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CONCISE
REPORT
Late
Expression
of M and
During
By Marja
The
M/N
cell
sialoglycoprotein.
drate
blood
groups
is needed
due
for
to amino
of
the
acid
for
carried
the
A.
activity.
but
We
M and
N blood
HE
M
AND
carried
to study
of the
human
protein,
erythrocyte
B,
glycophorin
blood
group.
cells,
group
antigens
major
the
The
irrespective
A
is
expressed
proerythroblasts,2
and
the corresponding
the carbohydrate
rin
A appear
identical,
M/N
antigenicities
oligosacchanides
because
are
Recently,
monoclonal
for
such
at which
bone
normal
was
essential
for
abolished
by
control
stainings,
rabbit
routine
normal
human
examinations
Alternatively,
thoracic
pieces
surgery
source
of bone
at
of bone
marrow
of ribs
which
Helsinki
marrow
by
further
Finnish
typing
Specific
En(a
-
Cross
rabbit
red
cell
cell
through
of M and
specific
membranes,
which
which
8161
4(8A2)
phonin
A,9
respectively,
against
were
Culture
Collection
(Rockville,
tane (Sigma,
St Louis)-treated
1 (July).
lack
produce
1985:
from
described.3
The
MGG.
of the
monoclonal
rabbit
anti-
(FITC)-conju-
mouse
first
IgG
and
normal
antibodies.
of antibody
of erythroid
cells,
cells
were
irrelevant
the
centrifuged
monoclonal
binding
bone
cells
were
was
counted.
onto
lgG
antibody
or antiserum
of staphylococci
bound
to different
marrow
cells
were
was
glass
slides
or normal
used
in
control
least
50
cells
stained
Ig instead
rosettes.
to the morphologically
At
and
rabbit
The
recognizable
of each
maturation
scored.
labeled.
oxidation
with
the cell-surface
Sialic
sodium
acid
residues
metapeniodate
glycoconjugates
were
were
radiolabeled
at 0 #{176}C
followed
by
by reduction
with NaB3H4.’2 Immunoprecipitations
of the surface-labeled
membrane lysates were made with monoclonal
antibodies
8 I 59 or 8 161,
followed by rabbit anti-mouse
Ig using
the protein
A containing
S
From
by the
prepared
N forms
American
as
with
A. The
the
Pathology
healthy
hy8159
of glycoType
Md). Ascites fluid produced
in PnisBALB/c
mice by the 8159 clone-
pp 233-236
the
with
were
absorption
antibodies
the
with
were
immuno-
.
Transplantation
and
No.
by
and
5 ROl
the
CA
Pathology.
Helsinki,
©
I 985
University
Finnish
theSigrid
26294-05
reprint
from
Society,
Foundation,
the
University
of
to Dr Le:fC.
Helsinki,
Departments
the
of
Academy
Helsinki,
National
25, 1985; accepted
requests
and
of Helsinki.
Cancer
Juselius
Bethesda.
Md.
Submitted
March
Address
Laboratory
Biochemistry,
Supported
Finland.
glycophonin
M and
obtained
from
was
by
Double
myeloid
performed
monoclonal
the
density
cells
Helsinki.
A antiserum
clones
No
was
were
smears
stained
smears
minutes.
isothiocyanate
analysis
stages
radioactively
a
England)
were
treated
in suspension
with rabbit anti-glycophorin
A antiserum
or
the monoclonal
antibodies
8159 and 8161
Cells with antibodies
bound to their surface antigens
were allowed to form rosettes with
protein
A containing
Staphylococcus
aureus
Cowan
I strain as
column.”
consent
Service,
as
cells
Sweden)
Ig-anti-Ig
N antigens
rendered
suspended
erythroid
informed
Transfusion
were
containing
and
and
Vol 66,
the
an
with
used
mononuclear
Uppsala,
cases,
were
Cheshire,
cytocentnifuge
monoclonal
instead
semiquantitative
stage
open
of
clone-
of 1:5,120.
the slides
by incubation
Fluorescein
For immunoprecipitation,
during
cells
the
anti-glycophonin
(6A7)
Blood,
marrow
during
used
maturational
transplantation.
resected
of Fc-receptor
obtained
Blood
described3
)
removal
obtained
Hospital
and
some
by passage
The
Red
previously
bnidoma
cells
were
volunteers.
In
by the
samples
before
been
(Pharmacia,
enriched
monocytic
donors
bone
(PBS),
Ficoll-Isopaque
centnifugation.’#{176}
Blood
were
had
The
saline
gradient
and
marrow
University
cells.
in phosphate-buffered
isolated
bone
the
followed
irrelevant
Ig were
number
METHODS
incubating
or 8161
dilution
8161
gated sheep anti-mouse
lgG and tetramethyl
rhodamine
isothiocyanate (TRITC)-conjugated
sheep anti-rabbit
Ig antisera
(Cappel
Laboratories,
Cochranville,
Pa) were used as second antibodies.
In
For
by sialylation.8
by
the
20 #{176}C)
for ten
-
a
cells were prepared
Products,
staining,
(
made
at
by the
at a dilution
Southern
A antiserum.
erythroid
from
(Shandon
8159
glycophonin
of the
Cells
erythrocytes
produced
from the bone marrow
immunofluorescent
For
with
AND
type
ascites
N erythrocytes
smears
a Shandon
antibodies
marrow.
MATERIALS
and
homozygous
staining
antibodies
which recognize
epitopes
have become available.9
We have
to determine
the stages
of erythroid
the M and N antigens
are expressed
in
antibodies
findings
Inc.
M
1:10,240,
fixed in cold methanol
M and N antigens
maturation
These
For morphological
analysis,
with May-Grunwald-Giemsa
(MGG).
ery-
marker
are
these
& Stratton,
homozygous
to
using
precursors.34
the M and N antigens
polymorphism
within
of sialic acids and can be regained
removal
used
the
was
abun-
recognizable
proerythroblast.
cytocentrifuge.
positions.5
Although
the compositions
of
moieties
of the M and N types of glycopho-
the
by Grune
Cytocentnifuge
terminal
5, while
the
it
already
maturation.
1985
S
agglutinated
surface
recognizable
it is a useful
erythroid
M/N
region of the glycophorin
A polypeptide.5
AM contains
serine and glycine
at positions
1
glycophorin
AN has leucine and glutamic
acid at
the amino
Glycophorin
on the
the
precursor.
1:5,120
sialoglycoof the
on
morphologically
both normal
and malignant
erythroid
The structural
differences
between
are based on the amino
acid sequence
and
minor
dantly
agglutinated
primarily
sialoglycoprotein
N-active
antiserum.
be explained
by our previous
observation
that
the
0-glycosylation
of glycophorin
A gradually
increases
during
expression
are
A
glycophorin
A molecules
are
present
on the earliest
morphologically
can
we report
that the M/N
weakly
or not
at all
normoblast
stage.
A,
anti-glycophorin
that
erythroid
antibodies
membrane.’
is
Glycophorin
of the earliest
membrane
throid
blood
N
is
portion
their
polyclonal
shown
specificity
NH2-terminal
A
and Leif C. Andersson
Using
red
carbohy-
monoclonal
groups
by glycophorin
human
M/N
the
used
during
normal
erythropoiesis.
Here
blood
group
activities
are
very
expressed
before
the
polychromatic
T
major
on Glycophorin
Differentiation
Carl G. Gahmberg,
0-glyosidic
in the
have
Erythroid
Ekblom,
by the
replacements
molecule.
specific
are
glycophorin
N Antigens
April
and
Institutes
of
of
by grant
Health.
22. 1985.
Andersson.
Haartmaninkatu
Department
3. SF
of
00290
Finland.
by Grune
& Stratton,
Inc.
0006-4971/85/6601-0035$03.00/0
233
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
234
EKBLOM,
Cowan I strain.’3 Electrophoresis
on 8% polyacrylamide
slab
gel was performed
according
to Laemmli.’4
The gels were fixed and
treated for fluorography
as described.’5
GAHMBERG,
AND
ANDERSSON
aureus
‘
A
.t
RESULTS
-....e
A
E%
homozygous
with the
Surface-labeled
immunoprecipitated
and 8161 and
were identified
the molecules
recognized
by polyacrylamide
slab
homozygous
no bands
N red cells,
anti-M
(8 1 61
),
were
obtained
M and N erythrocytes
monoclonal
antibodies
antibody
(Fig
the glycophorin
from
were
precipitated
with
the anti-N
and dimer
N
red
cells
to the dimeric
B from
phorin
both
(Fig
and monomeric
1C),
forms
cells
by
analyzed
(Fig 2).
strongly
throid
(Fig
3).
of glyco-
basophilic
more
mature
both
the
and
A
Cowan
and
the
weak
normoblasts.
erythroid
technique
the
rabbit
anti-glycophorin
anti-N
antibodies.
The
ery-
and
anti-N
proerythroblasts
Polychromatic
showed
reacted
normoblasts
anti-M
with
cells
was
recognizable
monoclonal
reactivity
marrow
antiserum
basophilic
normoblasts
strong
reactivity
and
with
the anti-M
the same
and
stage
In indirect
A antiserum
(Fig
4A).
reacted
nucleated
immature
the
anti-N
antibodies
of the erythroid
appeared
The
monoclonal
at approximately
differentiation.
immunofluorescence,
stained
large
nucleated
anti-M
rabbit
anti-glycophorin
erythroid
precursors
and
anti-N
antibodies
with
mature
erythrocytes
and
relatively
small
red cell precursors
(normoblasts),
while the largest
glycophorin
A-containing
cells did not stain with
monoclonal
antibodies
A and
the monoclonal
epitopes
recognized
by
BC
(Fig
4B).
5o
40
R anti-GP-A
::.
DE
-
x64).
and the
N antigens
1-rosetting
anti-glycophorin
A
earliest
morphologically
contrast,
showed
anti-M
S aureus
Fig 2.
The binding of monoclonal
anti-N antibody
81 61 to bone
marrow
cells as assessed
by the S aureus
Cowan
l-rosetting
technique.
Erythroid
cells are marked
by letters.
A. proerythroblast;
C, polychromatophilic
normoblast;
E. erythrocyte.
MayGr#{252}nwald-Giemsa
stain
(original
magnification
x 1 00; current
magnification
M and
the
proerythroblasts,
In
antibodies
and
the
Rabbit
with the
cells,
for
heterozygous
‘$
but
M and N red cells.
The binding
of the anti-glycophorin
A antiserum
monoclonal
anti-M
and anti-N
antibodies
to bone
erythroid
4
the
antibody
molecules
not from
MM red cells (Fig 1E). Similarly,
the anti-M
antibody reacted
with glycophorin
A from M cells (Fig I D).
In addition,
the anti-N
antibody
precipitated
two bands
corresponding
“
by these antibodies
gel electrophoresis.
In
1B).
Using
A monomer
homozygous
were
8 1 59
-
.-
10
0
z
-
!
0
.il&
50
40
8159
0
GPA-D---
-
GPA-D--
30
>-
20
I
10
GPA-M
GPB-M--
$
-
O
-
GPA-
-GPB-M
M-
$
-
GPB-D
-GPB-M
Fig 1 .
Polyacrylamide
slab gel to show specificity
of monoclonal
anti-M
and anti-N
antibodies.
(A) Surface
glycoprotein
pattern
of RBCs labeled
by the periodate/NaB3H4
technique;
(B)
pattern
of immune
precipitate
obtained
from 3H-labeled
NN cells
with anti-M
antibodies;
(C) pattern
obtained
from 3H-labeled
NN
cells with anti-N
antibodies;
(D) pattern
obtained
from 3H-labeled
MM cells with anti-M
antibodies;
(E) pattern
obtained
from 3Hlabeled
MM cells with anti-N
antibodies.
GPA-D.
glycophorin
A
dimer;
GPA-M.
glycophorin
A monomer;
GPB-D.
glycophorin
B
dimer;
GPB-M.
glycophorin
B monomer.
Note that GPA-M
and
GPB-D
have the same electrophoretic
migration
rates and that
GPB is N-active
irrespective
of the MN blood group.
Aa
Bb
Cc
Dd
Ee
Fig 3.
The binding
of rabbit
anti-glycophorin
A antiserum
(R
anti-GPA-A)
and monoclonal
anti-M
(81 59) and anti-N
(8161)
antibodies
to bone marrow
erythroid
cells. Vertical
bars indicate
the number
of staphylococci
per cell (mean ± SD). Aa. proerythroblast; Bb. basophilic
normoblast;
Cc. polychromatophilic
normoblast;
Dd. orthochromatic
normoblast;
Ee. erythrocyte.
Capital
letters
indicate
binding of the relevant
antibodies
and small letters
indicate
binding of control immunoglobulins
from the same species
as the antibodies.
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LATE
EXPRESSION
OF M AND
235
N ANTIGENS
that the anti-N antibody (8161)
N types of glycophorin.9
We
reactivity
reacted
Instead,
AM.
precipitated
glycophorin
In addition,
any crossof this antibody
with the
8161 antibody
specifically
anti-N
antibody
B from
described
the
AN.
the monoclonal
glycophorin
earlier
N erythrocytes
rin B, whose
both
from
M and
N types
immunoblotting
precipitated
of erythrocytes
studies.9
as
Both
contain
some N antigen
activity
first 23 amino acids are identical
glycophorin
the M and
both
not observe
in immunoprecipitation
glycophorin
with
did
M and
in glycophowith those of
AN.S
Glycophorin
A has been shown to be a specific
and early
erythroid
marker
present
in the membrane
of proerythroblasts.23
The epitopes
and anti-N
antibodies
clearly
cells
later,
by the monoclonal
anti-M
on the surface
of erythroid
recognized
appear
at the polychromatic
normoblast
demonstrated
aureus
Cowan
Carbohydrates
by the double
immunofluorescence
I-rosetting
technique.
are known
to contribute
determinants
recognized
by
the
is highly
glycosylated,
monoclonal
The relatively
determinants,
phorin
Fig 4.
erythroid
Double
cells.
immunofluorescence
(A)
Cells
staining
stained
with
of bone
rabbit
marrow
anti-glycophorin
A
antiserum
followed
by TRITC-conjugated
sheep anti-rabbit
The same field stained
with
monoclonal
anti-M
antibody
followed
by FITC-conjugated
sheep anti-mouse
lgG.
1g. (B)
8159
glycophorin
are
rin
molecules
A
The monoclonal
to react specifically
findings
from
the
and
N erythrocytes
.
VT,
Sci USA
of the
Robinson
observations.
and reported
consistent
changes
erythroid
with
early
of
than
by the rabbit
reflects
those
from
antigens
monoclonal
phorin
anti-glycophorin
in the glycosylation
The present
maturation.
our recent
erythroid
more
the present
results
leading
to expression
Since
changes
adequate
Sieff
Andersson
Glycophorin
tiation
in acute
5.
Furthmayr
immunochemical
1978
surface
orientation
erythrocyte
C,
Delia
Segrest
JP,
Scott
of the
membrane.
D,
HLA-DR,
differentiation.
Proc
52:379,
LC,
NatI
RE:
major
Acad
finding
precursors
mature
that
glycophoare less 0-
erythrocytes.”
show
that
the carbohydrate
of the M and N blood group
relatively
late during
erythroid
antibodies
reacting
with
epitopes
A to which
carbohydrates
contribute
for phenotyping
early erythroleukemias.
maturation,
on glycomay not be
occur
CG,
marker
Int J Cancer
Structural
analysis
Greaves
and
M:
glycophonin
1981
LC: Expression
erythroid
cells
of
Teerenhovi
of early
of the
in human
23:717,
comparison
genetic
variants.
L,
Vuopio
erythroid
P:
differen-
glycophonins
Nature
enzyme.
M and
N by influenza
Proc NatI Acad
Sadler
JE, Paulson
expression
of
human
254:2112,
1979
viruses
Sci USA
JC,
MN
44:182,
and
271:519,
6. M#{228}kel#{228}
0, Cantell
K: Destruction
of M and N blood group
receptors
of human red cells by some influenza
viruses. Ann Med
Exp Biol Fenn 36:366, 1958
7. Springer
GF,
Ansell
NJ:
Inactivation
of human erythrocyte
and
receptor-destroying
1958
Hill RL: The role of sialic
blood
group
antigens.
J
acid in the
Biol
Chem
9. Bigbee WL, Vanderlaan
M, Fong 55, Jensen RH: Monoclonal
antibodies
for the M- and N-forms
of human
glycophorin
A. Mol
Immunol
20:1353,
1983
B#{246}yum
A: Isolation
of mononuclear
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10.
1979
of
agglutinogens
8.
1978
surface
leukemia.
H:
on
Gahmberg
A as a cell
PAW,
289:68,
M, Andersson
(glycophonin)
Blood
Edwards
HLA-ABC
Nature
CG, Jokinen
sialoglycoprotein
bone marrow.
RL,
and
of cell-surface
erythroid
Jackson
1972
J,
3. Gahmberg
4.
TW,
human
69:1445,
Expression
major
Tillack
characterization
glycoprotein
during
with these
technique
identified
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Chemical
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8159 has been reported
of glycophorin
A.9 Our
of homozygous
M
are in agreement
an immunoblotting
Bigbee et al used
1
anti-M
antibody
with the M type
immunoprecipitation
of blood
group
M and N
to the appearance
of the glyco-
A during
results
oligosac-
oligosaccharides.’6
expression
apparently
glycosylated
DISCUSSION
late
1 5 0-glycosidic
as compared
A molecule
A antiserum,
of
and
NH2-terminal
mature
red cells
N-glycosidic
one
containing
at ASN-26
as
the S
to the antigenic
M/N-specific
antibodies
8159 and 8l6l.
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portion
of the glycophorin
A molecule
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charide
stage
and
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using
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(suppl
5)
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of cell surface
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human
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or plastic
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affinity
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the
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1985 66: 233-236
Late expression of M and N antigens on glycophorin A during erythroid
differentiation
M Ekblom, CG Gahmberg and LC Andersson
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