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Membrane Orientation of Rh(D) Polypeptide and Partial Localization of Its
Epitope-Containing Domain
By Kimita Suyama and Jack Goldstein
We have previously shown that the effects of various enzyme
treatments on Rh antigen-containing polypeptides in situ
could be monitored by an antibody preparationwhich recognizes only these polypeptides following Western blotting.
We now have preparedantibodiesthat specifically react with
either the N- or C-terminal ends of Rh-related proteins. Using
all three, we have established that the C-terminus of Rh(D)
polypeptide is at the cell surface, whereas its N-terminal
domain is situated at the cytoplasmic side of the red blood
cell membrane. Chymotrypsin digestion of ghosts derived
from (-D-/-D-)
cells that are devoid of Rh (C/c) and (E/e)
antigens produces three major Rh(D)-relatedfragments: the
20-Kd fragment contains the molecule’s C-terminal domain,
the 17-Kd fragment its N-terminus, and the 13-Kd fragment
neither.However, only the 17-Kd fragment forms an immunecomplex with human polyclonal anti-D, indicating that it
contains the Rh(D) antigenic domain. Other findings presented here provide further evidence for a unique folding of
Rh(D) polypeptide within the cell membrane and suggest
that Rh(C/c) and (E/e) polypeptides, when present, may
form complexes with it.
o 1992by The American Society of Hematology.
C
surface of the cell and its N-terminus at the cytoplasmic
surface. We found too that chymotrypsin can cleave Rh(D)
polypeptide into several fragments, one of which, the
17-Kd, contains both the N-terminus and the Rh(D)
antigenic epitope. Some evidence is also presented suggesting that Rh polypeptides may be present as complexes
within the membrane.
ONSIDERABLE progress has been made in defining
the structures of the antigen-containing components
of the Rh blood group systems, the most recent being two
reports of the cloning of an Rh-related protein.’,’ The most
commonly occurring antigenic alleles, Rh(D), (C/c), and
(E/e), are now known to be components of integral
membrane proteins3-’which, while apparently not glycosylated; contain a significant number of acylated fatty acids?
Despite the fact that Rh(D), (c), and (E) polypeptides
contain identical N-terminal amino acid sequences, they
exhibit slight differences in size when subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE).6.10v11
More importantly, a comparison of their oneand two-dimensional peptide “maps” indicates that while
these polypeptides possess considerable homology, they are
structurally di~tinct.’”’~
It is also of interest to note that the
predicted molecular mass, on the order of 45,500’,’for the
cloned product, is somewhat larger than the 28,000 to
33,000 reported for Rh protein that had been immunoprecipitated and subjected to SDS-PAGE.3-7There is some
evidenceI6 that this disparity is due to the anomalous
behavior of Rh protein in SDS-PAGE.
We have reported that enzymatic treatment of intact red
blood cells (RBCs)and ghosts can be used to cleave both
the extracellular and cytoplasmic domains of Rh polypept i d e ~ Using
. ~ ~ such treatment coupled with appropriate
antibodies, we are attempting to define the positions of the
Rh antigens as well as other structural or functional regions
of these proteins. Some of the studies described here
directly show that the Rh(D) polypeptide is so placed
within the cell membrane that its C-terminus is at the outer
From The Lindsley F. Kimball Research Institute of The New York
Blood Center, New York, hT
Submitted July 22,1991; accepted September 30,1991.
Supported by office of Naval Research ContractNo. N-00014-84-C0543.
Address reprint requests to Jack Goldstein, PhD, The New York
Blood Center, 310 E 67th St, New York, NY10021.
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.
8 1992 by The American Society of Hematology.
0006-4971I921 7903-OO26$3.00/0
808
MATERIALS AND METHODS
Materials. Fresh RBCs (cDEIcDE, cdelcde) were obtained
from The New York Blood Center, New York, NY,and rare RBCs
(-D-I-D-)
from The American Red Cross Blood Services, St
Paul, MN. Human polyclonal anti-D plasma was purchased from
Ser-Tech Biologicals (North Brunswick, NJ); goat antirabbit IgGHRP conjugate from Cooper Biomedical (Malvem, PA). Antihuman IgG-agarose, trypsin, chymotrypsin, phospholipase A2, papain, glucose oxidase, lactoperoxidase, N-b-tosyl-L-lysine
chloromechloromethylketone (TLCK), N-tosyl-L-phenyl-alanine
thy1 ketone (TPCK), and keyhole limpet hemocyanine were obtained from Sigma (St Louis, MO); nitrocellulose paper from
Schleicher and Schuell (Keene, NH); 4-chloro-1-naphthol from
Bio-Rad (Rockville Center, NY);iodine-125 (carrier free) from
Dupont (Boston, MA); N-ethylmaleimide, maleimidobenzoyl-Nhydroxysuccinimide ester from Pierce (Rockford, IL).
Enzymatic treatments of evthrocytes, unsealed ghosts, and sealed
inside-out vesicles. Unsealed ghosts were prepared as described
by Jennings et a],” and sealed inside-out vesicles by the method of
Steck and Kant.” Enzyrne treatments were as previously reported”
except for chymotypsin digestion, which was performed as follows:
Ghosts (20%) in 10 mmol/L phosphate buffer, pH 8.0, containing
150 mmol1L KCI were incubated with 50 FgImL chymotrypsin at
37°C for the desired time. Incubation was stopped by addition of
TPCK to a final concentration of 50 kg/mL and treated ghosts
washed three times with 30 vol of phosphate-bufferedsaline (PBS),
pH 7.4.
Isolation of Rh(D) epitope-containing polypeptide and cleavage
products from chymotrypsin-digested unsealed ghosts obtained from
labeling of intact RBCs
‘zsI-surfacelabeled -D-1-Dcells.
was performed as described by Moore et ai.) Preparation of ghosts
and their treatment with chymotrypsin were performed as described in the previous section. Isolation of immune complexeswas
performed as previously published” except that in the washing of
the complex a 0.4 mol1L urea-1% Triton X-100 step was included
between the 1% Triton X-100 and H,O treatments. The eluted
immune complexes were separated by SDS-PAGE using 14% gels.
The slab gels were dried and exposed to x-ray film for 2 days at
-70°C.
Preparation of antibody against N-terminal and C-terminal regions
Blood, Vol79, No 3 (February 1). 1992: pp 808-812
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PROBING THE STRUCTURE OF Rh(D) ANTIGEN IN SITU
809
of Rh polypeptides. The sequence of the first 15 amino acid
residues at the amino terminusof Rh(D), (c), and (E) polypeptides
has been found to be identica1.l" A peptide consisting of this
sequence plus a cysteine residue at its C-terminus was prepared.
Similarly, a peptide corresponding to the predicted amino acid
sequence at the C-terminus (residues 402 to 416) of a recently
cloned Rh-related protein' was synthesized with an additional
cysteine residue added to its N-terminus. Each peptide was
conjugated with keyhole limpet hemocyanin (KLH) as described by
Davies et al." Antibodies were raised against the conjugates in
female New Zealand white rabbits and their titers determined by
enzyme-linked immunosorbent assay (ELISA) using 200 ng of
peptide/well.*' The antibody preparations were affinity-purified
using an AminoLink column (Pierce) containing the immobilized
synthetic peptides. Antibody against N-terminal peptide (antiRhNt) was used in a dilution of 1:100 antibody against C-terminal
peptide (anti-Rh Ct) in a dilution of 1:25. Western blots were
performed as previously described."
RESULTS
We have previously shown that Rh(D) polypeptide can
be cleaved to yield a 30-Kd fragment when intact RBCs are
treated first with phospholipase A2 followed by papain, and
that the cleavage can be monitored by immunostainingwith
a rabbit polyclonal antibody preparation specific for Rhrelated proteins (anti-Rh).17Using this approach with cells
having the -D-/-Dphenotype and newly prepared
antibodies to the amino (anti-RhNt) and carboxyl (antiRhCt) ends of Rh proteins (see Materials and Methods), it
was found that while both reacted with Rh(D) polypeptide
obtained from untreated cells (Figs 1 and 2) only anti-Rh
Nt immunostained the 30-Kd digestion product (Fig lB,
lane 2, Fig 2A, lane 4), indicating that the C-terminus of the
Rh(D) polypeptide is at the outer surface of the cell and
removed by phospholipase-papain digestion. To demonstrate directly that the N-terminus of Rh(D) polypeptide is
A
x ~ 0 - 3I 2 3
MW
I '- -.-
g64
55
MW
xto-3I I
B
2 3
g64
55
Fig 1. Western blots of Rh(D) polypeptides obtained from either
untreated or phospholipase A2 and papain-treated -D-/ -D- intact
cells and trypsin-treated -D-/-Dghosts using anti-Rh (A) or
anti-RhNt (e). Total membrane proteins prepared from similar volumes of packed cells or ghosts were separated by SDS-PAGE using a
12% polyacrylamide gel, transferred t o nitrocellulose paper, and
reacted with anti-Rh (1:l.OOO dilution) or with anti-Rh Nt (1:lOO
dilution), followed by goat antirabbit IgG conjugated with honeradish peroxidase (1:2,OOO dilution). Lane 1, ghosts from nontreated
cells; lane 2, ghosts from cells treated with phospholipase A2
followed by papain (0.1%); lane 3, trypsin digestion of ghosts from
nontreated cells. Identical results were obtained using inside-out
vesicles prepared from -D-/ -D- cells.
96 4
964
29
a
Fig 2. Western blots of Rh(D) polypeptides obtained from either
untreated or phosphollpase A2 and papain-treated -D- / -D- intact
cells and trypsin-treated -D-/-Dghosts using anti-Rh and antiRhCt. Separation and immunostaining of membrane proteins were
the same as described in the legend t o Fig 1. (A) Lane 1, ghosts from
nontreated cells reacting with anti-Rh; lane 2, ghosts from cells
treated with phospholipase A2 followed by papain (0.1%) reacting
with anti-Rh; lane 3, ghosts from nontreated cells reacting with
anti-RhCt; lane 4, ghosts from cells treated with phospholipase A2
followed by papain (0.1%) reacting with anti-RhCt. (B) Lane 1, ghosts
from nontreated cells reacting with anti-Rh; lane 2, trypsin digests of
ghosts from nontreated cells reacting with anti-Rh; lane 3, ghosts
from nontreated cells reacting with anti-RhCt; lane 4, trypsin digests
of ghosts from nontreated cells reacting with anti-RhCt. Arrows show
32-Kd and 31-Kd regions. Identical results were obtained using
inside-out vesicles prepared from -D- / -D- cells.
at the cytoplasmic surface, ghosts or inside-out vesicles
were prepared from -D-/-Dcells and treated with
trypsin. As previously de~cribed,'~
such treatment results in
the formation of a double-banded region, slightly reduced
in size (Fig lA, lane 3). Neither of these 31- or 32-Kd bands
reacted with anti-RhNt (Fig lB, lane 3) but both were
immunostained by anti-RhCt (Fig 2B, lane 4), thus indicating that the small reduction in size is due to the loss of the
N-terminus from Rh(D) polypeptide.
Trypsin treatment of ghosts made from Rh-negative cells
(cde/cde) readily resulted in the digestion of Rh(c) and (e)
polypeptides with the production of two anti-Rh immunostaining bands having apparent molecular weights of approximately 20,000 and 17,000, respectively (Fig 3A, lane 2).
Anti-RhNt was found to react only with the 17-Kd region
(Fig 3A, lane 4), whereas the opposite was true for
anti-RhCt-it reacted specificallywith the 20-Kd region (Fig
3B, lane 4).
The time of digestion of ghosts with trypsin that yielded
the results shown in Fig 3 and those previously reported17
was 45 minutes. However, when a time-course study was
performed with ghosts from cde/cde cells, the 20-Kd and
17-Kd anti-Rh immunostaining regions appeared within 10
minutes after treatment (Fig 4A, lane 2). No other bands
were found even after 20 hours of digestion (data not
shown). In contrast, when ghosts from cDE/cDE cells were
subjected to the same digestion conditions, four anti-Rh
immunostaining bands were detected. The 17-Kd and
20-Kd bands appeared at the same time as in the Rhnegative study (Fig 4A) while the other two, located in the
31-Kd to 32-Kd region of the gel, were generated between
60 and 120 minutes of trypsin treatment (Fig 4B, lanes 5
and 6). These barely separable bands were produced much
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810
MW
SUYAMA AND GOLDSTEiN
A
obtained over the 4-hour digestion period (Fig 5). The
three of interest were detected by anti-Rh at approximately
the 20-Kd, 17-Kd, and 13-Kd regions of the gel. An almost
immediate formation of a 20-Kd immunostaining band was
observed (Fig 5, lane 2) which, by 4 hours of chymotrypsin
digestion, appeared as a doublet (Fig 5, lane 7). Two
further bands appeared by 10 minutes, one at about the
17-Kd region and the other at the 13-Kd region of the gel
(Fig 5, lane 3). Although both of these bands were only
weakly immunostained, particularly the smaller by anti-Rh,
they were discernible after 30 minutes of digestion as well
(Fig 5, lane 4). To determine which of these bands
contained the N- and C-terminal regions Of the
a
chymotryptic digest Of membranes from -D- / -D- cells
was separated by SDS-PAGE and tested with appropriate
antisera. As shown in Fig 6, anti-RhNt reacted with the
17-Kd band (Fig 6A, lane 4) and anti-RhCt immunostained
the 20-Kd region (Fig 6B, lane 4) while neither reacted with
the 13-Kd cleavage product.
To ascertain which, if any, of these fragments contained
Rh(D) antigenic epitopes, we radiolabeled those segments
of the Rh(D) polypeptide exposed on the cell surface by
treating intact -D-/-Dcells with '=I as described in
Materials and Methods. Membranes were prepared from
these radiolabeled cells and treated with chymotrypsin for
30 minutes. Immuno-complexing using human anti-D was
then performed and the radiolabeled digestion products
separated on SDS-PAGE and subjected to autoradiography. The results are presented in Fig 7. Untreated Rh(D)
polypeptide is represented by the band at the 33-Kd region
of the gel as well as aggregates of this polypeptide that are
present at the higher molecular weight regions and top of
the gel (Fig 7, lane 1). This is not unexpected because
several laboratories have found that isolated Rh(D) polypeptide readily aggregates?'.'' After 30 minutes of chymotrypsin treatment one cleavage product of approximately 17 Kd
in size was obtained (Fig 7, lane 2). Identical results were
obtained when monoclonal anti-D was used in place of the
polyclonal antibody (results not shown). These results
indicate that while the three major chymotryptic cleavage
products can be immunostained with rabbit anti-Rh, only
B
MW
XI 0-3
x,
-3
,
_I
I.)
Jp
29
.
.
18
12
12
Fig 3. Western blots of Rh polypeptides before and after trypsin
digestion of ghosts prepared from Rh-negative (cde/cde) cells using
anti-Rh, anti-RhNt, and anti-RhCt. Separation and immunostainingof
membrane proteins were the same as described in the legend to Fig 1.
(A) Lane 1, ghosts from nontreatedcells reacting with anti-Rh; lane 2,
trypsin digests of ghosts from nontreatedcells reacting with anti-Rh;
lane 3, ghosts from nontreated cells reacting with anti-RhNt; lane 4,
trypsin digests of ghosts from nontreated cells reacting with antiRhNt. (B) Lane 1, ghosts from nontreated cells reacting with anti-Rh;
lane 2, trypsin digests of ghosts from nontreated cells reacting with
anti-Rh; lane 3, ghosts from nontreatedcells reacting with anti-RhCt;
lane 4, trypsin digests of ghosts from nontreated cells reacting with
anti-RhCt.
earlier, within 15 minutes, with essentially all of the intact
Rh(D) polypeptide being cleaved when ghosts from -D-/
-D- cells were digested with trypsin (Fig 4C, lane 2). The
32-Kd region virtually disappeared by 120 minutes (Fig 4C,
lane 5), presumably degraded into fragments no longer
immunostainable with anti-Rh. The lack of formation of
31-Kd to 32-Kd fragments in digests of Rh-negative ghosts
and their presence in digests from both Rh-positive phenotypes indicates that the 31-Kd to 32-Kd region arose solely
from the cleavage of Rh(D) polypeptide with trypsin and
was not the precursor of the 17-Kd and 20-Kd immunostaining regions. These, in turn, must therefore have been
digestion products of Rh (C/c) and (E/e) polypeptides.
Because digestion of ghosts from -D-/-Dcells with
trypsin degraded the intact Rh(D) polypeptide only to
31-Kd and 32-Kd fragments, we replaced trypsin with
chymotrypsin and performed similar time-course studies
using these ghosts. A number of cleavage products were
Mw
A
x103 I 2 3 4 5 6
v---
55
Mw
do-3 I 2
55
B
3 4 5 6
Mw
C
xIO'~ I 2 3 4 5
55
36
12
Fig 4. Western blots of Rh polypeptides before and after trypsin digestion of ghosts at various incubation times prepared from Rh-negative
/-D-) cells. Separation and immunostainingof membrane proteins were the same as described in the
(cde/cde) and Rh-positive (cDE/cDE, 4legend to Fig 1. Arrow indicates the 31-Kd region. (A) Lane 1, ghosts from nontreatedcde/cde cells; lanes 2 through 6, trypsin digestion of ghosts
from nontreatedcde/cde cells for: lane 2,lO minutes; lane 3,20 minutes; lane 4,45 minutes; lane 5,60 minutes; iane 6,120 minutes. (B) Lane 1,
ghosts from nontreatedcDE/cDE cells; lanes 2 through 6. trypsin digestion of ghosts from nontreatedcDE/cDE ceilsfor: lane 2,lO minutes; lane
3.20 minutes; lane 4,45 minutes; lane 5,W minutes; lane 6,120 minutes. (C) Lane 1, ghosts from nontreated -D- / -D- cells; lane 2 through 5,
trypsin digestion of ghosts from nontreated -D- / -D- cells for: lane 2,15 minutes; lane 3,30 minutes; lane 4,W minutes; lane 5,120 minutes.
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PROBING THE STRUCTURE OF Rh(D) ANTIGEN IN SITU
MW
xIO-~ I
81 1
MW
2 3 4 5. 6 7
x~0-3 I
. .
18
12
4
Fig 5. Western blots of Rh(D) polypeptides before and after
chymotrypsin treatment at various incubation times of ghosts from
Rh-positive (-D-/ -D-) cells. Separation and immunostaining of
membrane proteinswere the same as described in the legend to Fig 1.
Arrow indicates the 13-Kd region. Lane 1, ghosts from nontreated
-D- / -D- cells; lanes 2 through 7, chymotrypsindigestion of ghosts
from nontreated -D-/-Dcells for: lane 2, 1 minute; lane 3, 10
minutes; lane 4,30 minutes; lane 5 , l hour; lane 6,2 hours; lane 7,4
hours.
the 17-Kd polypeptide fragment formed an immunecomplex with human anti-D, suggesting that it contains an
intact Rh(D) epitope.
DISCUSSION
The two reports’3 of the cloned Rh-related polypeptide
disagree as to the location of its C-terminus. However,
studies in other laboratories based on carboxypeptidase Y
digestions of isolated proteins’’ and whole cells” have
provided indirect evidence for the carboxyl-terminal end of
Rh polypeptides being situated at the surface of the RBC.
In earlier work, we had shown that Rh(D) polypeptide in
intact cells could be cleaved by papain if the cells were first
MW
XIO-3
I
-
A
2 3 4
MW
XIO-3 I
B
2 3 4
AIF“-
55
18
12
Y ’
18
12
Fig 6. Western blots of Rh(D) polypeptides obtained from either
untreated or chymotrypsin-treated-D-/ -D- ghosts using anti-Rh,
anti-RhNt, and anti-RhCt. Separation and immunostaining of membrane proteins were the same as described in the legend to Fig 1
except that a 14% gel was used for (E). (A) Lane 1, ghosts from
nontreated -D- / -D- cells reacting with anti-Rh; lane 2, chymotrypsin digest of ghosts from nontreated -D-/ -D- cells reacting with
cells reacting
anti-Rh; lane 3, ghosts from nontreated -D-/-Dwith anti-RhNt; lane 4, chymotrypsin digests of ghosts from nontreated -D-/-Dcells reacting with anti-RhNt. (6) Lane 1, ghosts
from nontreated -D-/-Dcells reacting with anti-Rh; lane 2,
chymotrypsin digest of ghosts from nontreated -D- / -D- cells
reacting with anti-Rh; lane 3, ghosts from nontreated -D-/-Dcells reacting with anti-RhCt; lane 4, chymotrypsin digest of ghosts
from nontreated -D- / -D- cells reacting with anti-RhCt.
Fig 7. Autoradiogram of Rh(D)
polypeptide and cleavage product obtained by immune-comDIex formation with polvclonal
anti-D of chymotrypin-treated
and untreated ghosts from ’=I
surface-labeled -D-/ -D- cells.
Separation and autoradiography
of labeledmembraneproteinsare
described in Materialsand Methods. Lane 1, untreated ghosts;
lane 2, ghosts treated with chymotrypsin for 30 minutes.
2
55
36
29
18
12
treated with phospholipase A2. Our current results using
antibodies that selectively recognize each end of Rh-related
proteins (anti-RhNt and anti-RhCt) show that following
such enzymatictreatment the C-terminus of Rh(D) polypeptide is removed, indicating that it is at the surface of the cell.
Similarly, trypsin digestion of ghosts or inside-out vesicles
results only in the loss of that part of the N-terminal region
reactive with anti-RhNt, signifying that it is positioned at
the cytoplasmic surface of the RBC membrane. Because
Rh(C/c) and (E/e) polypeptides in intact cells are unaffected by phospholipase A2 and papain, we cannot conclude, by using this approach, that they have the same
orientation as Rh(D) polypeptide. However, this appears
quite likely based on their, as well as Rh(D) polypeptide’s,
susceptibility to carboxypeptidase as discussed above, and
the fact that they all have identical N-terminal amino acid
sequences.
Rh (C/c) and (E/e) polypeptides respond differently in
situ to trypsin digestion than their Rh(D) counterpart.”
Ghosts or inside-out vesicles so treated produce two cleavage products observed in the 20-Kd and 17-Kd regions of
SDS-PAGE gels that are derived from (C/c) and (E/e)
polypeptides. Because the 17-Kd region contains the N-terminus and the 20-Kd region the C-terminus, both halves of
these molecules appear to be present. In contrast, tryptic
digestion products of Rh(D) consist only of the 31-Kd and
32-Kd bands that are devoid of the N-terminal region
reactive with anti-RhNt, but contain the C-terminus of the
molecule. These results further support our previous suggestion that Rh (C/c) and (E/e) polypeptides may be folded
differently within the cell membrane than Rh(D) polypeptide because the latter is cleaved at an entirely different site
with trypsin. Furthermore, the tryptic products of Rh(D)
polypeptide appear much earlier in digests of ghosts or
inside-out vesicles from -D-/-Dcells (15 minutes)
than from preparations of other Rh positive cells that
contain (C/c) and (E/e) antigens (60 to 120 minutes). It
may be that the presence of other Rh-containing polypeptides exerts a protective effect against trypsin digestion,
possibly by forming a complex with Rh(D) polypeptide
within the lipid bilayer.=
In contrast to trypsin treatment, digestion of -D-/
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SUYAMA AND GOLDSTEIN
812
-D- ghosts with chymotrypsin produces three major
Rh-related regions. The first located at 20 Kd contains the
molecule’s C-terminus, the second at 17 Kd its N-terminus,
while the third at 13Kd contains neither. However, only the
17-Kd fragment could be isolated by immune-complex
formation with human polyclonal anti-D. This region was
also found to have radioactive tryosine residues, indicating
it was part of the Rh(D) polypeptide that had been labeled
with ‘*’I at the extracellular membrane surface of -D-/
-D- cells. This is further evidence that the 17-Kd material
does contain the antigenic domain of the Rh(D) molecule.
Based on hydropathy analysis, it has been proposed that
the cloned Rh-related polypeptide can display from five to
six looplike regions at the surface of the cell.’*2If Rh(D)
polypeptide has a similar distribution, then the 17-Kd
fragment is sufficient in size to encompass the first three
from the N-terminal end of the molecule. Whether one or
more of these regions are part of the Rh(D) antigenic
epitope is the subject of current investigation.
ACKNOWLEDGMENT
We thank R. Lunn, R. Hurst, and A. Sun for their expert
technical assistance and the following for providing us with
-D-/-Dcells: Carolyn Sullivan of the American Red Cross
Blood Services, St Paul, MN and donor S.M.; Dr Celso Bianco and
the Frozen Blood Laboratory of the New York Blood Center.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1992 79: 808-812
Membrane orientation of Rh(D) polypeptide and partial localization of
its epitope-containing domain
K Suyama and J Goldstein
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