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Human Erythrocyte Acetylcholinesterase Bears the Yt" Blood Group Antigen
and Is Reduced or Absent in the Yt(a-b-) Phenotype
By Neeraja Rao, Carolyn F. Whitsett, Sarah M. Oxendine, and Marilyn J. Telen
The Cartwright (Yt) blood group antigens have previously
been shown likely to reside on a phosphatidylinositol-linked
erythrocyte membrane protein. In this study, an unusual
individualwhose red blood cells (RBCs) were of the previously unreported Yt(a-b-) phenotype were used, along
with normal Yt(a+) cells, to investigate serologically and
biochemically the relationshipof the Yt" antigen to known
phosphatidylinositol-linked erythrocyte proteins. Yt(a-b-)
RBCs expressed normal amounts of various phosphatidylinositol-linked proteins except acetylcholinesterase. Further, human anti-Yt" reacted with acetylcholinesterase in
immunoprecipitation and immunoblotting studies. Thus,
acetylcholinesterase is now identifiedas the protein bearing
the Yt blood group antigens.
0 1993 by The American Society of Hematology.
T
The Yt(a-b-) RBCs (donor WH) used in this study were identified
as having that phenotype by a number of different reference laboratories, and detailed serologic studies will be described elsewhere.
A number of different Yt" antisera were used in various parts of
this study. Two different antisera for Yta were used for immunochemical analyses: These were kind gifts from Rock Oyen (New York
Blood Center, New York) and from Kathy Skradski (Minneapolis
Memorial Blood Center, Minneapolis, MN). For some radioimmunoprecipitations, human antisera were partially purified by adsorption
onto random donor erythrocytes and eluted by using Elu-Kit11 (gift
of Gamma Biologicals, Houston, TX), according to manufacturer's
instructions. For negative controls, normal sera not containing detectable alloantibodies were similarly processed. A large excess of
albumin was then added to each eluted antibody to equalize protein
content and to prevent degradation of small quantities of purified
antibody.
All the monoclonal antibodies (MoAbs) used in this study have
been previously described. The anti-AChE antibodiesAE1 and AE2"
were obtained from hybridoma cells supplied by the American Type
Culture Collection (Rockville,MD). AE3 and AE4 antibodies'' were
the kind gift of Dr Douglas Fambrough (Johns Hopkins University,
Baltimore, MD). Anti-LFA-3" was a gift of Dr Timothy Springer
(Dana Farber Cancer Institute, Boston, MA), and anti-JMH antibody
H823,24and anti-DAF antibody 3.3 were gifts of Dr Robert Knowles
(Ortho Pharmaceuticals, Raritan, NJ). Anti-CD59 antibody 2/24mZ5
was provided by Dr Anne Fletcher (Red Cross Blood Transfusion
Service, Sydney, Australia).
Radioimmunoassays. Antibody reactivity with random donor
as well as the variant Yt(a-b-) E was measured by radioimmunoassay
All erythrocytes were assayed in
(RIA) as previously des~ribed.'~,'~
triplicate for antigen expression at comparable cell counts (determined
with an ELTI; Ortho Diagnostics, Raritan, NJ). Yt(a-b-) E, along
with cells from normal donors, were tested for their ability to bind
antibodiesto various PI-linked proteins such as DAF, LFA-3, MIRL,
and JMH as well as four MoAbs AE I , AE2, AE3, and AE4 to different
HE CARTWRIGHT (Yt) blood group system comprises
two antigens: Yta and Ytb.I The Yta antigen occurs in
99.7% of blood donors, while the antithetical antigen Ytb
occurs in only 8.1% of donors. Nevertheless, anti-Yta, made
by the 0.3% of the population that is Yt(a-b+), is not rare
and may have significant ability to destroy transfused homologous Yt(a+) red blood cells (RBCs). N o persons with
Yt(a-b-) RBCs have been previously described.
Until recently, little was known about the biochemistry of
the Yt antigens, except that they could be denatured by combinations of sulfhydryl reagents and proteases,' implying a
protein backbone. However, study of RBCs from patients
with paroxysmal nocturnal hemoglobinuria (PNH) has shown
that the Yt antigens were likely to reside on a phosphatidylinositol (PI)-anchored protein, as abnormal complementsensitive cells of PNH patients fail to express these antigens.'
It is well recognized that complement-sensitive PNH erythrocytes fail to express proteins anchored to the membrane
in this way, although the defect responsible for this deficiency
has not yet been defined.3
Human blood cells express a wide variety of PI-anchored
proteins subserving diverse functions! PI-anchored proteins
expressed by erythrocytes include decay accelerating factor
( D A F CD55),536membrane inhibitor of reactive lysis (MIRL;
CD59),7-9 lymphocyte function associated antigen-3 (LFA3; CD58),"." acetylcholinesterase (AChE),''-14 NAD+ glycohydrolase,15JMH protein (p76),I6 and the protein that bears
the Holley and Gregory antigens (Hy/Gy protein)." DAF
protein bears Cromer blood group antigens.18-20Membrane
inhibitor of reactive lysis (CD59), LFA-3 (CD58), AChE, and
NAD' glycohydrolase have not been previously shown to
bear blood group antigen activity.
The purpose of this study was to identify the PI-anchored
protein carrying Cartwright (Yt) blood group antigens and
to characterize a newly described Yt(a-b-) phenotype. Using
biochemical and serologic techniques to study both normal
Yt(a+b-) and rare Yt(a-b-) erythrocytes, we have now
demonstrated that the common Yta antigen resides on erythrocyte AChE.
MATERIALS AND METHODS
Blood cells and sera. For most studies, whole blood from healthy
normal donors was collected using tripotassium EDTA as anticoagulant and was stored in acid citrate dextrose at 4°C for up to 1
week. Erythrocytes (E) were then washed in phosphate-buffered saline
(PBS; pH 7.4) before use. Immunoprecipitation studies were performed on freshly drawn, unstored cell samples.
Blood, Vol81, No 3 (February I ) , 1993: pp 815-819
From the Department of Medicine, the Division of Hematology/
Oncology, Duke University Medical Center, Durham, NC; and the
Department of Pathology and Laboratory Medicine, Emory University
School of Medicine, Atlanta. GA.
Submitted August 31, 1992; accepted September 29, 1992.
Supported by Grant Nos. HL33572 and HL44042from the National
Institutes ofHealth (NIH). M.J. T. is the recipient ofResearch Career
Development Award HL02233 (NIH).
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.
0 1993 by The American Society of Hematology.
0006-49 71/93/8103-0007$3.00/0
815
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816
RAO ET AL
AChE epitopes. P3x63/Ag8 ascitic fluid, as well as buffer alone, was
used as negative control.
Enzyme assays. AChE activity was measured at room temperature using the assay of Ellman et alZ7with 1 mmol/L acetylthiocholine
iodide and 250 pmol/L 5,5’-dithiobis(2-nitro-benzoicacid) in 100
mmol/L sodium phosphate buffer, pH 7.4. After the reaction was
stopped with absolute alcohol, optical density (OD) was measured
spectrophotometricallyat 412 nm (Spectronic 1001; Milton Roy Co,
Rochester, NY).
Radioimmunoprecipitation. Erythrocytes were obtained from
EDTA-anticoagulated whole blood. They were washed three times
in PBS and were labeled with ‘’’I by using Iodo-Gen (Pierce, Rockford,
IL), as described previously.26Immunoprecipitation with anti-Yta
and AE4 antibodies was then performed according to previously described procedure^.^^^^^ For immunoprecipitation with human antisera, 5 X 10’ washed, radiolabeled cells were incubated with 400 pL
of adsorbed and eluted Yt” antibody or a similar preparation made
from nonreactive serum, for 1 hour at 22”C, washed, and then lysed
with hypotonic buffer.30Membranes from 5 X 10’ cells were then
solubilized in 400 pL of 0.015 mol/L NaC1, 0.05 mol/L Tris, 0.005
mol/L EDTA, pH 7.4 with 1% (vol/vol) Nonidet P-40 (Sigma, St
Louis, MO), 0.003 mol/L phenylmethylsulfonyl fluoride (PMSF),
and 0. I % gelatin (Rip buffer) for 30 minutes at 4°C. After centrifugation to remove unsolubilized material, supernates were then used
for immunoprecipitation. “Preclearing” was accomplished by two
incubations of this membrane protein solution with 200 pL of gelatinSepharose (Pharmacia Fine Chemicals, Uppsala, Sweden)suspended
in Rip buffer. Immune complexes were then precipitated by using
Sepharose beads conjugated to anti-human antibody (Cappel, West
Chester, PA). Elution of immunoprecipitated protein and analysis
by polyacrylamide gel electrophoresis(PAGE) and autoradiography
were then accomplished as described previously.28~29
Radioimmunoprecipitation and analysis with the use of MoAb AE4 and P3x63/
Ag8 ascitic fluid (negative control) were performed simultaneously
to control for AChE level of expression.13
Dot-blot analysis. Serial dilutions of purified AChE were applied
to nitrocellulose and allowed to bind for 2 hours at 22°C. The membrane was then washed with Tris-buffered saline containing 0.05%
Tween 20 and incubated with either human anti-Ytaor normal human
serum overnight at 4°C. Washing and detection of antibody binding
was then performed using an alkaline phosphatase-anti-IgG conjugate
and the 5-bromo-4chlorc-3-indolyl phosphate/nitro-blue tetrazolium
(BCIP/NBT)chromogenic substrate (Promega, Madison, WI).I6
RESULTS
Identijication of Yt(a-b-) propositus. A 60-year-old
white man, with cardiomyopathy and previous aortic valve
replacement, was undergoing evaluation for heart transplant.
He had no history of neurologic dysfunction. Routine pretransfusion antibody screening for RBC alloantibodies
showed presence of an antibody reactive with all cells but
the patient’s. Family members’ RBCs were incompatible. The
patient’s cells had a weakly positive ( 1 +) direct antiglobulin
test with anti-complement only, and extensive antigen typings
of the patient’s RBCs showed them to be Yt(a-b-) using
standard blood bank (agglutination) techniques. However,
during studies performed by several consulting labs (to be
reported elsewhere), very weak expression of Yt” was suggested by the fact that some, but not all, examples of antiYt” could be adsorbed onto and eluted from the patient’s
cells. The patient’s antibody reacted with all cells tested except
complement-sensitive PNH RBCs, suggesting that it recognized a PI-anchored structure. Chromium survival studies
confirmed apparent presence of anti-Ytab in that both
Yt(a+b-) and Yt(a-b+) cells had reduced survival: 74% and
12% of Yt(a+b-) cells survived I and 24 hours, respectively,
whereas 75% and 46% of Yt(a-b+) cells survived 1 and 24
hours; 96% of autologous cells survived at 24 hours.
Expression of PI-linked proteins by Yt(a-b-) erythrocytes. Expression of various PI-anchored proteins was examined by RIA, using a variety of monoclonal antisera. In
initial studies, Yt(a-b-) RBCs bound antibodies to LFA-3,
CD59, JMH protein, and DAF in amounts similar to those
bound by normal controls (Table 1). However, in the same
study, 4% as much AEl antibody to AChE was bound by
Yt(a-b-) cells as by random donor cells (Table 1). When
binding of other anti-AChE MoAbs was measured and compared with control cells, the amount of binding seen was
17%,8%, and 17% of normal for antibodies AE2, AE3, and
AE4. AE2 antibody recognizes an epitope that overlaps but
is not identical to that which binds AE 1, while the AE3 and
AE4 antibodies recognize two other independent epitopes of
AChE.” Antibody AE4 was then used to compare expression
of AChE by Yt(a-b-) cells with that of a larger number of
random donors. When compared with 13 additional normal
cell samples, the Yt(a-b-) cells bound only 10% as much
AE4 antibody as did the normal cells (64 cpm v 643 cpm k
35 SEM). Thus, Yt(a-b-) cells expressed a level of AChE
antigenic activity more than 4 SDs below the mean for normal
individuals.
AChE enzyme activity. To confirm that lack of AChE
antigenic activity correlated with lack of AChE enzyme activity, a colorimetric assay of AChE was p e r f ~ r m e d . ’ ~ . ~ ’
Yt(a-b-) cells expressed 15% as much AChE enzyme reactivity as did normal, sample age-matched controls (OD4,*of
0.058 v a mean of 0.385 for three normal controls). Thus,
the level of AChE enzyme activity correlated with AChE antigenic activity.
Immunochemical studies of Yt(a-b-) RBCs. To confirm
further that AChE was absent on Yt(a-b-) RBCs, immunoprecipitation studies were performed using variant and
normal cells in parallel. As shown in Fig 1A, AChE could be
immunoprecipitated by antibody AE4 from the lysate of
normal radiolabeled RBC membranes but not from Yt(a-b-)
cell membrane lysate.
Immunoprecipitation and dot-blot studies using human
anti- Yt”. Confirmation of the relationship between AChE
and the high-frequency antigen Yt” was obtained using human
anti-Yt” in immunoprecipitation studies. In experiments
performed using human anti-Yt” and murine monoclonal
anti-AChE in parallel (Fig IB), all three antisera immunoprecipitated a single protein band of approximately 160 Kd
Table 1. Expression of PI-Linked Proteins
by Yt(a-b-) Erythrocytes
Specific cpm Bound
Yt(a-b-)
Normal no. 1
Normal no. 2
LFA-3
MlRL
JMH
DAF
AChE
1,820
1,427
1,743
2,284
2,396
2,674
872
802
630
1,759
1,720
1,731
19
543
278
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ACETYLCHOLINESTERASE BEARS y r ANTIGEN
817
Mr x l o 3
Mr x 10"
200 -
200 92.5 -
92.5 -
69 -
.I
69 -
46 -
46 -
B
30 -
1
2
3
4
14.3 -
A
1
2
3
4
Fig 1. Immunoprecipitation studies using murine anti-AChE antibody AE4 and human anti-VP. (A) Antibody AE4 (lanes 1 and 3) and
control murine ascites fluid P3x63/Ag8 (P3) (lanes 2 and 4) were used to immunoprecipitate proteins from lysed radiolabeledVt(a-b-)
erythrocytes (lanes 1 and 2) and normal Vt(a+) erythrocytes (lanes 3 and 4). and the results were analyzed under nonreducingconditions.
AE4 precipitated AChE (Mr approximately 160,000) only from the Vt(a+) cells. (B) Two diRerent examples of human anti-VP (lanes 1 and
2). as well as nonreactive human serum (lane 3)and murine antibody AE4 (lane 4). were used in immunoprecipitation experiments with
normal Vt(a+) erythrocytes. Both examples of anti-VV precipitated a 160.000-Da band (lanes 1 and 2) of similar molecular weight to the
protein immunoprecipitated by antibody AE4 (lane 4). Nonreactive human serum (lane 3) did not precipitate a similar band, nor did murine
myeloma protein P3 (data not shown). The protein of approximately 92,500 Da in lanes 1 through 3 is band 3,nonspecilically immunoprecipitated by all human sera.
(analyzed under nonreducing conditions), whereas control
DISCUSSION
sera (normal human serum and murine P3 myeloma protein)
failed to precipitate such protein bands.
Because RBC membranes contain only very small numbers of AChE molecules, a fact that makes Western blotting
of membrane proteins for AChE difficult, and, because
human anti-Yt" immunoprecipitates protein only when
antibody is applied to intact cells, a third technique was
used to prove the identity of the protein recognized by
anti-Y t".
Physicochemically purified AChE'* was applied to nitrocellulose at varying concentrations, and the bound protein was reacted with both human anti-Yt" as well as normal human serum. The anti-Yt" reacted with AChE in this
system, whereas the normal serum did not (Fig 2).
This study shows that deficient expression of human
RBC AChE and normal expression of other PI-anchored
proteins are associated with the first reported example of
the Yt(a-b-) blood group phenotype. Furthermore, human anti-Yt', the antibody directed against the high-frequency Yt antigen, recognizes AChE in immunoprecipitation and immunoblotting experiments. Thus, this study
confirms previous work demonstrating that both the Yt'
and Ytb antigens were absent from the affected cells of
patients with paroxysmal nocturnal hemoglobinuria and
thus were probably resident on a PI-anchored membrane
protein.* While this report was being prepared, confirmation of this work was presented by Spring and An~tee.~'
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RAO ET AL
818
1
2
3
A
Fig 2. Dot-blot of AChE. Physicochemically puritied AChE was applied to nitrocellulose at varying
concentrations (decreasing from columns 1 to 3).
Row A was stained using human anti-Yr, while row
B
B was reacted with normal human serum containing
no identifiable alloantibodies. Anti-YV, but not normal human serum, reacted in a dose-dependent
manner with AChE.
In addition, recent mapping of the Yt blood group system
to chromosome 7q now provides a locus for the human
AChE gene.32
Extensive study and follow-up ofthe Yt(a-b-) propositus
(to be reported elsewhere) has shown that this patient developed the Yt(a-b-) phenotype in conjunction with an antibody probably directed to the AChE molecule. Over many
months, expression of Yt", as well as of AChE, increased, so
that Yta became detectable, although weakly so, by routine
blood banking methods 4 months after initial study. Quantitation of AChE at the end of the follow-up period showed
increased expression of AChE to 57% of normal levels concomitantly with appearance of the Yt" antigen. Loss of
expression of blood group antigens while an autoantibody to
the antigen or molecule is present has been described previously and is perhaps most common in the acquired JMHnegative phenotype.' However, it is important to note that
the function of AChE on RBCs is unknown and that isolated
hereditary erythrocyte AChE deficiency is at least theoretically
possible. Recent work has shown that RBC AChE uses a
small exon not expressed in other tissues; it is this exon that
provides the sequence necessary for posttranslational cleavage
and attachment of a PI anchor.33 Thus, a mutation in that
exon might be predicted to cause isolated AChE deficiency.
However, genetic studies have not been performed in the
current study because of the transient nature of the Yt(a-b-)
phenotype.
In summary, PI-anchored proteins have now been confirmed to bear antigens of at least four different blood groups:
Cromer, JMH, Holley/Gregory (Hy/Gp), and Cartwright
(Yt). In addition, Dombrock antigens appear to reside on a
PI-linked protein.2 Utilization of human antisera to these
antigens have thus far identified two apparently novel proteins, those bearing the JMH and Hy/Gp antigens. Further
use of human blood group antisera to identify and characterize membrane components should continue to help elucidate the nature and roles of RBC membrane proteins.
ACKNOWLEDGMENT
The authors thank Martha Rae Combs MT(ASCP)SBB and June
Godwin MT(ASCP) for help in confirming Yt' and Ytb typings, as
well as the many researchers (noted in the text) who contributed
antibodies for this study. In addition, we thank Dr Wendell Row
for the gift of purified AChE, and we thank Dr Palmer Taylor for
helpful discussions and for sharing unpublished data.
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ACETYLCHOLINESTERASE BEARS Yt' ANTIGEN
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1993 81: 815-819
Human erythrocyte acetylcholinesterase bears the Yta blood group
antigen and is reduced or absent in the Yt(a-b-) phenotype
N Rao, CF Whitsett, SM Oxendine and MJ Telen
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