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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1819-1826
RED CELLS
A study of the coregulation and tissue specificity of XG
and MIC2 gene expression in eukaryotic cells
Claude Fouchet,
Pierre Gane,
Martine Huet,
Marc Fellous,
Philippe Rouger,
George Banting,
Jean-Pierre Cartron, and
Claude Lopez
From Inserm U76, Institut National de la Transfusion Sanguine, and
Institut Pasteur, Paris, France, and the Department of Biochemistry,
University of Bristol, Bristol, United Kingdom.
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Abstract |
CD99, the product of the MIC2 gene, exhibits an
erythroid-specific quantitative polymorphism coregulated with the
polymorphism of the XG blood group gene. As a preliminary study of this
phenomenon, human XG and CD99 recombinant proteins were expressed in
murine RAG cells and analyzed by flow cytometry. Both proteins were
expressed independently and at a similar level in single and double
transfectants. Immunoprecipitation and Western blot analysis, using the
murine monoclonal antibodies NBL-1 and 12E7, revealed species of 26 kd (XG) and 32 kd (CD99), respectively. A putative 28-kd intracellular precursor of CD99 was also detected, as was a 26-kd species after neuraminidase treatment of CD99-expressing cells. No evidence of
association or complex formation between XG and CD99 proteins could be
proven, either on transfected RAG cells or on human erythrocytes. These
results were confirmed using somatic hybrids between single transfectants. These findings suggest that the phenotypic relationship between XG and CD99 is mostly regulated at the transcriptional level,
but they do not formally exclude some posttranscriptional effect.
Studies on the tissue specificity of XG expression showed that
surface expression of the XG protein could not be restored in somatic
hybrids between B-lymphoblastoid cell lines from Xg(a+) persons and fibroblasts (RAG) or erythroid (MEL) cells. RT-PCR analysis
of the transcripts revealed the existence of an XG mRNA in each
cell line, suggesting that the tissue-specific regulation of cell
surface XG expression occurs either at a quantitative transcriptional
level or is a posttranscriptional event. By Northern blot analysis,
XG transcripts were detected in erythroid tissues and several
nonerythroid tissues.
(Blood. 2000;95:1819-1826)
© 2000 by The American Society of Hematology.
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Introduction |
The human erythrocyte antigen Xga, a 26-kd
glycoprotein, was identified with an antibody found in a patient who
underwent multiple transfusions.1,2 It is inherited as a
sex-linked dominant character,2 and its phenotypic
frequencies are different in males and females. The polymorphism is
defined by the Xg(a+) and Xg(a ) phenotypes because no antigen
antithetical to Xga has been found. Approximately 89% of
females and 66% of males are Xg(a+).1 The
XG polymorphism could result from different amounts of the XG
protein on Xg(a+) and Xg(a) erythrocytes,3 but this
has yet to be proven. One of the 2 X chromosomes in a female is
inactivated early in embryonic development.4 However, unlike most X-linked genes, the XG locus escapes this
inactivation.1 The Xga antigen has also been
found on human fibroblasts in culture.5
The murine monoclonal antibody 12E7, raised against lymphocytes from a
patient with T-cell acute lymphocytic leukemia,6 recognizes
a cell surface glycoprotein of 32 kd, called CD99, which is encoded by
the MIC2 gene.7,8 The MIC2 gene is borne by
the X and Y chromosomes9 and is localized in the
pseudoautosomal regions of the short arms Xp and Yp,8 which
pair and exchange in male meiosis. The MIC2 locus on the X
chromosome is not subject to inactivation.10
The MIC2 gene is expressed in many tissues and shows an
erythrocyte-specific quantitative polymorphism that is coregulated with
the polymorphism of the XG gene.11 All Xg(a+)
persons are CD99-high expressers, and all Xg(a) females are
CD99-low expressers. Xg(a) males can be CD99-high or CD99-low
expressers. To explain this phenotypic relation, the existence of a
regulatory locus XGR, present on both X and Y chromosomes, has
been postulated.12 In this model, XGR controls the
expression of MIC2 and XG and is polymorphic with 2 alleles, A and B. The A allele induces
Xga antigen expression and high-level CD99 expression; the
B allele fails to express Xga and induces low-level
CD99 production. The coexpression of the 2 genes, XG and
MIC2, would lead to the cell surface production of the 2 proteins and would be controlled at the transcriptional level.3
MIC213,14 and XG15 genes have
been cloned, and their organization on the short arm of the X
chromosome has been (Xp) determined. The 2 genes encompass 150 kb of
DNA located on both sides of the pseudoautosomal boundary of Xp, and
both are oriented toward the centromere. The first 3 exons of
XG are situated in the pseudoautosomal region, approximately 10 kb downstream of MIC2 (10 exons), whereas the other 7 exons are
in the Xspecific region.
The CD99 and XG proteins are sequence related (48% homology) and share
some unusual motifs in a similar order, suggesting that the 2 genes
originated from the duplication of an ancestral gene. The predicted
structure of CD9916 and, by analogy, of XG defines an
integral membrane protein with a single transmembrane region, an
extracellular amino terminus, and a cytosolic carboxy terminus.
Immunochemical studies on Xg(a+) and Xg(a) erythrocytes demonstrated the quantitative polymorphism of CD99 at the cell surface
and indicated that the XG and CD99 proteins from Xg(a+) red blood cells
(RBCs) can be copurified with the 12E7 antibody17 or
coprecipitated with anti-Xga antibodies.18
These data suggested that the 2 proteins may be closely associated in
the erythrocyte membrane, possibly as a heterodimer.
To understand the molecular basis of the phenotypic relationship
between XG and CD99, we analyzed the coexpression of the XG and
MIC2 cDNAs in transfected mammalian cells, either in double transfectants or in somatic hybrids from single transfectants. We also
studied the tissue specificity of XG expression at the RNA and
protein levels.
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Materials and methods |
Subcloning of the XG and MIC2 cDNAs in the
expression vector pcDNA3 and its derivative pcDNA-Hyg
Full-length cDNAs were cloned by polymerase chain reaction (PCR)
from a human fetal liver cDNA library using published
information13-15 and sequenced on both strands by the
dideoxy chain termination method.19 The XG and
MIC2 cDNAs were subcloned as BamHI-EcoRI fragments in the pcDNA3 expression vector (Invitrogen, San Diego, CA)
to give pcDNA3-XG and pcDNA3-MIC2, respectively. For
cotransfection experiments, pcDNA3 was modified by replacement of the
selection marker as follows: the 1.9-kb NruI-HindIII
fragment from p220.2 (gift from Dr B. Sugden, University of Wisconsin,
Madison, WI), containing the hygromycin resistance gene
(hygR) under the control of promoter and
processing signals from the tk gene of HSV1, was filled in with
the Klenow fragment of Escherichia coli DNA polymerase I and
inserted between the blunt-ended SmaI and BsmI sites
(removal of the neomycin resistance cassette,
neoR) of pcDNA3 to obtain pcDNA-hyg. The
0.6-kb, filled-in BamHI-EcoRI XG or
MIC2 fragments were introduced into the EcoRV site of
pcDNA-hyg to give the plasmids pcDNA-hyg-XG and
pcDNA-hyg-MIC2, respectively. All plasmids used were propagated
in E. coli XL1 blue.
Cell lines and cell culture
RAG cells (mouse adenocarcinoma cell line) were maintained in IMDM
(Life Technologies, Paisley, UK) supplemented with 10% (vol/vol) fetal
calf serum (FCS). MEL cells (murine erythroleukemia) were obtained from
the American Type Culture Collection (Rockville, MD) and were cultured
in Dulbecco's modified Eagle's medium (DMEM; Life Technologies)
supplemented with 20% FCS. Epstein-Barr-derived B-lymphoblastoid cell
lines (B-LCLs) obtained from 2 healthy male donors CT and CL, typed as
Xg(a+) and Xg(a)/CD99-high expressor, respectively, were
established in our laboratory and maintained in IMDM supplemented with
20% FCS. NBL-1,3 a mouse hybridoma cell line producing a
monoclonal anti-Xga antibody, was cultured in RPMI 1640 supplemented with 10% FCS and HAT (Sigma, St. Louis, MO). Red blood
cells from Xg(a+) and Xg(a) donors typed as CD99-high or
CD99-low expressers were from the Centre National de
Référence pour les Groupes Sanguins (Paris, France).
Antibodies
The murine monoclonal anti-Xga was purified from
NBL-13 hybridoma supernatants by protein A-Sepharose
chromatography, as described.20 The 12E7 murine monoclonal
antibody6 was kindly provided as an ascites fluid by Dr
Peter Goodfellow (University of Cambridge, Cambridge, UK) and was
purified by the same procedure. Binding constants for 12E7 and NBL-1
antibodies were determined by Scatchard analysis as described
before.17A polyclonal antibody directed against residues 1 to 13 of the mature XG protein was produced in rabbits according to
standard protocol. The human anti-Xga (serum
Alo.) was from the Centre National de
Référence pour les Groupes Sanguins.
Stable transfection of RAG cells
RAG cells were transfected with the different expression vectors by
calcium phosphate coprecipitation.21 NeoR and
hygR transfectants were selected in IMDM plus 0.35 mg/mL
G418 (Geneticin; Life Technologies) or 1 mg/mL hygromycin (Life
Technologies), respectively. When less than 50% of the resistant cells
expressed the protein of interest, they were sorted as follows: the
cells were incubated with the appropriate monoclonal antibody (NBL-1 or
12E7) and then with magnetic beads covered with sheep antimouse IgG
(Dynal A.S., Oslo, Norway) according to the instructions of the
manufacturer. The sorted cells and resistant pools containing more than
50% expressing cells were then subjected to limiting dilution cloning
in 96-well microtiter plates.
Cell fusions (somatic cell hybrids)
All fusion experiments were performed with polyethylene glycol 400 (Merck, Darmstadt, Germany).22 Adherent hybrids generated by the fusion of RAG cells (hypoxanthine
phosphoribosyltransferase-deficient or HPRT) with B-LCLs (CT and
CL) were selected in DMEM plus HAT because B-LCLs are HPRT+ and
nonadherent. Hybrids between RAG transfectants were selected in IMDM
plus 0.35 mg/mL G418 and 1 mg/mL hygromycin. Hybrids between the clone
RAG-XG13 (neoR) and B-LCLs were selected in IMDM plus HAT
and 0.35 mg/mL G418. Before the fusion of MEL cells with somatic
hybrids RAG × B-LCLs (CT or CL), MEL cells and RAG × B-LCLs
were stably transfected with pcDNA3 and pcDNA-hyg, respectively. The
MEL/neoR × RAG B-LCL/hygR hybrids were
subsequently selected in DMEM plus HAT, 0.55 mg/mL G418, and 0.5 mg/mL hygromycin.
Flow cytometry analysis
Surface expression of the XG and CD99 antigens was determined after
incubation with NBL-1 or 12E7 monoclonal antibodies. The antibody-binding capacity was determined using calibration mouse IgG-coated beads (Qifikit; DAKO, Glostrup, Denmark) as standards. Before analysis, adherent cells were detached from plastic with 50 mmol/L HEPES (pH 7.3), 125 mmol/L NaCl, 5 mmol/L KCl, and 1 mmol/L EDTA
because the XG and CD99 proteins are sensitive to trypsin
treatment.17,23 Cell lines (5 × 105),
in a phosphate-buffered saline (PBS)/0.2% (wt/vol) bovine serum albumin suspension, were incubated for 1 hour at 4°C with
antibodies used at saturation. After they were washed with PBS, cells
were incubated for 30 minutes at 4°C with R-phycoerythrin
(RPE)-conjugated F(ab')2 fragment of goat antimouse
immunoglobulins (DAKO). TO-PRO-1 iodide (Interchim, Montluçon,
France)-positive cells (dead cells) were excluded from analysis.
Fluorescence was measured on a FACScan flow cytometer (Becton
Dickinson, San Jose, CA).
Northern blot analysis
Total RNAs from cell lines were extracted by the TRIZOL reagent
(Life Technologies), and 20 µg was resolved by electrophoresis on a
6% (wt/vol) formaldehyde, 1% (wt/vol) agarose gel and transferred to
nylon filters Zeta-probe GT (Bio-Rad, Hercules, CA). Hybridization with
the 32P-labeled XG or MIC2 cDNA probes and
stringent washes were performed as described.24 Human
multiple-tissue Northern blots (Clontech, Palo Alto, CA) containing
polyA+ RNA from different human tissues were hybridized with the XG
or MIC2 cDNA probes in the ExpressHyb solution (Clontech),
according to the manufacturer's instructions.
Western blot analysis
Membrane proteins from 107 cells (transfectants, somatic
hybrids) were solubilized with 1% (wt/vol) Triton X-100 in ice-cold 10 µmol/L Na-phosphate (pH 7.4), 150 mmol/L NaCl (PBS) containing the
protease inhibitors 1 mmol/L 4-(2 aminoethyl)-benzenesulfonylfluoride (Pefabloc-SC; Boehringer Mannheim, Germany), 10 µg/mL leupeptin (Sigma); and 5 µg/mL pepstatin (Sigma) for 30 minutes at 4°C with gentle shaking. After centrifugation at 37,000g for 45 minutes, one-fortieth supernatant (cell lysate) was subjected to sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by
immunoblotting with NBL-1 or 12E7 monoclonal antibodies, essentially as
described,17 except that incubation with antibodies and
washings were performed in PBS, 5% (wt/vol) skimmed milk and PBS, and
0.1% (wt/vol) Tween, respectively. Specifically bound antibodies were
detected by chemiluminescence with the ECL reagent (Amersham, Bucks,
UK). For neuraminidase treatment, 107 transfected cells or
5 × 109 RBCs were resuspended in 1 vol
receptor-destroying enzyme (RDE; or neuraminidase from Vibrio
cholerae; Boehringer Mannheim) diluted (0.1 or 0.25 U) in 50 mmol/L
Na-acetate (pH 5.5), 150 mmol/L NaCl, 9 mmol/L CaCl2, and
100 µg/mL bovine serum albumin and were incubated for 30 minutes at
37°C. After 3 washes in PBS, transfected cells were lysed and
proteins were analyzed as above. Membranes from treated or untreated
RBCs were prepared,25 and protein content was
determined.26 Fifty micrograms was analyzed by SDS-PAGE and
immunoblotting with NBL-1 or 12E7. Specifically bound antibodies were
detected by chemiluminescence with the ECL reagent (Amersham).
Immunoprecipitation analysis
Intact erythrocytes (109 cells) or cell lines
(107 cells) were sodium iodide 125-labeled by IODO-GEN
(1,3,4,6-tetrachloro-3 -6-diphenylglycoluril; Pierce, Rockford,
IL) as described before.20 RBC membranes were prepared and
incubated with 10 µg NBL-1 (or the anti-Xga serum Alo.)
or 12E7 overnight at 4°C, whereas labeled cells were incubated for
1 hour with the antibodies. After membrane solubilization (RBCs and
cell lines), a preformed complex of protein A-Sepharose (10% wt/vol)
and of rabbit antimouse IgG (10 µg) in PBS containing 1% (wt/vol)
Triton X-100 was then added to the mixture (100 µL) for at least 1 hour at 4°C. The immune complexes were washed on sucrose
gradients,20 and specifically bound proteins were eluted from the final pellet by boiling for 5 minutes in 10 mmol/L Tris-HCl (pH 6.8), 1 mmol/L EDTA, and 5% (wt/vol) SDS separated by SDS-PAGE and
detected by autoradiography.
Reverse transcription-PCR analysis of the XG transcripts
Ten micrograms total RNAs from cell lines was reverse transcribed
using the first-strand cDNA synthesis kit (Pharmacia, Uppsala, Sweden).
The XG fragment (551 bp) was amplified by a nested PCR between
primers XG1 (sense primer, positions 36 to 16
from the translation initiation codon) and XG4 (antisense
primer, positions +571 to +548), then between primers XG2
(sense primer, positions 6 to +15) and XG3 (antisense
primer, positions +545 to +522). All reactions were performed in the
Expand High Fidelity PCR System (Boehringer Mannheim) under the
following conditions: 30 cycles of 1 minute at 94°C, 1 minute at
60°C, 1 minute at 72°C, and a final elongation for 7 minutes at
72°C. Specific products were analyzed by agarose gel
electrophoresis and hybridization with an internal
[32P]-labeled oligonucleotide probe.
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Results and discussion |
Cell surface expression of the XG and MIC2 cDNAs in RAG cells
and somatic cell hybrids
Full-length cDNAs corresponding to the known coding sequences of
XG15 and MIC227 were cloned by
PCR from a human fetal liver cDNA library. The sequence of the
XG cDNA was identical to the published sequence, whereas
sequence analysis of the MIC2 cDNA revealed 1 base substitution
G493A changing Asp to Asn at position 165 of the CD99 protein
(cytosolic tail). Whether this change reflected a polymorphism of the
population was not investigated.
Murine fibroblastic RAG cells were first stably transfected with either
XG or MIC2 cDNA in either pcDNA3 or pcDNA-hyg
expression vectors. NeoR and hygR cells
expressing either XG or CD99 proteins at their surfaces were selected
and cloned as described in "Materials and methods." Flow
cytometry analysis revealed positive staining with either NBL-1 or 12E7
antibodies on transfected cells, indicating that the XG and CD99
proteins, respectively, are readily transported to the plasma membrane
of RAG cells. This is the first report showing the expression of
recombinant XG protein at the surface of transfected cell lines,
whereas CD99 has already been produced in mouse L cells.28
The relative amount of XG and CD99 expressed at the surface of the
transfectants was estimated through the antibody-binding capacity of
the monoclonal antibodies NBL-1 and 12E7, respectively. Preliminary
experiments by Scatchard analysis indicated that the binding constant
(Kd) for 12E7 was 2.5 × 10 8
mol/L,17 whereas the Kd for NBL-1 was
0.5 × 108 mol/L (data not shown).
Antigen-site determination by flow cytometry analysis was performed
using saturation conditions for each antibody (antibody concentration
equal to 10 times the Kd values), using calibrated mouse
IgG-coated beads. Accordingly, the estimated number of XG and CD99
copies on these cells is an approximate extrapolation from the maximum
number of antibody molecules bound to the cells (assuming an ideal
molar 1:1 ratio of antibody bound per antigen). Although absolute
values of XG and CD99 on each transfectant is difficult to compare (see
below), it is clear that in all cases more molecules of CD99 than XG
were expressed on the transfectants (and red cells), but the exact
ratio between the 2 values is uncertain. These estimates of the number
of XG and CD99 molecules at the cell surface also assumed that the
antigenic determinants were fully accessible to mAbs on intact cells.
The clones described in Table 1 represent
the best XG and CD99 expressers among a total of 24 clones tested for
each transfection. All clones apparently express a higher level of CD99
than XG at the cell surface. When cDNAs were expressed from the pcDNA3
vector (selection with G418), the difference was approximately 10-fold
(XG13, 3 × 104 copies/cell; MIC22,
3.1 × 105 copies/cell), whereas with the pcDNA-hyg
vectors (hygromycin selection), there was a 30-fold difference (h.XG66,
7.8 × 104 copies/cell; h.MIC11,
2.5 × 106 copies/cell). Such a difference was also
seen with erythrocytes from Xg(a+) persons, in whom the average numbers
of XG and CD99 molecules per cell are approximately 102 and
103, respectively (unpublished results). More molecules
were expressed on transfectants when hygromycin rather than neomycin
selection was used, and this increase was greater for the MIC2
cDNA (XG copies/cell increase only 2-fold between XG13 and h.XG66;
CD99 copies/cell increase 10-fold between MIC22 and h.MIC11). The
higher level of CD99 expression, compared with XG, was apparently not caused by the toxicity of XG affecting cell growth because we found
that the average doubling rate of clones that expressed XG (XG13 and
h.XG66) or did not express XG (MIC22 and h.MIC11) was similar (5 doublings per 24 hours).
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Table 1.
Relative levels of the XG and CD99 antigens at the
surface of transfected RAG clones and somatic hybrid derivatives
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The XG13 and MIC22 clones were next cotransfected with the plasmids
pcDNA-hyg-MIC2 and pcDNA-hyg-XG, respectively, and new clones were obtained under G418 plus hygromycin selection. Again the
same characteristics of expression were observed as with the single
transfectants (see Table 1) a higher cell surface level of CD99 than
XG in every clone and a higher expression of the MIC2 gene when
expressed from the pcDNA-hyg vector than with the pcDNA3
(neoR) vector. The number of XG or CD99 antigen copies/cell
was equivalent between double and single transfectants for the same
vector, pcDNA3 or pcDNA-hyg (Table 1), suggesting that the level of
cell surface expression of these proteins is not interdependent because
the coexpression of 1 protein (XG or CD99) has apparently no effect on
the cell surface expression of the other. The same result was obtained
with double transfectants of the clone h.XG66 with pcDNA3-MIC2 or of the clone h.MIC11 with pcDNA3-XG (not shown).
To further confirm these data, we generated somatic cell hybrids by
fusion of the individual clones and analyzed the expression of the 2 proteins (Table 1). Within experimental limits, there was no
significant variation in the number of XG or CD99 molecules between
single transfectants (eg, XG13, 3 × 104 XG
copies/cell; h.MIC11, 2.5 × 106 CD99 copies/cell)
and double hybrids (eg, XG13 × h.MIC11, 1.5 × 104
XG copies/cell and 1.8 × 106 CD99
copies/cell). Single hybrids used as controls also showed equivalent
amounts of surface proteins (XG13 × hygR,
2.1 × 104 XG copies/cell;
neoR × h.MIC11, 1.9 × 106 CD99
copies/cell).
Transcripts and genomic analysis of transfectants
The XG and MIC2 cDNAs are under the control of the
cytomegalovirus promoter in the expression plasmid pcDNA3. To explain
the difference in the cell surface expression of XG and CD99, we
estimated the transcription efficiency of each plasmid in the RAG
transfectants by measuring the number of integrated plasmid copies and
the approximative level of each transcript. Southern blot analysis of
genomic DNA indicated that the expression plasmids were integrated at
approximately 1 copy per cell in each transfectant (not shown),
excluding gene dosage as the principal cause of the apparent difference
in levels of cell surface expression of CD99 and XG. Next, RNA from the different clones and somatic hybrids was extracted and analyzed by
Northern blot. A major transcript of the expected size (1 kb XG
plus vector sequences) and a minor transcript (3 kb) were detected with
the XG cDNA probe (Figure 1A),
whereas a single band of 1 kb (expected size) was revealed with the
MIC2 probe (Figure 1B). The minor 3-kb XG species might
be a product generated by an alternative termination of transcription
downstream of the specific sequences of the bovine growth hormone gene
(BGH) present in the pcDNA3 and pcDNA-hyg vectors.
Semiquantitative analysis by densitometry (not shown) indicate that the
hybrids XG13 × hygR and XG13 × hMIC11
expressed fewer XG transcripts (Figure 1A) and fewer XG copies
(Table 1) than the other clones. Similarly, the transfectants MIC22 and
the hybrids MIC22 × hygR and MIC22 × h.XG66
expressed fewer MIC2 transcripts (Figure 1B) and fewer CD99
copies (Table 1). In all cases, therefore, the relative amounts of
XG and MIC2 mRNA correlated well with the number of
XG and CD99 molecules found at the cell surface, as determined by
flow cytometry.
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| Fig 1.
Northern blot analysis of the XG and MIC2
transcripts from transfected RAG clones and somatic hybrid derivatives.
Twenty micrograms total RNA from the different cell lines were
hybridized under high-stringency conditions with either the XG
(A) or MIC2 (B) cDNA probes. Identical amounts of RNA were
deposited in each lane, as determined by densitometry analysis of
ribosomal RNA under ultraviolet light illumination (not shown).
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The differences in cell surface expression of XG and CD99 seen with all
transfectants and hybrids might have resulted from some difference in
the transcription efficiency (plasmid integration-site dependency), the
stability of the mRNA or posttranscriptional events, or a combination
of these factors. This may also be the case for the 10-fold difference
between XG and CD99 molecules at the surface of Xg(a+) erythrocytes,
though the genes are under the control of different promoters in human
erythroblasts. Altogether, these data did not reveal any obvious
mechanism of coregulation of XG and CD99 at the protein level because
there was no change in the steady state levels of XG and CD99
expression when they were expressed together. This is in accordance
with a transcriptional mechanism of control of the coexpression of the
2 genes,3 but it does not exclude some form of
translational or posttranslational regulation of cell surface
expression of the 2 proteins.
Characterization of the XG and CD99 proteins in recombinant cells
and in erythrocytes
Western blot analysis.
Solubilized proteins from the different clones and somatic cell hybrids
were separated on SDS-PAGE and immunoblotted using the NBL-1 or 12E7
monoclonal antibodies. NBL-1 reacted with a single band corresponding
to a protein of 26 kd in XG13, XG13-h.MIC15, and XG13-h.MIC19 extracts,
whereas 12E7 recognized a major species of 32 kd in MIC22 and the
double transfectants (Figure 2A). On the
blots, which were loaded with the same amount of total membrane proteins, the staining intensity of the bands was rather similar (Figure 2A, left and middle panels). However, the exposure times were
much longer for NBL-1 blots (5 minutes) than for 12E7 blots (3 seconds), suggesting that more CD99 than XG molecules were present in
the transfectants. This result correlates well with those obtained by
flow cytometry and Northern blot analysis (see above). The results from
Figure 2A clearly indicate that the 26- and 32-kd components are
related to XG and CD99, respectively, because no signal could be
detected with lysates from nontransfected (murine) RAG cells that did
not express either of these proteins. Indeed, examination of the
evolutionary conservation of the 2 genes on zoo blots showed that
XG (unpublished results) and MIC214 are
undetectable in nonprimate species. The point mutation in our
MIC2 cDNA (see above) did not affect the reactivity of
recombinant CD99 with 12E7 because the Asp165Asn substitution occurs in
the cytosolic tail of the protein and the epitope recognized by 12E7 has been mapped to the luminal domain of the protein.16 The 26-kd protein detected by NBL-1 is present in Xg(a+) RBCs (female, CD99
high-expressor) but not in Xg(a) RBCs (male, CD99-high
expressor), whereas the 32-kd species detected by 12E7 is present in
Xg(a) RBCs and Xg(a+) RBCs, which are CD99-high
expressers (Figure 2B). However, this protein is undetectable in RBCs
from an Xg(a), CD99-low expressor (see Figure
3B). These results strongly suggest that
the presence or absence of the 26-kd component (XG) determines the
basis of the Xg(a+)/Xg(a) polymorphism and that the quantitative polymorphism of CD99 is coregulated with XG.
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| Fig 2.
Western blot analysis of the XG and CD99 proteins from
transfected RAG clones, somatic hybrid derivatives, and erythrocytes.
Proteins from cell lysates (RAG transfectants or somatic hybrids) or
erythrocyte membranes were subjected to SDS-PAGE on a 12% (wt/vol)
polyacrylamide gel, transferred to nitrocellulose sheets (Schleicher
and Schuell, Dassel, Germany), probed with either NBL-1 or 12E7
monoclonal antibodies (as indicated below the blots), and revealed by
chemiluminescence (ECL reagent, Amersham). (A) Western blot analysis,
using indicated antibodies, of lysates from single or double
transfectants (names above blots; details of transfectants given in
Table 1) or nontransfected RAG cells. Right-hand panel represents
Western blot analysis with monoclonal antibody 12E7 after treatment of
the clone XG13-h.MIC15 (CD99-expressing, RAG transfectant) with 0, 0.1 (RDE1), or 0.25 (RDE2) units of neuraminidase (as described in
"Materials and methods"). Exposure times to radiographic films
were 5 minutes for NBL-1 blots, 3 and 6 seconds for 12E7 blots on
middle and right hand panels, respectively. (B) Western blot analysis,
using indicated antibodies, of membrane lysates from erythrocytes from
Xg(a) (male, CD99-high expressor) or Xg(a+) (female) (exposure
time, 1 minute). Right-hand panel represents Western blot analysis with
monoclonal antibody 12E7 after treatment of Xg(a+) (female)
erythrocytes with 0, 0.1 (RDE1), or 0.25 (RDE2) units of neuraminidase
(as described in "Materials and methods") (exposure time, 10 minutes). (C) Western blot analysis, using indicated antibodies, of
lysates from somatic hybrids of the single transfectants h.XG66
(XG-expressing) and MIC22 (CD99-expressing) (details of transfectants
given in Table 1) (exposure time, 6 minutes). The size (kd) of the
detected bands was determined using rainbow-colored protein molecular
weight markers.
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| Fig 3.
Immune precipitation of the Xga and CD99
antigens from transfected RAG cells and erythrocytes.
(A) Intact nontransfected RAG cells or the clone XG13-h.MIC15 were
125I-labeled and incubated with NBL-1 or 12E7 antibodies,
as noted below the autoradiograph. Solubilized complexes were
precipitated, separated on SDS-PAGE, and autoradiographed. (B)
Erythrocyte surface proteins from Xg(a+) (female), Xg(a) (male,
CD99-high expressor) or Xg(a) (female, CD99-low expressor)
donors were 125I-labeled. Cell membranes were prepared,
incubated with NBL-1 (or the anti-Xga serum Alo.) or 12E7
antibodies, and solubilized complexes were treated as in A. The size
(kd) of the detected bands was determined using rainbow-colored protein
molecular weight markers (Amersham).
|
|
In previous immunoblot studies with human RBC, diffuse bands ranging
from 24 to 29 kd, probably resulting from heterogeneity of
glycosylation of the XG protein, were detected in membranes prepared
from Xg(a+) erythrocytes using either human anti-Xga
antisera or rabbit polyclonal antibodies,3,18,29
whereas a faint band at approximately 26 kd was revealed with
NBL-1.3 The CD99 glycoprotein was detected as a single
32-kd band with the 12E7 antibody in such studies.18,27,28
Examination of the immunoblot with 12E7 (Figure 2A, middle panel)
revealed a minor 28-kd band in clones expressing high levels of CD99
(double transfectants XG13-h.MIC15 and XG13-h.MIC19) but not in clone MIC22 (except after long exposures). A 28-kd molecule has also been
reported after neuraminidase (RDE) treatment of CD99 immunoprecipitates from thymocyte surface proteins30 or in lysates from
untreated or RDE-treated RBCs.27 This protein was assumed
to be the intracellular unsialylated precursor of the
membrane-associated polypeptide. The double transfectant XG13-h.MIC15
was treated with neuraminidase (RDE) before solubilization of proteins
and immunoblotting with 12E7 (Figure 2A, right panel; exposure time 6 seconds). With 0.1 U neuraminidase (RDE 1), the 28-kd band was present
in greater amounts than it was in untreated cells, and a new band of
approximately 26 kd (not identical to the XG band) became detectable.
When the cells were treated with 0.25 U neuraminidase (RDE 2), a
partial shift from the 28- to the 26-kd species was observed. Whether this 26-kd component is a more completely desialylated species or a
degradation product was not investigated. Treatments with higher
amounts of neuraminidase (0.5 U) or for a longer period of time (16 hours) did not allow a complete shift of the 32-kd component to
molecules of lower molecular mass (not shown), perhaps because of the
high number of CD99 molecules (2 × 106) at the cell
surface and incomplete removal of sialic acid residues. It is also
possible that there is heterogeneous terminal sialylation of CD99, with
only a subset of molecules susceptible to cleavage by the specific
neuraminidase used in these studies.
A Western blot of RDE-treated RBCs with 12E7 revealed 2 faster
migrating bands of 30 and 28 kd, the latter accumulating in higher
amounts with 0.25 U than with 0.1 U enzyme (Figure 2B, right panel).
Similar results were obtained by Latron et al,17 except
that the reported apparent molecular mass of the different species was
28 kd for untreated RBCs and 25 and 22 kd for RDE-treated RBCs. The
26-kd species obtained after neuraminidase treatment of the RAG clone
XG13-h.MIC15 expressing CD99 (Figure 2A) probably is the unsialylated
precursor equivalent of the intracellular 28-kd molecule in
RBCs27 (Figure 2B). The difference in size might correspond
to differences of glycosylation between molecules synthesized in human
(RBC) and murine (RAG) cells. A 29-kd intracellular protein recognized
by the 12E7 antibody and probably not related to the 32-kd CD99 has
been found in human and mouse cells, including RAG cells.28
The 29-kd protein was weakly detected in all RAG clones and
nontransfected cells by longer exposures of the Western blots (not shown).
NBL-1 and 12E7 also recognized XG and CD99 proteins in somatic cell
hybrids of RAG transfectants (Figure 2C). Interestingly, though only
the 32-kd band was revealed by 12E7 in the clone MIC22, the 28-kd
species was also detected in lysates from both
MIC22 × hygR (Figure 2C, right panel) and
MIC22 × h.XG66 hybrids (seen only on long exposure), where CD99
expression was higher (Table 1). These data strongly suggest that the
28-kd molecule is the intracellular, partially sialylated precursor of
the 32-kd CD99 antigen.
Immunoprecipitation studies.
To determine whether the XG and CD99 proteins are associated in the
plasma membrane and could be detected by coprecipitation analysis, the
double transfectant XG13-h.MIC15 (expressing high levels of these
proteins; Table 1) was 125I-labeled and incubated with
NBL-1 or 12E7, and the immunoprecipitates recovered in the cell lysates
were analyzed by SDS-PAGE (Figure 3A). Each antibody immunoprecipitated
the corresponding protein (26 and 32 kd, respectively) but neither
NBL-1 nor 12E7 coprecipitated CD99 or XG, respectively. Additional
proteins of 52 and 65 kd were also precipitated with NBL-1 and 12E7,
respectively. These higher molecular weight proteins could correspond
to SDS-resistant homodimers of XG and CD99 because there is some
evidence that CD99 exists as a dimer at the erythrocyte
surface.17 Immuno-cross-blot analysis failed to detect CD99
or XG proteins in the immunoprecipitates from NBL-1 or 12E7,
respectively (not shown).
The absence of coprecipitation of the XG and CD99 molecules from the
double transfectant cell line could result from differences of protein
conformation or from another unidentified factor, in nonerythroid
cells, that would prevent a close association between these molecules.
Accordingly, immunoprecipitation studies were carried out using NBL-1
(or the human anti-Xga serum Alo.) or 12E7 and membrane
proteins prepared from Xg(a+) and Xg(a) erythrocytes
125I-labeled to a similar extent
(3 × 108 cpm/ packed RBCs). The results (Figure 3B)
show that XG and CD99 were clearly precipitated by their respective
antibodies from the Xg(a+) RBCs (female), but again no coprecipitation
of the 2 proteins occurred. Neither XG nor CD99, however, could be
precipitated from the Xg(a) RBCs (female, CD99-low expressor),
and only CD99 was precipitated from the Xg(a) RBCs (male,
CD99-high expressor), thus confirming the absence of detectable XG and
the quantitative polymorphism of CD99 in Xg(a) RBCs. Higher
molecular weight bands on the gels are probably nonspecific because
they were detected in all precipitates. The XG protein from Xg(a+) RBCs
was also immunoprecipitated with a rabbit polyclonal antibody (raised
in our laboratory) directed against the same peptide as for
NBL-1,3 but again CD99 was not present in the precipitate
(not shown).
Immunoblotting Xga immunoprecipitates (obtained using NBL-1
or serum Alo.) obtained from nonlabeled RBCs with 12E7 did not reveal
any bands corresponding to CD99 (32 kd), as found by Petty and
Tippett.18 Moreover, immunoblotting 12E7 immunoprecipitates with NBL-1 did not reveal any band corresponding to XG (26 kd) (data
not shown). Using biotin-labeled RBCs, Petty and Tippett18 found that 1 human anti-Xga serum could coprecipitate a
32-kd component (tentatively assigned to, but not formerly identified
as, CD99), from Xg(a+) RBCs. Our studies indicate, however, that
although the NBL-1 antibody could efficiently immunoprecipitate the XG
protein, there was no detectable 32-kd component in the precipitate
(Figure 3B).
We draw the following conclusions from these experiments: (1) XG and
CD99 can be expressed independently in cell transfectants; (2) there is
no physical association between XG and CD99 detectable by
coprecipitation of the 2 proteins from Xg(a+) membrane preparations or
from transfected RAG cells, using either NBL-1 (and the
anti-Xga serum Alo.) or 12E7; and (3) the molecular basis
of the Xg(a) phenotype is most likely caused by the lack of the
XG protein on Xg(a) erythrocytes either from a CD99-low
expressor (Figure 3B) or a CD99-high expressor (Figures 2B, 3B).
Tissue specificity of XG expression
To elucidate the mechanisms of tissue-specific regulation of
XG expression, we constructed somatic cell hybrids between
B-LCLs from Xg(a+) (CT) and Xg(a) (CL) male donors (CD99-high
expressers) and murine fibroblastic RAG cells. We then examined, by
flow cytometry, whether a reactivation of XG protein expression at the
cell surface would occur. The CD99 protein, but not the XG protein, is
present at the surface of B-LCLs of persons of both phenotypes (Table 2). RAG cells express neither XG nor CD99
proteins (not shown). Our results indicate that the CD99 protein, but
not the XG protein, is present on the surface of somatic hybrids
RAG × CL or RAG × CT (Table 2). We then fused these
hybrids with MEL cells to test whether an erythroid environment would
restore a cell surface expression of XG, but again no Xga
antigen appeared at the plasma membrane. Finally, somatic
hybrids between B-LCLs and the XG13 clone were constructed to
investigate the influence of a nonerythroid context on XG protein
expression. No effect was detected because the numbers of XG
molecules/cell (2 × 104 and
1.6 × 104) were similar to those observed on the
different somatic hybrids of RAG transfectants, including the XG13
clone as 1 fusion partner (see Table 1).
To determine whether the tissue-specific regulation of XG
expression occurs at the transcriptional level, we analyzed (by RT-PCR)
the XG transcripts from the B-LCLs and the different somatic hybrids, using primers bordering the coding sequence of the XG cDNA (Figure 4). The transcripts from each
cell line generated a specific band of the expected size (551 bp) and
of normal sequence compared to the transcripts from erythroid cells
(not shown). Thus, the absence of XG molecules at the surface of these
nonerythroid cells might be explained by a low transcription rate of
the gene or by some posttranscriptional event altering the stability or translation of the messenger RNA, glycosylation of the protein, or its
translocation to the cell surface. We reasoned that as a hybrid
partner, MEL cells should have brought the necessary erythroid factors
for cell surface expression of XG, but this may not be true because the
hybrid cells may have eliminated some MEL chromosomes (not determined)
encoding these factors. An alternative possibility is that human B-LCLs
do not possess the program (sequence of events including the putative
erythroid factors) needed for cell surface expression of XG; therefore,
the supply of erythroid factor(s) by the MEL cells is ineffective.
Indeed, only adult hemoglobin was produced in fetal nonerythroid
(fibroblasts or lymphoblasts) × MEL cell hybrids.31
View larger version (20K):
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| Fig 4.
RT-PCR analysis of the XG transcripts from B-LCLs
and somatic hybrid derivatives.
Total-cell RNA from the indicated cell lines was reverse transcribed
and PCR-amplified to obtain a fragment of 551 bp corresponding to the
coding sequence of the XG cDNA. The PCR products were
electrophoresed on a 1.2% (wt/vol) agarose gel, transferred to a nylon
membrane (Hybond N+; Amersham), and hybridized with a
32P-labeled internal oligonucleotide probe. C, control PCR
without template cDNA. The size (bp) of the PCR products was checked
with EcoRI-HindIII-digested  DNA markers.
|
|
Finally, we studied the tissue distribution of XG gene
expression by Northern blot analysis (Figure
5). Two major transcripts of 2.3 and 1.0 kb
and 1 minor species of 3.8 kb were detected in erythroid tissues
(thymus, bone marrow, and fetal liver) and several nonerythroid tissues
(heart, placenta, skeletal muscle, prostate, thyroid, spinal cord, and
trachea). No XG mRNA was revealed in peripheral blood
leukocytes, whereas we found a specific band by RT-PCR analysis on
transcripts from B-LCLs (Figure 4), indicating that the XG gene
is expressed at a low level in these cells. XG mRNA was also
found by Northern analysis in human skin fibroblasts15 and
by RT-PCR analysis in other adult (lung, kidney, testis) and fetal
(spleen, adrenal glands, brain, pancreas, small intestine) tissues.3 Only a few lymphoid cell lines gave a weak
positive signal.3 MIC2 transcripts of 1.2 kb and 2 minor species of 3.6 and 6.2 kb were detected in all the tissues tested
for XG mRNA expression (not shown), as expected, because the
CD99 antigen is present on most human cells.32 The 1.2-kb
transcript was also found in several T-lymphocytic and monocytic cell
lines and in peripheral blood lymphocytes.27
View larger version (24K):
[in this window]
[in a new window]
| Fig 5.
Tissue distribution of the XG transcripts.
Human multiple-tissue Northern blots from Clontech were hybridized
under high-stringency conditions with the 32P-labeled
XG cDNA probe and autoradiographed.
|
|
| |
Conclusion |
The expression data obtained with the transfected RAG cells and
somatic hybrid derivatives are consistent with a transcriptional regulation of XG and MIC2 gene expression because no
influence of either protein on the surface production of the other was
found. Cloning, sequencing, and functional tests of promoter and
upstream regulatory regions of the XG gene, contained within
the 10-kb stretch downstream of the MIC2 gene, may help to
elucidate the molecular basis of the coexpression of these 2 genes and
by extension of the XG polymorphism and the quantitative
polymorphism of MIC2. Indeed, according to the model proposed
by Goodfellow et al,12 the putative XGR polymorphic
regulatory locus might be located between the 3' end of
MIC2 and the 5' end of XG. However, as long as
formal proof of this model is not given, this regulatory element can be
anywhere else within the 100-kb region, from the 5' end of
MIC2 to the pseudoautosomal boundary. We could not confirm a
physical association between the XG and CD99 proteins at the cell
surface of RBCs or transfected RAG cells. If such an association exists, it may be of low affinity and may be disrupted during analysis.
Future investigation along this line will be carried out by the
2-hybrid system.33 In addition, further analysis on the
tissue distribution of the XG protein and precise identification of
which cells express this protein may help to clarify its biologic role.
| |
Acknowledgments |
We thank Anne-Marie D'Ambrosio (INTS, Paris) for the production of
Epstein-Barr virus-positive B-lymphoblastoid cell lines, Peter
Goodfellow (University of Cambridge, UK) for the gift of mAb
12E7, and Dr PierreYves Le Pennec (CNRGS, Paris) for the gift of serum Alo. We also thank Nicole Souleyreau (Institut
Pasteur, Paris, France) for the transmission of the cell fusion
method and Patrick Lambin and Martine Debbia (INTS, Paris)
for immunochemistry analysis. We thank Patricia Hermand and Pascal
Bailly (INTS, Paris) for helpful discussions.
| |
Footnotes |
Submitted October 12, 1998; accepted October 20, 1999.
C.F. was supported in part by Ortho-Clinical Diagnostics.
Reprints: Jean-Pierre Cartron, Inserm U76, Institut National de
la Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015, Paris, France;
e-mail: cartron{at}infobiogen.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Race RR, Sanger R.
Blood Groups in Man. 6th ed. Oxford, UK: Blackwell Scientific; 1975:579.
2.
Mann JJ, Cahan A, Gelb AG, et al.
A sex-linked blood group.
Lancet.
1962;i:8.
3.
Ellis NA, Tippett P, Petty A, et al.
PBDX is the XG blood group gene.
Nat Genet.
1994a;8:285[Medline]
[Order article via Infotrieve].
4.
Lyon MF.
X-chromosome inactivation and developmental patterns in mammals.
Biol Rev.
1972;47:1[Medline]
[Order article via Infotrieve].
5.
Fellous M, Bengtsson B, Finnegan D, Bodmer WF.
Expression of the Xga antigen on cells in culture and its segregation in somatic cell hybrids.
Ann Hum Genet.
1974;37:421[Medline]
[Order article via Infotrieve].
6.
Levy R, Dilley J, Fox RI, Warnke R.
A human thymus-leukemia antigen defined by hybridoma monoclonal antibodies.
Proc Natl Acad Sci U S A.
1979;76:6552[Abstract/Free Full Text].
7.
Goodfellow P, Banting G, Levy R, Povey S, Mc Michael A.
Human X linked antigen defined by a monoclonal antibody.
Somat Cell Genet.
1980;6:777.
8.
Goodfellow P, Banting G, Sheer D, et al.
Genetic evidence that a Y-linked gene in man is homologous to a gene on the X chromosome.
Nature.
1983;302:346[Medline]
[Order article via Infotrieve].
9.
Goodfellow PJ, Darling SM, Thomas NS, Goodfellow PN.
A pseudoautosomal gene in man.
Science.
1986;234:740[Abstract/Free Full Text].
10.
Goodfellow P, Pym B, Mohandas T, Shapiro LJ.
The cell surface antigen locus, MIC2X, escapes X-inactivation.
J Hum Genet.
1984;36:777.
11.
Goodfellow PN, Tippett P.
A human quantitative polymorphism related to XG blood groups.
Nature.
1981;289:404[Medline]
[Order article via Infotrieve].
12.
Goodfellow PJ, Pritchard C, Tippett P, Goodfellow PN.
Recombination between the X and Y chromosomes: implications for the relationship between MIC2, XG and YG.
Ann Hum Genet.
1987;51:161[Medline]
[Order article via Infotrieve].
13.
Darling SM, Banting GS, Pym B, Wolfe J, Goodfellow PN.
Cloning an expressed gene shared by the human sex chromosomes.
Proc Natl Acad Sci U S A.
1986;83:135[Abstract/Free Full Text].
14.
Smith MJ, Goodfellow PJ, Goodfellow PN.
The genomic organization of the human pseudoautosomal gene MIC2 and the detection of a related locus.
Hum Mol Genet.
1993;2:417[Abstract/Free Full Text].
15.
Ellis NA, Ye TZ, Patton S, German J, Goodfellow PN, Weller P.
Cloning of PBDX, an MIC2-related gene that spans the pseudoautosomal boundary on chromosome Xp.
Nat Genet.
1994b;6:394[Medline]
[Order article via Infotrieve].
16.
Banting GS, Pym B, Darling SM, Goodfellow PN.
The MIC2 gene product: epitope mapping and structural prediction analysis define an integral membrane protein.
Mol Immununol.
1989;26:181[Medline]
[Order article via Infotrieve].
17.
Latron F, Blanchard D, Cartron JP.
Immunochemical characterization of the human blood cell membrane glycoprotein recognized by the monoclonal antibody 12E7.
Biochem J.
1987;247:757[Medline]
[Order article via Infotrieve].
18.
Petty AC, Tippett P.
Investigation of the biochemical relationship between the blood group antigens Xga and CD99 (12E7 antigen) on red cells.
Vox Sang.
1995;69:231[Medline]
[Order article via Infotrieve].
19.
Sanger F, Nicklen S, Coulson AR.
DNA sequencing with terminating inhibitors.
Proc Natl Acad Sci U S A.
1977;74:5463[Abstract/Free Full Text].
20.
Bloy C, Blanchard D, Lambin P, et al.
Human monoclonal antibody against Rh(D) antigen: partial characterization of the Rh(D) polypeptide from human erythrocytes.
Blood.
1987;69:1491[Abstract/Free Full Text].
21.
Chen C, Okayama H.
High-efficiency transformation of mammalian cells by plasmid DNA.
Mol Cell Biol.
1987;7:2745[Abstract/Free Full Text].
22.
Couillin P, Mollicone R, Grisard MC, et al.
Chromosome 11q localization of one of the three expected genes for the human alpha-3-fucosyltransferases by somatic hybridization.
Cytogenet Cell Genet.
1991;56:108[Medline]
[Order article via Infotrieve].
23.
Habibi B, Tippett P, Lebesnerais M, Salmon C.
Protease inactivation of the red cell antigen Xga.
Vox Sang.
1979;36:367[Medline]
[Order article via Infotrieve].
24.
Church GM, Gilbert W.
Genomic sequencing.
Proc Natl Acad Sci U S A.
1984;81:1991[Abstract/Free Full Text].
25.
Steck TL, Kant JA.
Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes.
Methods Enzymol.
1974;31:172[Medline]
[Order article via Infotrieve].
26.
Markwell MAK, Haas SM, Bieber LL, Tolbert NE.
A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.
Anal Biochem.
1978;87:206[Medline]
[Order article via Infotrieve].
27.
Gelin C, Aubrit F, Phalipon A, et al.
The E2 antigen, a 32 kd glycoprotein involved in T-cell adhesion processes, is the MIC2 gene product.
EMBO J.
1989;8:3253[Medline]
[Order article via Infotrieve].
28.
Banting GS, Pym B, Goodfellow PN.
Biochemical analysis of an antigen produced by both human sex chromosomes.
EMBO J.
1985;4:1967[Medline]
[Order article via Infotrieve].
29.
Herron R, Smith GA.
Identification and immunochemical characterization of the human erythrocyte membrane glycoproteins that carry the Xga antigen.
Biochem J.
1989;262:369[Medline]
[Order article via Infotrieve].
30.
Aubrit F, Gelin C, Pham D, Raynal B, Bernard A.
The biochemical characterization of E2, a T cell surface molecule involved in rosettes.
Eur J Immunol.
1989;19:1431[Medline]
[Order article via Infotrieve].
31.
Takegawa S, Brice M, Stamatoyannopoulos G, Papayannopoulou T.
Only adult hemoglobin is produced in fetal nonerythroid x MEL cell hybrids.
Blood.
1986;68:1384[Abstract/Free Full Text].
32.
Goodfellow P.
Expression of the 12E7 antigen is controlled independently by genes on the human X and Y chromosomes.
Differentiation.
1983;23(suppl):S35.
33.
Fields S, Song O.
A novel genetic system to detect protein-protein interactions.
Nature.
1989;340:245[Medline]
[Order article via Infotrieve].
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Abstract
Introduction