer (10 mmol/L 3-[cyclohexylamino]-l-propanesulfonic
acid [pH lo], 10% methanol), andthe filters were stained with
0.1% Coomassie Blue R-250 in 40% methanol/lO% acetic acid and
destained in 50% methanol. Filter strips containing EOS47 protein
were digested with trypsin, fragments were separated by reversephase high-performance liquid chromatography (HPLC; Applied
Biosystems model 170A; Applied Biosystems, Foster City, CA), and
individual peptides were sequenced by automated Edman degradation using an Applied Biosystems sequencer (model 477A) connected to an online phenyl thiohydantoin analyzer (Applied Biosysterns model 120). Two peptides with the highest homology to human
melanotransfenin, EOSTRIS-41 and EOSTRIS-42, were used for
degenerate oligonucleotide primer synthesis.
Sequence comparisons were performed using FASTA searches of
the Genembl and Swissprot databases. Evolutionary trees were derived using the Clustal V software and Pileup alignments of sequences retrieved from the Swissprot database. FASTA and Pileup
are part of the Wisconsin package Version 8.0 (Genetics Computer
Group Inc, Madison, WI). Clustal V software is publicly available
through the EMBL FI’P site (ftp.embl-heidelbergde).
Polymerase chain reaction (PCR) and library construction. Degenerate, PCR primers were designed based on the sense sequence
for the EOSTRIS-41 peptide (5’-GCIGTIGTIGTIMGICCIGAYACIGAYAAYACIGCIGTI’lTYCAM-3‘) and
the
antisense sequence for the EOSTRIS-42 peptide (5’-TCICCICCIGCIGGIACNAGNCCRTA-3’). Poly(A)+ RNA was prepared from HDlOO total
RNA (see below) using oligo(dT)-celluloseZxand then converted to
single-stranded cDNA using a IZAP-cDNA synthesis kit (Strategene,
La Jolla, CA) according to the manufacturer’s instructions. PCR of
single-stranded cDNA was performed using Taq polymerase (Pharmacia) and buffer conditions recommended by the manufacturer.
Amplification was performed by 30 cycles of 30 seconds of denaturation at 95”C, 30 seconds of template annealing at 40°C and 90
McNAGNY ET AL
seconds of elongation at 72°C. using an “Intelligent Heating Block”
(Biometra, Goettingen, Germany). PCR products were cloned into
plasmids using a TA cloning kit (InVitrogen, San Diego, CA) and
transformed into Escherichia coli strain XI-l blue, and 2 positive
clones were identified by restriction map andor sequence analysis
of 20 recombinants.
AnHDlOO cDNA phagemid library was constructed from 5 pg
poly(A)+ RNA using a XZAP-cDNA synthesis kit (Strategene) according to the manufacturer’s instructions andwas packaged into
viable phage using Gigapack Gold I1 (Strategene) packaging extracts. One million recombinant phage from the unamplified library
were plated on XL-l M W ’ bacterial host strain and these were
screened by hybridization with a ”P-labeled EOS47 PCR fragment
probe” (see below). Ten recombinants were identified and plaquepurified by two further rescreens, and plasmids were produced by
in vivo excision usingthe protocols recommended bytheAZAPcDNA synthesis kit manufacturer (Strategene). Inserts were sequenced by the EMBL DNA sequencing service usingan EMBL
automated sequencer or its commercial counterpart (A.L.F. DNA
sequencer; Pharmacia).
Nucleic acidhybridization.
For Northern blot analysis, total
RNA wasprepared by lysis and fractionation in guanidinium/acetate/
phenolkhloroform as described by Chomczynski and Sacchi.” Approximately 10 pg of each RNAwas resolved on a 1% agaroseformaldehyde gel and blotted onto nylon membranes (Genescreen;
Dupont, Newtown, CT) as described by Sambrook et al.’* Hybridization of radiolabeled probes and removal of unbound probe was performed in NaHPOJSDS buffer, as described previou~ly.~~
All hybridization probes were labeled with [a-”P] dCTP by random hexamer priming as described by Feinberg and Volgelstein.’”
The following cDNA fragments were used as probes: a 0.45-kb PCR
fragment of EOS47 (see above); a 1.5-kb Pst I fragment of EOS47
type 1, 2, and 3 clones; a 0.45-kb 3’ Sac I fragment specific for
EOS47 type 2 clones; a 0.3-kb 3’ Spe I fragment specific for EOS47
type I clones; and a GAPDH-specific probe.”
Immunohistologic analysis, immunojuorescence, and peroxidase
staining. Dissected tissues were imbedded in Tissue-Tek (Miles),
snap frozen in liquid NZ. and stored at -80°C. Frozen sections (4
pm) were cut using a -20°C microtome, fixed on glass slides for 5
minutes in acetone, and dried at -20°C. The tissues were covered
for 20 minutes with 100 mL of 10% calf serum in PBS and then
stained with 100 mL MoAb followed by biotinylated goat antibodies
to mouse IgG. Tissues were incubated for 30 minutes at room temperature, washed three times with PBS, and then soaked 20 minutes
in methanol containing 0.3% H2OZto block endogenous peroxidases.
The methanol-treated sections were washed with PBS, incubated for
30 minutes with 1 0 mL of avidin-biotin peroxidase complex reagent
(Vector Labs, Burlingame, CA), and exposed to 0.5 mg/mL of 3,3‘diaminobenzidine substrate (Sigma, St Louis, MO) inPBS containing 0.03% H202.After developing for IO minutes, the sections
were washed with PBS, counterstained with 1%methyl green,
washed briefly in H 2 0 , dehydrated in graded alcohols, and mounted
for microscopic analysis.
For PI-PLC digests and indirect immunofluorescence analysis, 1
x lo6 cells were washed twice in PBS and resuspended in 500 mL
of Hanks’ balanced salt solution (HBSS; GIBCO, Life Technologies,
Petersburg, MD) containing 2 mmol/L HEPES (pH 7.4) in the presence or absence of 0.35 U of PI-PLC (Sigma). Cells were washed
2 times in PBS and stained with EOS47,” MEP17 (anti-VLA-2)”””
or MYL51/2” MoAbs or with normal mouse serum, as described
previously.’3 All flow cytometric analyses were performed using a
Becton Dickinson FACScan (Becton Dickinson, Franklin Lakes,
NJ). Cell sorting was performed using Facstar Plus (Becton Dickinson) and Epics Coherent (Coulter, Hialeah, E)cytometers.
Eosinophil peroxidase staining was performed by modification of
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MELANOTRANSFERRIN AS AN EOSINOPHIL MARKER
1345
clone, suggesting an unsplicedmessage (Fig 1A). These data
suggest thatthe type 3 cDNAcorresponds to an unprocessed
precursor that gives rise to the type 1 and 2 messages via
alternative polyadenylation and splicing; when polyadenylation occurs at the most 5' site, the type 1 message is exclusively produced, whereas downstream polyadenylation
RESULTS
allows in frame splicing of the type 2 tail to occur.
Primary structure of the EOS47 protein. The predicted
Production of the HDlOO cell line, purijication of the
amino
acid sequence for EOS47 type 1 clones is shown in
EOS47 protein, and peptide sequencing. Pools of primary
Fig 1B. The 738 amino acid sequence shows striking homolE26-transformed progenitor clones were serially passaged
ogy to members of the transferrin supergene family. Highest
until cell lines were established and subcloned(see Materials
homology
(61.1% identity) was observed with human melaand Methods and Metz and Graf"). One of these cell lines,
notransfemn,
the only transfemn family member known to
named HD100, expresses several phenotypic markers charoccur in a membrane-boundf ~ r m ?This
~ , ~homology
~
begins
acteristic of E26-transformed progenitor lines, butis unusual
just after the putative 19 amino acid signal peptide and ends
in that it also expresses high levels of the EOS47 antigen
abruptly at the beginning of a carboxy-terminal stretch of
and, unlike most E26-transformed progenitor lines, fails to
27, largely hydrophobic, amino acids (Fig l b and data not
differentiate on treatment with TPA'2~13*20
(unpublished obshown).
servations). This cell line was used as a source of EOS47
Despite their membrane localizations, both the human and
protein. For this purpose, proteins from approximately 1 X
avian
proteins lack optimal transmembrane sequences due
10"HDlOO cells were solubilized by detergent lysis and
to
proline
and hydrophilic residues within the relatively hyEOS47 waspurified to homogeneitybyimmunoaffinity
drophobic C-terminal domain (Fig 1B and Rose et a135). In
chromatography followed by preparative gel electrophoresis
addition, both proteins lack charged residues beyond this
and electroblotting. Purified EOS47 was then digested with
potential
transmembrane stretch that could serve to anchor
trypsin, and peptides were separatedby reverse-phase HPLC
them
on
the
cytoplasmic face of the endoplasmic reticulum,
and sequenced.
Golgi, and plasma membrane. In the case of human melanoAFASTA search of the Swissprot data base withthe
transferrin, it has been shown that the hydrophobic tail
is
resulting peptide sequences showed three with greater than
cleaved
and
a
glycosyl-phosphatidylinositol
anchor
(GPI)
is
58% sequence identity to human rnelanotran~ferrin."*~~
added
cotranslationally
or
posttran~lationally.~~.~~
To
test
if
Based on the sequence of two peptides, EOSTRIS-41 and
EOS47 shares this posttranslational modification, HDlOO
EOSTRIS-42, degenerate oligonucleotides were synthesized
cells were stained for surface EOS47 and VLA-2expression
and used to PCR amplify and subclone a 453-bp cDNA
(a known transmembrane protein) before and after treatment
probe (see Materials and Methods). This probe was used to
with phosphoinositide-specific phospholipase C(PI-PLC),
screen an HDlOO cDNA library cloned in a AZap bacterioan enzyme thatspecifically cleaves GPI-linked tails from
phage vector. Ten positive clones were identified and subproteins.39As shown in Fig 2, pretreatment of HDlOO with
cloned from 1 X lo6recombinants in an unamplified HDlOO
PI-PLC removes virtually all of the detectable EOS47 from
library. Based on restriction mapping and sequencing of 5'
the cell surface, whereas no change is observed for VLA-2
and 3' termini, these clones can be separated into three cateexpression. This would indicate that EOS47is also expressed
gories (Fig 1A). Type 1 clones (8 clones) display identical
as a GPI-linked molecule.
restriction maps and 3' untranslated regions and vary onlyin
The carboxy-terminal tail encoded by type 2 clones is
the length of their 5' termini (probably due to heterogeneous
shown in Fig 1C. The alternative tail replaces the last 27
stopping of reverse transcriptase during cDNA synthesis).
amino acids of the coding sequence with a 10 amino acid
The longest of these clones (2,802 bp) was sequenced and
stretch of hydrophilic amino acids, suggesting that this mesfound to contain a predicted 5' untranslated region of 172
sage encodes a secreted form of themolecule(Fig1C).
bp followed by an open reading frame of 2,214 bp and a 3'
Consistent with this observation, expression of the type 2
untranslated region of 404 bp (Fig 1A and B, anddata base
clone in insect cells using a baculovirus vector results in
access no. X91908). The end of this 3' untranslated region
high level production of EOS47 in the culture supernatant
contains an imperfect polyadenylation signal sequence AT(data not shown). Soluble forms of human melanotransferrin
TAAA (consensus AATAAA)followed 18 bplater by a
have also been detected previously in melanoma culture supoly(A) tail. The second type of cDNA clone (1 clone) has
pernatants, but it was proposed that these were not the result
an alternative 3' tail that begins within the last 5 1 bp of the
of variable mRNA processing?'
coding region and ends with the poly(A) tail (Fig 1A and
Although the calculatedmolecularweight
of EOS47
C). The 3' untranslated region of this clone contains two
(80,912 Daltons) is significantly less than the 1 0 0 - k D apparperfect polyadenylation signals 13 and 31 bp upstream of
the poly(A) tail. These alternative 3' termini should result
ent molecular weight observed for the antigen purified from
in a different coding sequence at the carboxy-terminus of
avian eosinophils,'* this discrepancy may be accounted for
the protein (Fig 1C and see below). The third type of cDNA
by glycosylation at one of the six potential N-linked glyco(one clone) is identical to the type 1 cDNAs throughout its
sylation sites (one of which is shared with human melanosequence up to the polyadenylation site but then contains a
transferrin). The protein contains 28 cysteine residues, all of
unique 72-bp sequence followed by the 3' tail of the type 2
which are perfectly conserved with melanotransfemn, sug-
the procedure described by K a p l o ~ ?which
~
allows the analysis of
peroxidase expression in viablecell^.'^ Myeloperoxidase activity has
not been detected in several detailed studies of avian granulocytes
andgranularperoxidasehasprovento
be an exclusive marker of
the eosinophil lineage in chickens.""
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McNAGNY ETAL
1346
A
SP
Type1
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Type2
4
Type3
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R1
I
R2
R1
I
R2
R1
I
R2
P
SP
1.0 kb
I
B
1bMetLys
Z3bArgTrp
SerSer
GluAsn
ValLeuTyrLeuLeuLeuVal
Cys Thr Met Ser Asn G l nG l u
45bAla G I ~I I Leu
~ ProProLeu
67bLysAspTyrLeu
GIU
89bGly LeuLys
Pro ValVal
G l yG l u
His AlaAla
Leu Ser Lys Cys LysAspMet
cysMet
GIU
A l a Asp Thr Val Thr Leu
111bVal Val A r g LysGly
GIY
GIU
GlnGlu
I I ~
Tyr GlnAlaGlyLysGluTyr
I l e Gly Thr Ser X T y r A l a Val A l a
Asn Ser LeuLysGly
Val Pro Val G ~ YTyrLeu11e
Cys ASP Leu Pro Lys A l a Val Ser AspTyr
Leu Ser Leu G l u A r g Val
Ser Asn A l a Phe Thr Gly
Ser AI^ ~ ~ a l A scys
n Thr 1GlnMet
G l yA r gT r pI l e
ValTyrAsp
Serl-iThrle
1 3 3 b l l e l A s n A r g Thr [ A l a GIY TrpAsn
155bMetGty
I
PheSer
Val A r g Ser Cys H i s Thr Gly
ASP Ser GIY A r g Leu Pro Ala
A l a SerCys
Val ProGly
Thr Asn
SerSer
GI y G l n AsnLys Cys
177bSer A l a Ser TyrPro Thr Ser Leu Cys GI n Leu Cys LysGlyAsp
199bGlnGly Asn Ser GlnGluGlnTyrTyrAsp
X S e r G l y A l a Phe A r g Cys Leu A l a G l u G l y A l a
H i s Ser ThrVal
Z Z l b G l yG l uV a lA l a
Phe ValLys
243bTrpAlaGlnGln
Phe A r g Ser LysAsp
265bThrGluTrpArg
Thr Cys His Leu A l a A r g Val P r o A l a A r g A l a
287bAspGly
Phe Asp Ser Thr A l a TyrGlyAlaGln
A r g Thr Leu Ser Thr
Leu Cys Argl-lThr
Thr A l a Val Ph&Ginku- Leu Asn Gln Gly Gln Gln
309bPhe GlnMet
331bLeuVal
353bAlaLeu
P r oG l u Asn Thr AspGly
Phe GlnLeu
A l a AspVal
Val ValVal
a P h e Asn Asp Val G l y A l a G l n
AsnLeuMet
Phe A r gA s p
Ser Thr Thr Lys
A l a ValThr
Ser G l n AsnTyr
G l n A l a T r p LeuGlyAspGluTyr
Leu HisGlyMetGln
Ser Cys Asp Pro Asn Thr Leu P r oG l u Ser LeuAsn T r p Cys Val Val Ser Thr G l uG l u
375b11eTrpLysCys’GIyGluMetGly
Thr A l a Phe A r g Ser LysAsnLeuLysProGlu
3 9 7 b l l e Ser A l aL y s
Cys MetGluMet
419bLeu ~
AreProsp-Thl
G
Thr LysGluGlu
I l e G l n Cys
I l e Asp ValVal
Ala
I l e Tyr I l e A l a GIy&s_Ih~Tyrr~eu-Val P r o A l a A l a G l ~ L u A e r _
l Val
y Asp
441bme- Ser A l aG l u
I l e G l n LysLysGlu
AspAsnAsnAsn
Ala
Tyr A l a Val A l a LeuValLys
GI yLysLys
463bAl a Phe Thr I l e Asn AspLeuLys
485bTrp Asn I l e P r o I l e G l y MetLeuValLysLys
ArglAsnPro
Ser IAsn
SerCys
H i s Thr Gly Leu Gly Arg
Thr AI a GI y
Gly Phe I l e Asn Pro Arg Asp Cys Asn I l e P r o
507bGlnAla Val Ser G l u Phe Phe Ser A l a SerCys
Val P r o Ser A l aG l uG l nG l y
Asn Tyr P r o Ser
Gly AsnAsnLys
CysSer
A l a SerSer
G l nG l u
529bThrLeu Cys GlnLeu Cys I l e G l y AspAsnAsn
551bArgTyrTyr
Ser X A s n G l y A l a Phe A r g Cys Leu A l a G l u A s p A l a G l y A s p
Val A l a Phe Val
573bLys H i s Ser Thr Val Phe GluAsn
Thr AspGlyLysAsn
Thr G l u Ser T r p A l a A r g
AspLeuLys
59SbSer Ser Gly Phe GInLeuLeu
Cys A r g A s n G l y A l a A r g A l a G l u
Val Thr Gln Phe A l a G l n Cys
His ProAsp Thr Asn I l e Phe A l a LeuTyr
617bHisLeuAlaArg
Val P r o A l a - A l a I l e
MetVal
AI^ G I ~
GIU Tyr Phe ~ ~ y l A s
Asn
n S e r l A s n A r g Asn G I Phe
~
LYS Met Phe
639bGly LeuLeu
ASP LYS
661bAsp SerSer
A l a Phe GlnGlyLysAspLeu
6 8 3 b G l uG l uA r gA r g
70SbThr P r oG l n
Thr Tyr A l a G l u T r p
CysSer
G l yA l aG l y
727bPro Phe I l e I l e LeuGlyGln
C
Type 1
Type 2
I I e Phe LysAsp
Leu Gly Ser GluTyr
AsnLysLeu
Leu G l nG l y
I l e GlnGln
Ser A l a Val Lys I l e Val Pro Val
Val Glu Ser LeuGluGlyMetGln
H i s LeuLeuVal
I l e Thr Phe Val
Leu Gly
705
738
. . . MQTPQCSGAGNKLI QbHLLVI TFVPFI I LGQLQG
IIIIIIIII
. . . MQTPQCSGAVSPELCFQKR
gesting a similar overall tertiary structure (Fig 3). Like melanotransfenin, EOS47 contains two repeated domains that,
by analogy with other vertebrate transfenin family members,
should correspond to two independent iron-binding domains
(Fig 1B and3A). Structural analysis of vertebrate transfemns
indicates that four amino acids in each repeat are crucial
ligands for coordinating iron, Asp-60, Tyr-92, Tyr-193, and
His-253 (using the human lactofenin numbering system),
and that anadditional Arg residue at position 143 is required
for binding of a CO:- anion (Fig 3A and B).36 Human mela-
R,
.. .
R2
Fig 1. Structure and coding
capacityof
the EOS47
cDNA
clones. (AI Schematic representation ofEOS47cDNAclones.
Solid lines indicate5‘- and 3’-untranslated regions; stippled lines
indicate alternative 3”untranslated regions present in type 2
and type 3 clones. PAS indicates
polyadenylationsignals, with an
asterisk indicating an imperfect
signal (see text). Boxes indicate
codingregions. SP,signal p e p
tide; GPI, putative glycosylphosphatidylinositolanchor signal; S, putative secreted-form
tail; R1 and R2, amino- and carbow-terminal iron-binding repeats,respectively.
IB) Amino
acid sequence of EOS47 protein
derived from type 1 cDNA sequence. The hydrophobic signal
peptide and
carboxy-terminal
domains are underlined, as is the
division between putative ironbinding repeats. Dashed lines indicatepositions of EOSTRIS-41
and EOSTRIS-42 peptides that
were used to produceprimers
for PCR cloning. Boxes indicate
potential N-linked glycosylation
sites. Bold and underlined amino
acids indicate positions of ironbinding amino acids deduced by
alignment with human melanotransferrin, serum
transferrin,
and lactofenin. (Cl Carboxy-termina1 amino acid alignments for
putative GPI-linked and secreted
forms of €OS47 encoded bytype
1 and type 2 cDNAs,respectively.
notransferrin has only one functional iron-binding domain
per molecule, presumably due to a point mutation of Asp to
Ser in the carboxy-terminal iron-binding domain and mutation of Arg to Serin the anion binding site (Fig 3B).” EOS47
contains a similar point mutation of Asp to Gly in the carboxy-terminal iron binding site and an additional mutation
of His to Glu within the same domain (Fig 3B). This second
mutation is a relatively conservative substitution and, although it has never been observed in any vertebrate transferrin, it is present in the amino-terminal domain of two
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
1347
MELANOTRANSFERRINAS A N EOSINOPHIL MARKER
VLA-2
EOS47
+
PLC
-
PLC
Control
mAb
Fig 2. Analysis of EOS47 attachment to the
p~asmamembrane.
HD100 c e l l s were treated with
PI-PLC or mock-treated.The presence of cell rutface
EOS47 or VIA-2 on each preparation
was then evaluated by antibody staining and FACS analysis.
Relative Fluorescence Intenstty (log)
Expression pattern of EOS47 mRNA and protein in cell
lines and tissues. EOS47 mRNA expression was analyzed
in a number of hematopoietic cell lines. A major transcript
of 2.8 kb and a minor transcript of 3.5 kb were observed in
the antigen-positive cell lines HDlOO and HLXOM-GATAl + (an eosinophil cell line recently produced in our lab'*)
but not in antigen-negative B-, T-, myeloid, erythroid, or
progenitor cell lines (Fig 5A). Similar sized bands were detected with type l and type 2 specific probes, although the
type 2 probe yielded considerably weaker hybridization with
the 2.8-kb band (data not shown). These results suggest that
the 3.5-kb transcript corresponds to the unspliced type 3
clone that contains untranslated regions detected byboth
type 1 and type 2 probes (Fig 1A).
Analysis of RNA from tissues of 2-day-old and 5-weekold chickens showed transcripts in intestine, liver, and kidney, but not in bone marrow, brain, bursa of Fabricius (the
primary site of B lymphopoiesis in birds), eye, Harderian
gland (an ocular associated tissue rich in plasma cells in
birds), heart, muscle, and spleen (Fig 5B). The lack of detect-
insect transferrins which have retained the ability to bind
iron (Fig 3B).4'342In the amino-terminal domain of EOS47,
there is a nonconservative mutation of the neutral His to
positively charged Arg that should ablate the ability of this
position to coordinate iron (Fig 3B). The data therefore suggest that the amino-terminal domain of EOS47 may also
have a lower affinity for iron or that the protein may have
evolved a completely novel ligand specificity.
Computer alignment of the primary amino acid sequence
of EOS47 with 11 transferrin family members from various
species and construction of an evolutionary tree for transferrin family members shows a distinct segregation of human
melanotransferrin and EOS47 (Fig 4). This cosegregation
of EOS47 and human melanotransfenin suggests again that
EOS47 represents the avian homologue of the human protein
and that the divergence of melanotransferrins from serum
transferrins and lactofemns occurred before the divergence
of birds from mammals. This also indicates that melanotransfenin is highly conserved during evolution and probably
exerts an indispensable function in higher vertebrates.
A
S
I
Repeat 1
Repeat 2
H
B
IRON AND ANION COORDINATING AMINO ACIDS
Protein
5aur.x
€OS47
Avisn
Melanotransterrin
Human
R
e
m
a
!1
D
Y
R
Y
E
ReDeatP
B
Y
R
Y
Q
D
Y
R
Y
H
S
Y
S
Y
SBNm Transtenin * Vertebra!e
D
Y
R
Y
H
D
Y
R
Y
H
Laclotranstenin+
Vertebrate
D
Y
R
Y
H
D
Y
R
Y
H
Tranabmn Q
Invertebrate
D
Y
R
Y
Q
D
Y
R
Y
H
*
+
0
bovine. porcine. equiw. and avian 8aNm tmndenin
invariant in human. bovine. and m u d ladofenin
invariant In human, mbbii.
ssquem from mck roach transtsnin
H
Fig 3. Structure of EOS47
andputativeiron-binding sites
(A) Diagram of repeat structure
of EOS47 protoin. Hatched boxes
indicate hydrophobic aequmces
(signal peptida and GP1 signal).
Vettical linesindicatecysteine
residues.Arrowheadsindiccrte
putativeironcoordinatingresidues. (B)Putative iron and anion
coordinatingreddues in transferrin family members. Bold and
underlined
residues
indicate
variations from the consensus
sequence.
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McNAGNY ET AL
1348
rather than an enrichment of eosinophils (data not shown),
confirming that the purification of eosinophils using EOS47
antibody is not due to nonspecific binding of mouse IgG.
Despite the increase in eosinophils in EOS47-enriched
bone marrow, a small percentage of eosinophils was still
detectable in EOS47-depleted fractions, suggesting that not
all bone marrow eosinophils express the antigen. This observation was confirmed in experiments in which eosinophils
were enriched by Percoll density fractionation and subsequently stained for EOS47 expression (Fig 6C and D). Bone
marrow eosinophils tended to sediment in density fractions
from 1.06 to 1.09 g/mL, whereas peripheral blood eosinoEOS47
MothTf
phils banded in fractions from 1.09 to 1.11 g/mL. In the
high-density fractions from bone marrow, the frequency of
Fig 4. Evolutionarytree of transferrins.EOS47,EOS47/chi&en
melanotransferrin; HuMTf, human melanotransferrin; HuSTf, human EOS47+ cells exceeded that of peroxidase’ cells. Because
serum transferrin; RaSTf,rat serum transferrin; PoSTf, porcine serum E26-transformed progenitors undergoing eosinophil differtransferrin; EqSTf, equine serum transferrin; XeSTf, Xenopus serum
entiation in vitro usually express EOS47 before peroxidase,
transferrin; ChSTf, chicken serum transferrin; HuLTf, human
ladoferthese cells may represent committed eosinophil precursors
rin;MuLTf,murineladoferrin;BoLTf,bovineladoferrin;MothTf,
that have not yet acquired peroxidase
However,
Manduca sexta transferrin.
consistent with the cell sorting experiments, several fractions
contained more peroxidase+ cells than EOS47+ cells, indicating that a small proportion of mature eosinophils lack expresable expression in bone marrow and spleen probably reflects
sion of the antigen (Fig 6C and D). In fact, none of the
the low frequency of antigen positive eosinophils in these
peripheral
blood fractions enriched for peroxidase+ eosinotissues (see below). Although expression of human melanophils contained significant numbers of
EOS47’
cells
transferrin in liver and embryonic intestine has been reported
previously, expression in kidney has not been o b ~ e r v e d . ~ ~ . (>OS%,
~ ~ . ~ see Fig 6D). Similar results were obtained from
both young (8 day) and juvenile (5 week) chickens (data not
To further delineate the cell types expressing EOS47 in
shown). This suggests that, before or soon after leaving the
kidney, intestine, and liver, protein expression was analyzed
bone marrow, eosinophils lose expression of the antigen.
by immunohistology of frozen tissue sections (Fig 5C). In
intestine, EOS47 protein is expressed at high levels on the
DISCUSSION
apical face of epithelial cells on the brush borders of the
In this study, we have cloned and analyzed several cDNAs
villi. In young animals (5 days posthatching), the staining is
encoding the eosinophil-specific protein EOS47 and further
most prominent in the crypts, suggesting an association with
characterized its distribution on normal avian cells. The folimmature enterocytes, but in older animals the antigen is
uniformly expressed throughout the length of the villi, suglowing observations suggest that the EOS47 antigen is the
avian homologue of human melanotransferrin. First, the progesting expression throughout enterocyte maturation (Fig
teins exhibit 6 1% sequence identity, including conservation
5C). In kidney, EOS47 is also expressed by brush borders in
of all 28 cysteine residues (many of which are not conserved
the thick-walled proximal tubules and is absent on glomeruli,
with lactoferrin or serum transferrin), indicating similar prodistal tubules, and collecting tubules. EOS47 is also weakly
tein folding and tertiary ~tructure.~’
Second, of the known
expressed by sinusoid endothelial cells in the liver (Fig 5C).
transferrin supergene family members, only EOS47 and huWe previously reported the expression of EOS47 by a
man melanotransferrin occur predominantly in membranesmall percentage (2% to 4%) of peroxidase+ and peroxidasebound forms and in both cases this is due to posttranslational
cells in bone marrow and spleen.” These results were conor cotranslational addition of a GP1 anchor, a relatively unfirmed in experiments in which bone marrow was separated
common posttranslational modifi~ation.~’,~~
Expression of
into EOS47+ and EOS47- fractions by fluorescence-actilow levels of soluble melanotransferrin has also beenrevated cell sorting and these fractions were assessed for the
ported for human melanoma cells, but whether these arise
frequency of eosinophils by staining for eosinophil peroxidue to RNA processing or posttranslational processing has
dase. The results obtained from one of three such experinot been resolved.37 Our data would indicate that, in birds,
ments are shown in Fig 6A and B. From a starting population
expression of a soluble form of the molecule may in fact be
of 1.3% EOS47+ bone marrow cells, one round of sorting
regulated at the level of alternative polyadenyiation and preresulted in a population which was 3 1.1% EOS47+ and these
mRNA splicing. Such a mechanism has previously been
were then resorted to greater than 70% positive. The percentshown to govern the production of secreted versus transage of peroxidase+ eosinophils was dramatically increased
membrane Ig heavy chain in which there is a switch in
in all EOS47-enriched fractions, from 0.9% in unsorted bone
the efficiency of cleavage and polyadenylation during the
marrow to 15.9% after the first round of sorting and to 34.9%
maturation of B cells into plasma
after the second round. Control sorts were performed in parIn addition to their shared biochemical and structural propallel using nonspecific isotype-matched control antibody or
erties, EOS47 and human melanotransferrin also showan
the MYL51/2 antibody that is weakly expressed by avian
overlapping distribution in nonhematopoietic tissues, immyeloblast^.^^ In bothcases these sorts resulted in a depletion
XeSTf
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B
Fig 5. Expression pattern of EOS47 (A) Northern
blot analysis of RNAs from EOS47- and EOS47+
(HD100and HWM-GATA-l) hematopoieticcell lines
(approximately l0 p g total RNAllane) using EOS47
PCR probe. Cnwchybridizcltion with 18s ribosomal
RNA represents aninternal control for RNA loading.
(B)Northern blot analysis of RNAs from 2-day-old
chicken tissues (approximately 10 p g total RNA/
lane) using the EOS47 f s t I probe. Small box shows
resultsobtainedby hybridizationwith GAPDH probe.
(C) lmmunohrtologic analysis of EOS47-reactive
cells in X d a y o l d intestine (original magnifkation
x 200),2dayold kidney (original magnification x
100). and 2-dayold liver (original magnification x
430). Arrows indicate staining on brush borders of
intestinal villi, brush borders of proximal tubules in
kidney, and sinusoidal cells in liver, respectively.
A
E EOS47-
1.06
' Relativ;Fluordscance
1.07
1.08
1.09
EOS47++
1.10
' Intensity
7
Percol1 Fraction ( g / c d )
Fig 6. Analysis of EOS47+cells in bone marrow and blood. (A) Fluorescence-activatedcell sorting of EOS47+ cells from 2-dayoldchicken
bone marrow. Closely spaceddotted lines, control stain; widely spaced dotted lines, EOS47 staining before sorting;solid lines, EOS47 staining
(B)The percentage of peroxidase+ cellsin various
after one round of sorting. The percentage positive
of
cells above background are indicated.
EOS47-sorted fractions. Uns, unsorted bone marrow; EOS47-, EOS47depleted fractions; EOS47+, bone marrow cells after one of sorting
(31.1% EOS47+),EOS47++, bone marrow cells after two sequential sorts for EOS47(>70%EOS47+).(C and D) Percentage of EOS47+ or
peroxidase+ cellsin density fractionated bone marrow (Cl or peripheral blood (Dl. Cells from a 2day-old chick were separated in 0.01 glmL
stepwise P m l l density gradients; hatched bars,the percentage of EOS47+ cells determined by flow cytometry (solid bars, the percentage of
peroxidase+ cells). (E)Light micrograph EOS47++and EOS47- sorted fractions from (B)after granular-peroxidase stain.
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
1350
plying conservation of function. Thus, both proteins are expressed on the brush border linings of intestinal epithelial
cells and on the apical surface of sinusoidal cells in the
liver.38.43.44 However, our results also suggest that EOS47
has a broader pattern of expression because it is expressed
by intestinal epithelia in juvenile birds long after hatching,
whereas in humans melanotransferrin has been reportedto be
fetal intestine-specifi~.~~~~~~~
It also remains to be determined
whether human melanotransferrin, like EOS47, is expressed
by early eosinophils and by brush border cells of the proximal tubules in kidney.
In previous experiments, we have shown that in vitro differentiation of hematopoietic precursor cell lines results in
high level expression of this antigen by eosinophilic precursors just before the expression of eosinophil peroxidase and
that the expression then gradually decreases as these cells
mature and terminally differentiate.l2.I3In thepresent studies,
we have shown a corresponding distribution on normal eosinophils and expression by eosinophils and presumptive
precursors in bone marrow but notby circulating eosinophils
in peripheral blood. Although several molecules have been
described to be specifically expressed in eosinophilic granules (such as eosinophil peroxidase, eosinophil cationic protein, major basic protein, and eosinophil-derived neurot ~ x i n ) to
, ~ our knowledge this is the first description of an
eosinophil-specific membrane protein in any species. Thus,
EOS47/melanotransferrin should be a valuable reagent for
the isolation and study of eosinophil precursors, particularly
if a similar expression pattern is found on mammalian eosinophils. In this regard, it will be of interest to see if melanotransferrin is also a marker of newlyformed mammalian
eosinophils and their precursors (manuscript in progress).
Because these cells comprise a very minor subpopulation of
total bone marrow cells, the expression of melanotransferrin
in mammals could easily have been overlooked in previous
studies.
The reported tumor-specific nature of the human melanotransferrin protein, the observation that melanoma patients
can develop an antibody response against this antigen, and
the finding that a recombinant vaccinia virus expressing melanotransferrin can generate a T-cell response and protective
immunity to melanoma cells in mice have led to the promotion of this protein as a potential target for immunotherapy
of melan~ma!~-~'Yet, despite the attention this molecule has
attracted in tumor models, relatively little is known about
its function. Because all rapidly proliferating cells require
iron for ribonucleotide reductase and other enzymes essential
to cell division, it has been proposed that melanotransfemn
acts as an alternative iron adsorption mechanism to the classic transferridtransfenin receptor mediated pathwayand
thereby augments the proliferation of tumor ~ e l l s . How~~~~'
ever, studies performed to address this hypothesis have
yielded inconclusive or even conflicting r e s ~ l t s ,and
~ ~sev*~~
eral lines of evidence suggest that high affinity iron-binding
might not be the primary function of the molecule. First,
human melanotransferrin has acquired point mutations that
inactivate one iron-binding domain and thereby lower the
molecule's overall avidity for iron and reduce its efficiency
as an iron transporter.@ Similarly, the avian molecule has
MCNAGNY ET AL
acquired deleterious mutations in both iron-binding pockets,
which suggests that it has evolved away from high-affinity
iron-binding. Second, growth of HDlOO cells in the presence
of iron-chelating agents or hemin has no effect on the levelof
the avian melanotransferrin expression, whereas these agents
have been shown to have profound effects on the expression
of a number of molecules involved in iron uptake and storages4 (unpublished observations). Similarly, despite the proposal thatmelanotransfemn bypasses the transferrin receptor
pathway for iron uptake, neither avian nor human RNAs for
melanotransfenin contain iron-responsive elements (IRES),
which have proved to be the key regulators of transferrin
receptor expre~sion.'~
Lastly, the highlevel expression of
the antigen in the proximal tubules of kidney also argues
against this idea. Undernormal circumstances, free iron
never reaches the proximal tubules in kidney because it remains bound to transferrin that is retained in the glomerulus
during blood filtration. Thus, this distribution does not correlate with an iron binding function.
What then is the primary function of melanotransferrin?
An intriguing possibility is suggested by the recent cloning
and analysisof another transferrin-like protein known as
saxiphilin.55-57 This amphibian serum protein was first identified
on the basis of its ability to specifically bindsaxitoxin, a low
molecular weight neurotoxin. Although saxiphilin displays a
high overall homology to serum transfemns, it lacks all but
one of the known iron-ligating amino acids and has probably
evolved a novel specificity for exogenously or endogenously
produced toxins.56 Thus, therea is
precedence fornovel ligand
specificities in transferrin-like molecules. By analogy, melanotransferrin may have evolved to bind and inactivate toxic
substancespresent in intestineorgeneratedduringkidney
filtration or eosinophilmaturation.Furtherexperimentsare
required to elucidate the molecule's true function.
Because of its restricted distribution within the hematopoietic system to early eosinophils, analysis of the regulatory
elements that govern the expression of melanotransferrin
may provide insights into mechanisms governing eosinophilspecific gene expression. Recently, we have shown that the
overexpression of the GATA-1 transcription factor in avian
myelomonocytic cells is sufficient to convert these cells into
eosinophils." Similarly, overexpression of the CEBPP transcription factor (NF-IL-6 or NF-M) in multipotent hematopoietic precursors results in eosinophilic differentiati~n.~~
In
these experiments, the expression of EOS47 represented one
of the first detectable phenotypic changes, suggesting that
the gene is a direct target of GATA- 1, CEBPP, and perhaps
additional transcription factors. It is noteworthy that GATA1 and CEBPP are coexpressed in eosinophils, although they
are expressed individually in a number of other hematopoietic lineage^.'^^^^^^^ This suggests that a combinatorial effect
of these factors may be required for eosinophilic differentiat i ~ n . ' *We
. ~ ~are presently analyzing the EOS47 promoter in
an attempt to define how these and other factors influence
eosinophil differentiation.
ACKNOWLEDGMENT
We thank Ulrich Muhlner for help in sequence analysis of EOS47
mRNA subtypes; Klaus Hexel and Anne Atzberger for cell sorting
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MELANOTRANSFERRINAS AN EOSINOPHIL MARKER
analysis; Tony Houthave for peptide sequence analysis; Drs Des
Higgins, Toby Gibbs, and Gemt Vriend for assistance in protein
modeling, sequence alignments and analysis; and Drs Micheline
Chu, Jon Frampton, Holger Kulessa, Claus Nerlov, and Michael
Sieweke for discussions and suggestions and critical evaluation of
this manuscript. We also are indebted to Hilary Davies-Riick and
Servane Leillard for help in preparation of the manuscript.
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From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
1996 87: 1343-1352
The eosinophil-specific cell surface antigen, EOS47, is a chicken
homologue of the oncofetal antigen melanotransferrin
KM McNagny, F Rossi, G Smith and T Graf
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