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Relationship of the Human Erythrocyte Wrb Antigen t o an Interaction Between
Glycophorin A and Band 3
By Marilyn J. Telen and Joel A. Chasis
The Wrb antigen is a high-frequency human erythrocyte
antigen invariably absent from En (a- 1 erythrocytes, which
lack glycophorin A. However, glycophorin A from En (a+)
W r (a b-) red cells has an amino acid sequence identical
to that of glycophorin A from W r (b+) erythrocytes.
Evidence has suggested that the Wrb antigen may require
the interaction of glycophorin A with either a lipid moiety
or with another erythrocyte-integral membrane protein,
band 3. We have investigated the role of band 3 in Wrb
expression using murine monoclonal antibodies (MoAbs)
with Wrb specificity. These antibodies reacted by radioim-
munoassay (RIA) only with cells expressing both glycophorin A and band 3. In immunoprecipitation studies, Wrb
antibodies immunoprecipitated both band 3 and glycophorin A, while antibodies specific for band 3 or glycophorin precipitated only the protein with which they were
reactive. These data strongly suggest that band 3 is the
other membrane component necessary for expression of
Wrb and that band 3 and glycophorin A are closely associated in the erythrocyte membrane.
0 1990 by The American Society of Hematology.
T
been identified. Their red cells contain normally glycosylated
glycophorin A, which is normal by sodium dodecyl sulfate
(SDS) polyacrylamide gel analysis and which possesses an
identical amino-acid sequence to glycophorin A from cells of
the common W r (a- b + ) phen~type.~.'
These findings, along
with the observation that highly purified glycophorin A in
aqueous solution does not bind Wrb antibodies: suggest that
additional membrane components might play a role in the
expression of Wright antigens. In light of prior studies, band
3 appeared to be a possible candidate for this role. Dahr et a14
had reported that when red cell membranes were solubilized
in nonionic detergent, glycophorin A aggregated with band
3 . Moreover, protein diffusion measurements, antibody crosslinking studies and freeze-etch electron microscopy studies,
have suggested that band 3 and glycophorin A associate with
each other in the lipid
To determine whether Wrb antigen expression is dependent upon both glycophorin A and band 3, we studied the
ability of Wrb-specific MoAbs to bind to cells that express
one or both of these proteins. In addition, we used immunoprecipitation studies to determine what protein species were
bound by these antibodies. We found that the Wrb epitope
appears to involve both band 3 and glycophorin A, suggesting
that these two membrane proteins are closely associated in
the erythrocyte membrane.
+
H E WRIGHT BLOOD group is composed of two
antigens-Wr" and Wrb-that are most likely allelic.'
The Wrb antigen is a high-frequency erythrocyte antigen,
while the Wra antigen is rare, occurring in approximately 1 in
every 1,500 individuals. Although previous studies have
shown that these antigens are related to expression of the
transmembrane sialoglycoprotein glycophorin A, the nature
of this relationship has remained unclear. En (a-) erythrocytes, which lack glycophorin A, do not express the Wr" and
Wrb antigens.* The hypothesis that these antigens are resident on glycophorin A was further strengthened by the
results of Ridgwell et
who radiolabeled erythrocyte
glycophorins using peri~date-~H-borohydride
and who demonstrated immunoprecipitation of glycophorin A by a murine
monoclonal antibody (MoAb) with anti-Wrbreactivity. Dahr
et aI4provided additional evidence for the role of glycophorin
in Wrb expression by showing that a Triton extract containing glycophorin A, when incorporated into liposomes, bound
anti-Wrb.
However, important evidence indicates that Wr" and Wrb
do not simply constitute polymorphisms of glycophorin A.
Numerous studies have confirmed the original observation
that the gene for the low-frequency antigen Wr" segregates
independently from the genes encoding the M and N
amino-acid sequence variants of glycophorin A.5s6Also, two
individuals with the phenotype En ( a + ) W r ( a + b - ) have
MATERIALS AND METHODS
From the Division of Hematology/Oncology. Department of
Medicine. Duke University Medical Center. Durham, NC; the
Department of Medicine and Cancer Research Institute, University
of California. San Francisco; and the Division of Cell and Molecular Biology. Lawrence Berkeley Laboratory, Berkeley, CA.
Submitted November 13, 1989; accepted April 19. 1990.
Supported by Grant No. CH-371 from the American Cancer
Society and by Grant No, ROI HL 33572 from the National
Institutes of Health. M.J.T. is the recipient of Grant No. RCDA
KO4 HL 02233 from the National Institutes of Health.
Address reprint requests to Marilyn J. Telen, MD. Box 3387,
Duke University Medical Center, Durham, NC 27710.
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 I734 solely to
indicate this fact.
o I990 by The American Society of Hematology.
0006-4971/90/7604-0011$3.00/0
842
Antibodies. Three MoAbs with Wrbor Wrb-like specificity were
used. Antibody E6 has been previously described as having specificity for erythrocyte band 3"; its Wrb specificity has since been
confirmed within the present investigation and also by the First
International Workshop on Monoclonal Antibodies Against Red
Cells and Related Antigens (Paris, 1989). Antibodies 10-22 and
4-21 were provided by Dr Margaret Nichols (Organon Teknika,
Durham, NC); the specificity of these antibodies for the Wrbantigen
was also confirmed by multiple laboratories within the aforementioned workshop. For cell binding and radioimmunoprecipitation
assays, antibody E6 was used as unprocessed murine ascitic fluid,
while antibodies 10-22 and 4-21 were used as unprocessed tissueculture supernates. None of these three antibodies were reactive by
immunoblotting techniques.
Other antibodies used included previously described antibodies to
glycophorin A [ 10F7" and E4"], glycophorins A and B [E3"], band
3 [TEIOlo], as well as murine ascitic-fluid and tissue-culture
supernate from the murine myeloma cell line P3x63/Ag8 (P3)."
For studies of the effect of antibody binding on erythrocyte
Blood, VOI 76, NO 4 (August 15), 1990: pp 842-848
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843
ERYTHROCYTE W R ANTIGEN
~
EXPRESSION
membrane deformability, antibodies were first purified using affinity
chromatography. Affinity columns were prepared by coupling Affi10(Bio-Rad Laboratories, Richmond, CA) with purified glycophorin
A, kindly provided by Dr Heinz Furthmayr (Stanford University,
Stanford, CA). After coupling, the ability of these columns to absorb
out antibodies to glycophorin A was confirmed using 10F7, a
well-characterized antiglycophorin antibody. Antibodies were bound
to the columns in phosphate-buffered saline (PBS), pH 7.4, and
eluted in HC1-glycine, pH 2.2. Column fractions were monitored
spectrophotometrically.
Cell lines, media, and reagents. K562” and HELI4 erythroleukemia cell lines, obtained from Dr Russell Kaufman, Duke University, and from the American Type Culture Collection (Rockville,
MD), respectively, were grown in Dulbecco’s modified Eagle’s
medium ( G i b , Grand Island, NY) with 10% fetal calf serum
(FCS; MA Bioproducts, Walkersville, MD).
Cell preparation. Whole-blood samples were drawn from normal individuals after obtaining informed consent. Erythrocytes used
for radioimmunoassays (RIAs) and radioimmunoprecipitation studies were collected in acid citrate dextrose and then washed in PBS
prior to use; erythrocytes used for deformability studies were washed
in 5 mmol/L Tris, 140 mmol/L NaCl, pH 7.4. Cell suspensions were
counted using an ELT-8 (Ortho Diagnostic Systems, Inc, Raritan,
NJ). When thawed cells with rare blood group phenotypes (eg,
MkMk)were used, thawed control cells were tested simultaneously.
Immunoassay and immunoprecipitation. The reactivity of red
blood cells (RBCs) with MoAbs was tested quantitatively by RIA
under conditions of saturating antibody concentrations, as previously
described.” All red cells were used in RIAs at similar concentrations
(-4 x 108/mL) in each assay. All RIAs were performed in triplicate. Nucleated cells were assayed for reactivity with various
antibodies at concentrations of 2 x 107/mL.The binding of antibodies to nucleated cells was detected by subsequent binding of
fluorescein-labeled goat antimouse IgG (TAG0 Inc, Burlingame,
CA), as analyzed by a FacScan (Becton-Dickinson, San Jose, CA).
Immunoprecipitation experiments were done as previously
described.’0,’6Briefly, cell surface proteins of intact erythrocytes
were radiolabeled using Iodo-Gen (Pierce Biochemical Co, Rockford, IL). Erythrocyte membrane ghosts were then prepared by the
method of Dodge et al” and solubilized in Tris-buffered saline
containing 1 mmol/L ethylene diamine tetra-acetic acid (EDTA),
0.1% gelatin, and either 1% deoxycholate or 1% Nonidet-P40.
Generally, 5 pL of ascitic fluid or 50 pL of tissue culture supernate
containing MoAbs were added to lysate prepared from 4 x108
erythrocytes. Immune complexes were isolated using formalin-fixed
staphylococci-bearing Protein A, in the case of E6, or similar
Staphylococci precoated with antimouse Ig antibodies in the cases of
10-22 and 4-21. Results were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography.
Deformability studies. To measure red blood cell deformability,
washed red cells were resuspended in dextran (40,000 mol wt, 25
g/100 mL in 10 mmol/L phosphate buffer, pH 7.4; Pharmacia Fine
Chemicals, Uppsala, Sweden) and examined by ektacytometry, a
laser diffraction method previously
In brief, suspended
cells were exposed in the ektacytometer to an increasing shear stress
(0 to 125 dynes/cm*) and the change in their laser diffraction
pattern from circle to ellipse measured. This photometric measurement produced a signal-designated deformability index that quantitated cell ellipticity. By an automatic image analysis system, the
deformability index was recorded as a continuous function of applied
shear stress. Analysis of the curve generated by the ektacytometer
provided a measure of deformability.
To examine the effect of antibodies on red cell deformability, red
cells were suspended to 0.5% hematocrit in MoAb in dextran. The
suspensions were incubated for 30 minutes at room temperature and
then examined in the ektacytometer.
RESULTS
Reactivity of MoAbs with variant erythrocytes. Initial
serologic investigations using antibody E6 revealed that it
reacted equally well with all random donor erythrocytes.
However, continued studies demonstrated that E6 failed to
react with En (a-) and MkMkerythrocytes, two types of red
cells known to lack glycophorin A and glycophorins A and B,
respectively (Table 1). This dependence on glycophorin A for
antibody reactivity prompted us to investigate whether E6
was an anti-Wrb. We therefore measured the binding of E6 to
the extremely rare red cell phenotype En (a+) Wr(a+b-)
(M.Fr.).20 As shown in Table 1, E6 failed to react with En
( a + ) Wr (a+ b-) erythrocytes. These findings demonstrated that antibody E6 had Wrbspecificity.
We then compared the reactivity pattern of antibody E6
with that of antibodies 10-22 and 4-21, two other murine
MoAbs with Wrb or Wrb-like specificities. RIA results
obtained with these three antibodies are summarized in
Table 2. Antibodies 10-22 and 4-21, like antibody E6,
reacted with En (a+) Wr (a-b+) erythrocytes. Antibody
10-22 reacted with En ( a + ) Wr (a-b+) erythrocytes and
not with En ( a + ) Wr (a+b-) cells, implying that, like
antibody E6, antibody 10-22 possessed Wrb specificity.
However, antibody 4-21 demonstrated extremely weak reactivity with the rare En ( a + ) Wr (a+b-) (M.Ft.) cells,
suggesting that its specificity was more complex.
Agglutination studies, performed in part in cooperation
with the First International Workshop on Monoclonal Antibodies Against Red Cells and Related Antigens, also demonstrated that while all three antibodies agglutinated normal
erythrocytes 3 + to 4+ (at Coombs phase using antimouse Ig
serum), E6 and 10-22 failed to agglutinate En (a-) Wr
(a-b-) cells, which totally lack glycophorin A. Interestingly, antibody 4-21 showed a different serologic specificity
in that it strongly agglutinated En (a-) cells (which were
Wr (a-b-) as defined by human antisera). These observations were confirmed by the American Red Cross Reference
Laboratory, South Florida Region, which found that antibody 4-21 reacted well in indirect agglutination assays with
three unrelated examples of En (a-) Wr (a-b-) erythro-
Table 1. Reactivity of MoAb E6 W i t h Normal and Rare Phenotype
Erythrocytes
Cell Phenotype
Antibody Used
Wr ( b + P
Wr (b+)
M’M‘
En (a-I
E6
p3t
5,181
245
5,723
273
163
245
273
273
Wr (a+b-l
290
328
Reactivity measured by cpm bound: cpm bound = cpm ‘251-labeled
F(ab’), sheep antimouse Ig.
*Reactivity with two representative Wr (b+) erythrocyte samples is
demonstrated. More than 30 such samples have been tested. Moreover,
no difference has been found between cpm bound by Wr (a - b ) and Wr
(a+b+) erythrocytes (M.J. Telen, unpublished data, 1988 to 1989).
tMurine ascitic fluid from the myeloma cell line P3 x 63/Ag8, used to
prepare MoAbs.
+
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Table 2. Reactivity of MoAbs W i t h Normal W r (b+) and En (a+)
W r (a b - 1 Erythrocytes
+
Mr x ~ O - ~
Antibody U s e d
Cell PhenotvM,
Wr
Wr
Wr
Wr
(b+)t
(b+)
(b+)
(a+b-) (M. Fr.)
E6
4-2 1
10-22
P3.
3.694
3,643
3,276
233
3,542
3.871
3,674
416
3.564
3,013
3,178
244
275
301
211
291
Reactivity measured by cpm bound: cpm bound = cpm 'z51-labeled
F(ab'), sheep antimouse Ig.
'Murine ascites fluid from the myeloma cell line P3 x 63/Ag8, used in
preparation of MoAb-producing cell lines. Tissue culture supernate from
the same cell line gave comparable results.
tReactivity with three representative Wr (b+) erythrocyte samples is
demonstrated. More than 3 0 such samples have been tested.
cytes but failed to agglutinate En ( a + ) Wr (a-b-) erythrocytes (P. Issitt, personal communication). These data suggested that there might be a Wrb-related structure present on
glycophorin A-deficient cells but not recognized by human
Wrballoantisera. Antibody 4-21 therefore appeared to recognize an epitope related to Wrb expression but not abolished
by the lack of glycophorin A.
Reactivity of MoAbs with erythroleukemia cell lines.
To examine whether band 3 played a role in Wrb expression,
the binding of the three Wrb antibodies to K562 and HEL
cells was quantitated by flow cytometric analysis of immunofluorescence. These two erythroleukemia lines were used
because they have been shown to express glycophorin A but
not band 3.2'.22As shown in Table 3, antiglycophorin antibodies E3 and E4 reacted with both K562 and HEL cell lines,
confirming that expression of glycophorin A was easily
detectable on these cells. In contrast, MoAbs E6, 10-22, and
4-21 showed minimal or no reactivity with either the K562 or
HEL cell lines. These data suggest that band 3 may be
necessary for the binding of Wrb antibodies.
Immunochemical characterization of proteins reactive
with MoAbs. Immunoprecipitation assays using antibody
E6 and erythrocyte membrane proteins solubilized in 1%
deoxycholate, a slightly ionic detergent, resulted in precipitation of band 3 (Fig IA). However, it has previously been
shown, using radiolabeled erythrocyte membrane proteins
solubilized in the nonionic detergent NP-40, that addition of
E6 resulted in specific immunoprecipitation of large quantities of band 3 as well as somewhat lesser amounts of
glycophorin A." Concurrent immunoprecipitation experiTable 3. Reactivity of MoAbs W i t h Human Erythroleukemia
Cell Lines
% Cells Reactiveby
Immunofluorescence
Antibody
E3 (antiglycophorin A
E4 (antiglycophorin A)
E6 (anti-Wrb)
10-22 (anti-Wrb)
4-2 1 (anti-Wrb)
K562
+ B)
69.7
28.1
12.0.
0
0
HEL
75.1
55.1
4.0'
0
0
*E6 reactivity with K562 and HEL cells, when graphed by cell number
and fluorescence intensity, appeared comparable with simultaneously run
negative controls because cells counted as positive were only very weakly
so.
200-
I
]Band 3
-GPA2
92.56946-
-G
30-
PA
-GPB
IT
m
A
Mr x
200-
J Band 3
92.5-
-GPA,
6946-
--PA
30B
-GPB
E3
E6 TElO P3
Fig 1. Immunoprecipitation by antibody E6 of band 3 and
glycophorin from erythrocyte lysates made with various detergents. (A) Antibodies E6 (anti-Wrb) and E3 (antiglycophorins A and
B) were used t o immunoprecipitate radiolabeled erythrocyte
membrane proteins solubilized in a buffer containing 1% deoxycholate. Normal W r (b+) erythrocytes were used as a source of
membrane proteins. Antibody E3 precipitated glycophorins A
(GPA,, dimer; GPA, monomer) and B (GPBI but no band 3, while
antibody E6 precipitated band 3 but no detectable quantity of
glycophorin. (6) Antibodies E3, E6. and TE-10 (antiband 3) were
used t o precipitate proteins from radiolabeled erythrocyte lysates
prepared in 1% Nonidet P-40. Again, antibody E3 only precipitated
glycophorins A and B, while antibody TE-10 precipitated only band
3 (along w i t h band 3 dimer). However, antibody E6 precipitated
both band 3 and glycophorin A.
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ERYTHROCYTE W R ANTIGEN
~
EXPRESSION
845
ments using antibody TEIO, directed against band 3, produced no similar coprecipitation of glycophorin A (Figure
1 B). Of note is the difference in width of band 3 immunoprecipitated by E6 and TE-IO. The narrowness of the band
observed after treatment with E6 suggests that only a subset
of band 3 molecules was immunoprecipitated by the antiWrb antibody. E3 antibody, specific for glycophorins A and
B, precipitated glycophorins A and B but no band 3 from
NP-40 lysates. Numerous MoAbs to glycophorin A used
under identical conditions did not precipitate detectable
quantities of band 3. Hence neither band 3 nor glycophorin A
were nonspecifically precipitated under these experimental
conditions. We therefore compared the ability of the three
Wrb-related murine MoAbs to precipitate membrane proteins solubilized in NP-40. As shown in Fig 2, antibodies
10-22 and 4-21 produced the same pattern of immunoprecipitated bands as antibody E6. At this time it is unclear why
there is a difference in the relative quantities of band 3 and
glycophorin A precipitated by the three antibodies; however,
the presence of both polypeptides in the precipitates implies
an association between band 3 and glycophorin A.
It is interesting to note that antibody 4-21 produced the
same immunoprecipitation pattern as antibodies E6 and
10-22, further suggesting that the antigen recognized by
antibody 4-21 is related to Wrb expression. To examine in
more detail how antibody 4-21 binds to erythrocytes that
lack glycophorin A, we performed immunoprecipitation
experiments using En (a-) W r (a-b-) erythrocytes. In
these studies antibody 4-21 precipitated small amounts of
band 3 (data not shown). These results imply that the
observed agglutination of En (a-) erythrocytes by antibody
4-21 was produced by antibody binding to the transmembrane protein band 3. Considered together, the results of
Mr x lo-’
200 -
92.56946-
-
\ Band3
J
-GPAz
-GPA
30-GPB
Fig 2. Immunoprecipitation of band 3 and glycophorin A by
Wrb antibodies. When ’261-radiolabeled erythrocyte membranes
were solubilized in a buffer containing 1% Nonidet P a , Wrb
antibodies E6, 10-22. and 4-21 all precipitated both band 3 and
glycophorin A, while the nonreactive murine myeloma protein P3
failed to precipitate radiolabeled protein.
experiments with antibody 4-21 suggest that band 3 plays a
role in Wrb expression.
Eflect of antibody binding on membrane deformability.
Previous studies have shown that binding of antibodies with
specificity for glycophorin A induced a decrease in erythrocyte membrane deformability, while binding of antibodies to
A, B, and Rh blood group antigens, which recognize other
cell-surface components, had no effect on def~rmability.’~.~~
Therefore, we measured the effect of E6 binding on red blood
cell deformability. When the deformability index was plotted
as a continuous function of applied shear stress, the curve
generated reached a plateau a t approximately 0.7 for normal
red cells, as seen in Fig 3A. Antibody E6 binding induced a
decreased deformability index. To quantitate the extent of
decreased deformability induced by E6 binding, the shear
stress was plotted on a log scale and the deformability index
on a linear scale (Fig 3B). Because the lines are parallel, one
can see that red blood cells treated with E6 required 43-fold
greater shear stress than nontreated control cells to reach
equivalent deformation a t all points along the curve. This
result indicates that E6-treated red cells had 0.025 xnormal
deformability. Doubling the amount of E6 did not further
change the deformability, indicating that we had achieved
the maximum effect of E6 binding on deformability (data not
shown).
The deformability of E6-treated erythrocytes was then
compared to that of cells treated with 10F7, a wellcharacterized antibody specific for glycophorin A (Fig 4).
While untreated control cells had a deformability of loo%,
erythrocytes pretreated with E6 had a relative deformability
of 2.5%, and those pretreated with antibody 10F7 had a
relative deformability of 8.9%. Thus E6 induced a dramatic
decrease in deformability similar to that observed with
ligands to glycophorin A. When E6 and 10F7 were incubated
with En (a-) erythrocytes, which lack glycophorin A,
erythrocyte deformability did not decrease significantly (Fig
4). These observations are consistent with the binding and
immunochemical data and imply that E6 requires the presence of glycophorin A for binding to intact cells.
To determine whether the rigidity-inducing effect of E6
could be absorbed out with glycophorin A, we passed the
antibody over a glycophorin A affinity column. N o antibody
bound specifically to the column, as determined by monitoring the acid eluate spectrophotometrically. Furthermore, the
ability of the E6 preparation to decrease deformability was
maintained after passage over the column. When we tested
the effects of antibodies 10-22 and 4-21 on deformability, we
found that they, like E6, decreased deformability and that
this effect was also not altered by passage over a glycophorin
A affinity column (data not shown). We conclude from these
data that E6, 10-22, and 4-21 do not bind to glycophorin A in
the purified form and may require glycophorin A and band 3
in their native, conformational relationship for binding.
Whether the antibody-induced decrease in membrane deformability is mediated through band 3 or through glycophorin A
remains unclear, in part because the effect on deformability
of band 3-specific antibodies is unknown. This set of experiments has been impossible to perform because, to our
knowledge, no band 3 antibody has been produced that binds
to intact, nonenzymatically treated erythrocytes.
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846
TELEN AND CHASIS
A
Control
l
R B C t E6
X
W
a
z
B
0.71
I
/Control R E C
0.3
/
RBCtE6
0.2
g 0.2
LL
:O0.1. l /
I
I
50
I
100
200
,
I
I
I
I , ,
500 1000
SHEAR STRESS (dynes/cm2)
O 0 I lY
I
40
80
!
I
I
I
120 160 200 240 280
320
S H E A R STRESS (dynes /cm2 1
I
I
360
400
Fig 3. Deformability of erythrocytes after incubation with E6 antibody. In panel A the deformability index is plotted against the shear
stress applied to erythrocyte membranes. Erythrocytes without antibody achieved a maximum deformability index of approximately 0.7,
while erythrocytes incubated with antibody E6 achieved maximum deformability index of 0.5. Results did not change when the amount of
antibody used was increased (data not shown), indicating that these results represent the maximal antibody effect. In panel E,
deformability index is plotted on a linear scale, while shear stress is plotted on a log scale. The data produce two linear curves, indicating
that erythrocytes incubated with E6 required 43 times more shear stress to reach a deformability equivalent to that of cells not incubated
with antibody. This relationship was constant at all levels of shear stress tested.
antibody 4-21. On the one hand it is similar to antibodies E6
DISCUSSION
and 10-22 in that it immunoprecipitates both band 3 and
The Wr" and Wrb antigens have presented an interesting
glycophorin A, it decreases membrane deformability, and it
puzzle to serologists for many years. MoAbs E6 and 10-22
does
not bind to K562 and HEL cell lines. On the other hand,
were useful probes for exploring the determinants of Wrb
although
antibody 4-21 has been reported by several laboraantigen expression, since both demonstrated anti-Wrbspecitories to fail to react with Wr (a+b-) erythrocytes, this
ficity in that they reacted with random donor erythrocytes
study, as well as others (eg, participants in the First
but failed to react with Wr (a+b-) and En (a-) erythroInternational Workshop on Monoclonal Antibodies Against
cytes. The finding that neither of these antibodies bound to
Red
Cells and Related Antigens), found that it reacted
K562 and HEL human erythroleukemia cell lines, which do
weakly when tested with fresh rather than frozen, thawed Wr
not express band 3, indicated that the presence of glyco( a + b-) cells. Furthermore, in deformability studies using
phorin A in apparently normal orientation on the cell surface
Wr (a+b-) erythrocytes, no change in deformability was
was not sufficient for Wrb expression. Although glycophorin
seen with antibodies E6 and 10-22, while a slight decrease in
A on K562 and HEL cells may be ~nderglycosylated,*~*~~
deformability was observed with 4-21, confirming weak
abnormal glycosylation is an unlikely explanation for the
reactivity of antibody 4-21 with Wr (a+b-) cells. Most
absence of Wrb, since the glycosylated domains of glycointeresting, however, is the normal reactivity of 4-21 with En
phorin A are not essential for expression of Wrbantigem4The
(a-) erythrocytes, suggesting that if antibody 4-21 does
binding data obtained with K562 and HEL cells thus support
recognize a determinant related to the Wrb antigen, it must
the hypothesis that a second membrane moiety is required
recognize one independent of glycophorin A expression. Due
for expression of Wrb and that band 3 may be this compoto the extreme rarity of En (a-) red cells and the necessity of
nent. The presence of both band 3 and glycophorin A in the
fresh cells for such studies, we were able to perform only one
immunoprecipitates further implies a role for both of these
immunoprecipitation experiment with En (a -) red cells.
polypeptides in Wrb expression.
When antibodies 4-21 and TE-10 were used in parallel,
At this time we do not fully understand the specificity of
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ERYTHROCYTE W R ANTIGEN
~
EXPRESSION
Normal erythrocytes
Control
+E6
+E6
adsorbed
with GP a
047
En(a-)
+10F7
Control
erythrocytes
+E6
+lOF7
Fig 4. Effect of antibody binding on normal and En (a-)
erythrocytes. When erythrocytes were incubated in buffer alone
or in buffer containing antibody, erythrocytes treated with antibody E6 had a relative deformability of 2.5%. and those treated
with antibody 10F7 (a well-characterized antibody to glycophorin
A) had a relative deformability of 8.9%. When En (a-) erythrocytes were tested in similar experiments. antibodies E6 and 1OF7
caused no significant decrease in deformability.
TE-10 precipitated large amounts of band 3 protein while
4-21 precipitated small amounts of band 3. We conclude
from this very preliminary data that the 4-21 epitope may be
on band 3 but that in the absence of glycophorin A, antibody
4-21 has low affinity for its epitope under the experimental
conditions used.
Taken together the antibody binding, immunoprecipitation, and membrane deformability data support the hypothesis that band 3 and glycophorin A are both involved in Wrb
antigen expression and that a polymorphism of band 3, which
affects band 3-glycophorin A interaction, may constitute the
primary polymorphism responsible for expression of the Wrb
antigen. For this model to be valid, molecules of band 3 and
glycophorin A must be in close proximity to one another
either at the external or cytoplasmic surface of the lipid
bilayer or within the bilayer. An association between the two
polypeptides has been suggested by Nigg et al,’ who reported
that antiglycophorin A antibodies markedly reduced the
rotational diffusion of band 3 and concluded that band 3 and
glycophorin A might form a complex in the erythrocyte
membrane. Although there is considerable knowledge about
the structural organization of both band 3 and glycophorin
A, little is known about the spatial relationships between
these two proteins. Band 3 associates with the cytoplasmic
skeletal protein network through an interaction between its
aminoterminal domain and the skeletal protein, ankyrin.”
However, not all copies of band 3 can associate with ankyrin,
since there are approximately 1 million copies of band 3 per
erythrocyte and only 2 x lo5copies of a n k ~ r i n . * ~Although
**~
protein-protein interactions of the remaining copies of band
3 have not been fully characterized, one potential interaction
is an association with the skeletal protein 4.1 .30 Interestingly,
protein 4.1 has a high-affinity binding site on glycophorin
A.”.” Since both band 3 and glycophorin appear capable of
interaction with the same skeletal protein component, at least
a certain number of copies of each of these two transmembrane proteins may be in close proximity to one another. The
possibility that only certain subpopulations of each of these
polypeptides is involved in Wrbexpression is supported by our
finding that anti-Wrb precipitates contain a narrower band 3
on SDS gels than the immunoprecipitates obtained with the
band 3-specific antibody TE-IO. If, indeed, molecules of
glycophorin A and band 3 are adjacent to one another, an
electrostatic interaction might occur between negatively
charged sialic acid residues on the extracellular domain of
glycophorin A and positively charged extracellular band 3
loops between helices 5-6,7-8 and 9-10.33.34
Conclusive evidence that a polymorphism of band 3 is
responsible for expression of the Wrb antigen must await
sequencing of band 3 or its gene from W r (a-b+) and Wr
( a + b-) erythrocytes or nucleated cells, respectively. However, the data in the present study strongly suggest that band
3 and glycophorin A are both involved in Wrb antigen
expression and that these two proteins are closely associated
in the erythrocyte membrane.
ACKNOWLEDGMENT
The authors are grateful to Dr Margaret Nichols (Organon
Teknika, Durham, NC) for contributing antibodies 10-22 and 4-21,
to Dr Heinz Furthmayr (Stanford University, Stanford, CA) for
supplying purified glycophorin A, to Dr Ronald Jensen (Lawrence
Livermore Laboratories, Livermore, CA) for antibody 10F7. and to
Dr Peter D. Issitt for sharing unpublished data and for providing
rare red cells for binding studies.
REFERENCES
1. Wren MR, Issitt PD: Evidence that WP and Wrbare antithetical. Transfusion 28:113, 1988
2. lssitt PD, Pavone BG, Wagstaff W, GoldfingerD: The phene
types En (a-), Wr (a-b-),and En (a+), Wr (a+b-), and further
studies on the Wr and En blood group systems. Transfusion 16:396,
1076
.,.-
3. Ridgwell K. Tanner MJ, Anstee DJ: The Wrb antigen, a
receptor for Plusmodiumfalcipurum malaria, is located on a helical
region of the major membrane sialoglYcoProteinof human red blood
cells. Biochem J 209:273, 1983
4. Dahr W, Wilkinson S. Issitt PD. Beyreuther K, Hummel M,
Morel P High frequency antigens of human erythrocyte membrane
sialoglycoproteins: Ill. Studieson the En’FR, Wrband Wr”antigens.
Biol Chem HoppeSeyler 367:1033. 1986
5. Holman CA: A new rare human blood group antigen (WP).
Lancet 2:119, 1953
6. Pavone BG, lssitt PD, Wagstaff W: Independence of Wright
from many Other
group systems. Transfusion
1977
17:479
7. Wren M R 7
PDv Dahr w: Additional data On the
relationship of Wrb to Wr“: Antigen measurement using enzymelinked antiglobulin tests and further data on the structure of Wrb.
Transfusion24:425, 1984(abstr)
8. ~i~~ EA, B~~~C, ~
i M, Cherry
~ RJ: ~Band 3-glycophorin
~
d
A association in erythrocyte membranes demonstrated by combining
protein diffusion measurementswith antibody-inducedcross-linking.
Biochemistry 19:1887, 1980
9. Makler MT, Piper RC, Coull B: Erythrocyte membrane
changes associated with ligands which bind to the glycophorins. Adv
Biosci 67:251, 1987
~
~
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
848
10. Telen MJ, Scearce RM, Haynes B F Human erythrocyte
antigens. 111. Characterization of a panel of murine monoclonal
antibodies that react with human erythrocyte and erythroid precursor membranes. Vox Sang 52:236, 1987
1 1 . Bigbee WL, Vanderlaan M, Fong SSN, Jensen RH: Monoclonal antibodies specific for the M- and N-forms of human
glycophorin A. Mol Immunol20:1353, 1983
12. Galfre G, Howe SC, Milstein C, Butcher GW, Howard JC:
Antibodies to major histocompatibility antigens produced by hybrid
cell lines. Nature (Lond) 266550, 1977
13. Lozzio BB, Lozzio CB: Properties of the K562 cell line
derived from a patient with chronic myeloid leukemia. Int J Cancer
19:136, 1977
14. Martin P, Papayannopoulou T: HEL cells: A new human
erythroleukemia cell line with spontaneous and induced globin
expression. Science 216:1233, 1981
15. Chow FL, Telen MJ, Rosse W F The acetylcholinesterase
defect in paroxysmal nocturnal hemoglobinuria: Evidence that the
enzyme is absent from the cell membrane. Blood 66:940, 1985
16. Telen MJ, Palker TJ, Haynes B F Human erythrocyte antigens. 11. The In (Lu)gene regulates expression of an antigen on an
80-kilodalton protein of human erythrocytes. Blood 64599, 1984
17. Dodge JT, Mitchell C, Hanahan DJ: The preparation and
chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys 100:119,1963
18. Mohandas N, Clark MR, Jacobs MS, Shohet SB: Analysis of
factors regulating red cell deformability. J Clin Invest 66563, 1980
19. Chasis JA, Mohandas N: Erythrocyte membrane deformability and stability: Two distinct membrane properties that are independently regulated by skeletal protein interactions. J Cell Biol 103:343,
1986
20. A d a m J, Broviac M, Brooks W, Johnson NR, Issitt PD: An
antibody, in the serum of a Wr ( a + ) individual, reacting with an
antigen of very high frequency. Transfusion 11:290, 1971
21. Fukuda M: K562 leukemic cells express fetal type (i) antigen
on different glycoproteins from circulating erythrocytes. Nature
(Lond) 285:405,1980
TELEN AND CHASE
22. Gahmberg CG, Jokinen M, Anderson LC: Expression of the
major red cell sialoglycoprotein, glycophorin A, in the human
leukemia cell line K562. J Biol Chem 254:7442, 1979
23. Chasis JA, Mohandas N, Shohet SB: Erythrocyte membrane
rigidity induced by glycophorin A-ligand interaction: Evidence for a
ligand-induced association between glycophorin A and skeletal
proteins. J Clin Invest 75:1919, 1985
24. Chasis JA, Reid ME, Jensen RH, Mohandas N: Signal
transduction by glycophorin A: Role of extracellular and cytoplasmic domains in a modulatable process. J Cell Biol 107:1351, 1988
25. Ekblom M, Gahmberg CG, Anderson LC: Late expression of
M and N antigens on glycophorin A during erythroid differentiation.
Blood 66:233,1985
26. Liszka K, Majdic 0, Bettelheim P, Knapp W: Glycophorin A
expression in malignant hematopoiesis. Am J Hematol 19219,1983
27. Bennett V: The molecular basis for membrane-cytoskeleton
association in human erythrocytes. J Cell Biochem 18:49,1982
28. Low PS: Structure and function of the cytoplasmic domain of
band 3: Center of erythrocyte membrane-peripheral protein interactions. Biochem Biophys Acta 864:145, 1986
29. Sheetz MP: Membrane skeletal dynamics: Role in modulation of red cell deformability, mobility of transmembrane proteins,
and shape. Semin Hematol20:175, 1983
30. Pasternack GR, Anderson RA, Let0 TL, Marchesi VT:
Interactions between protein 4.1 and band 3. J Biol Chem 260:3676,
1985
3 1. Anderson RA, Lovrien RE: Glycophorin is linked by band 4.1
protein to the human erythrocyte membrane skeleton. Nature
(Lond) 307:1887,1980
32. Anderson RA, Marchesi VT: Regulation of the association of
membrane skeletal protein 4.1 with glycophorin A polyphosphoinositide. Nature (Lond) 318:295,1985
33. Jay D, Cantley L: Structural aspects of the red cell anion
exchange protein. Ann Rev Biochem 5 5 5 1 1, 1986
34. Kopito RR, Lodish HF: Structure of the murine anion
exchange protein. J Cell Biochem 29:1, 1985
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1990 76: 842-848
Relationship of the human erythrocyte Wrb antigen to an interaction
between glycophorin A and band 3
MJ Telen and JA Chasis
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