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Myosin Content and Distribution in Human Neonatal Erythrocytes Are Different
From Adult Erythrocytes
By Francine C. Colin and Stanley L. Schrier
Neonatal erythrocytes (N-RBC) are differentfrom adult erythrocytes (A-RBC). N-RBC are larger, less deformable, and
undergo enhanced spontaneous and drug-inducedendocytosis. The reticulocyte population of N-RBC is also different,
consisting primarily of the youngest (Rl)reticulocytes that
are motile and capable of receptor-mediated endocytosis.
Processes such as motility could require a contractile system.
Myosin, a contractile protein, was identified in both A-RBC
and N-RBC. We proposed to compare myosin content and
distribution in A-RBC and N-RBC by immunofluorescence
and enzyme-linked immunosorbent assay (ELISA) using a
monospecific polyclonal rabbit antimyosin. There was bright
immunofluorescence on 44% of N-RBC with some heteroge-
neity contrasting with a barely detectable fluorescence on
A-RBC. ELISA measurements showed that A-RBC had 4,315
myosin copies/RBC, whereas N-RBC had 10,855copies/RBC
(or 2.5times as much). ELISA measurements of white ghosts
showed that A-ghosts contained 1,250 copies of myosin/
RBC (29% of total) whereas N-ghosts contained 3.4times as
much at 4,320 copies/RBC (39% of total). Therefore, N-RBC
not only have more myosin, but the amount that is membraneassociated is disproportionately increased. It is proposed
that such differences in amount and distribution of myosin
could account for some of the unusual properties of neonatal
RBC indicated.
o 1991 by The American Society of Hematology.
N
lished.'O,'l Accordingly, we prepared an affinity-purified,
antihuman platelet myosin antibody. We used immunofluorescence to determine if specific subpopulations of RBC
would have different contents of myosin. Then, to precisely
determine the amount of cytoplasmic and membraneassociated myosin, we used a very sensitive double-antibody
enzyme-linked immunosorbent assay (ELISA) technique.
EONATAL ERYTHROCYTES (N-RBC) are different from adult erythrocytes (A-RBC) in that they
are less deformable,' and undergo more spontaneous endocytosis' and more drug-induced endocyto~is.~
The reticulocytes in cord blood are generally motile R1 reticulocytes4
that are capable of receptor-mediated endocytosis.' Certainly, the property of motility would require a contractile
system.
Myosin has been extensively studied in nonmuscle cellsc9
and is known to have a role in shape change and cell
motility. Human erythrocyte myosin was recently identified
and
It is composed of a heavy chain of 200 Kd
and two light chains of 19.5 Kd and 26 Kd. It forms bipolar
filaments with a head that binds to actin and possibly
protein 4.1," and a rodlike tail. The quantity of myosin in
RBC is 100 times less than in platelets.'' Fowler et a1'" used
a polyclonal antiplatelet myosin antibody to follow the
purification of myosin from RBC and showed that the heavy
chains of RBC and platelet myosin are structurally homologous. Wong et all' also purified myosin from RBC and
showed that it reacted with eight of eight of a panel of
monoclonal antibodies raised against platelet myosin. We
had previously shownI3 that there is more myosin in
neonatal RBC membranes than in adult RBC membranes.
To further explore this observation and consider the possible role of myosin in some of the unique properties of
neonatal RBC noted previously, we proposed to study the
content and distribution of myosin in N-RBC and A-RBC.
The structural homology and immunologic cross-reactivity
of platelet and RBC myosin had already been estabFrom the Division of Hematology, Stanford University School of
Medicine, Stanford, CA.
Submitted January 4, 1991; accepted July 25, 1991.
Supported by National Institutes of Health Grant No. DK32094.
Address reprint requests to Stanley L. Schrier, MD, Division of
Hematology, S161, 300 Pasteur Dr, Stanford Universiw School of
Medicine, Stanford, CA 94305-5112.
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 I991 by The American Society of Hematology.
0006-4971I91 /78II-0003$3.00/0
3052
MATERIALS AND METHODS
Materials
Swine serum was purchased from GIBCO Laboratories (Grand
Island, NY).Fluorescein-isothiocyanate (FITC) swine antirabbit
antibody, ct cellulose and crystalline cellulose (Sigmacell-50),
alcian blue, alkaline phosphatase, and Sigma 104-105 phosphatase
substrate were purchased from Sigma Chemical Co (St Louis,
MO). Methanol and acetone were obtained from Merck, Em
Science (Gibbstown, NJ).
Methods
Blood samples. N-RBC were obtained from placental vessels
just after normal full-term vaginal delivery. A-RBC were obtained
by venipuncture from healthy adult volunteers and was anticoagulated with heparin. All samples were drawn according to a protocol
approved by the Stanford Medical Committee for the Protection of
Human Subjects in Research. RBC were extensively washed and
column-filtered through cellulose as previously described to remove platelets and white blood cells (WBC). Ghosts were prepared
as described," using a ratio of 40 vol of 5 mmol/L phosphate lysis
buffer pH 8.0 to 1vol of RBC and performing three such washes.
Purification of platelet myosin and preparation of antimyosin
antibody. Human platelet myosin was purified exactly as previously described.I4 Polyclonal antibodies to this human platelet
myosin were raised in rabbits using the human platelet myosin
extract, and the antisera were then affinity-purified against a
column of the purified antigen. Myosin and antibodies were stored
at 4°C. The antibody specificity was checked by immunoblotting.
Western blotting on washed sonicated RBC and on ghosts was
performed as de~cribed'~
(Fig l), except that 8% polyacrylamide
gels were used, 5% bovine serum albumin (BSA) was used as
blocking agent, and no swine serum was used (Fig 1). The antibody
used in this study is different than the one we used in our initial
investigation" and was diluted in BSA before use.
Immunofluorescence. A 111,000 dilution of washed RBC was
fixed in 0.2% glutaraldehyde in phosphate-buffered saline (PBS)
pH 7.4 for 60 minutes at 4°C and then washed once with 5 mmollL
Blood, Vol78, No 11 (December 1). 1991: pp 3052-3055
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3053
NEONATAL RBC MYOSIN
RBC
Ghosts
m
N
A
A N
~-
Myosin
4
triethanolamine and twice with PBS. Then the fixed RBC were
placed on alcian blue-coated coverslips. After drying, the RBC
were permeabilized by dipping into methanol-acetone (1:l vol/vol)
for 1 minute. Nonspecific sites were blocked with a 5% swine serum
in PBS pH 7.4. Then the slides were incubated with the antimyosin
antibody for 1 hour at room temperature, followed by an FITCconjugated swine antirabbit antibody for 30 minutes at room
temperature. Between each step, extensive washes in PBS, pH 7.4,
were performed. Samples were examined on a Zeiss microscope
with a fluorescent filter and a Zeiss MC 63 photomicrographic
camera (Carl Zeiss, Germany).
ELISA. The method of double-antibody ELISA was performed exactly as described.I5 Platelets and WBC were removed by
cellulose filtration.I6 Then wells were coated with the antimyosin
antibody (10 Fg protein/well, 4-hour incubation at 37°C). Standards of purified platelet myosin or sonicated N- and A-RBC and
ghosts in incremental amounts were added for a I-hour incubation
at 37°C. The plates were then incubated with alkaline phosphataseconjugated affinity-purified IgG antihuman platelet myosin for 2
hours at 37°C. The reaction was quantified with phosphatase
substrate and the readings were performed at 405 nm. Between
each step washes were performed in PBS-0.05% tween 20-0.1%
BSA.
Because the high concentration of hemoglobin and other compounds in sonically lysed RBC could interfere with the detection of
myosin by the antiplatelet myosin, myosin standard curves were
also performed with the addition of the actual amounts of sonically
disrupted adult and neonatal RBC used in the assays. In parallel
plates, the myosin content was independently determined in
A-RBC and N-RBC. The myosin curves in the presence of the
lysate were still linear with a recovery of 64% using adult RBC and
70% using neonatal RBC.
RESULTS
Fig 1. Western blotting of washed sonicated RBC and of ghosts
from neonatal and adult RBC was performed using our antiplatelet
myosin antibody. Equal numbers of RBC and equal amounts of
membrane protein were applied t o the gels and run in parallel pairs.
The RBC and ghosts were run at different times; therefore, the length
of the gels are not comparable. To detect myosin in RBC, the gels have
t o be heavily overloaded and this probably account for the appearance of nonspecific bands in the midportion of the gels. The immunoreactive myosin band in the ghosts appears just below ankyrin as
visualized in the amido black-stained nitrocellulose transfer papers
(not shown), and thus has a molecular weight of 200 Kd, consistent
with myosin heavy chain.
ADULT RBC
To detect the presence and analyze the distribution of
myosin in the population of N-RBC compared with A-RBC,
immunofluorescence staining of these two types of RBC
was performed on four sets of A- and N-RBC. The
immunofluorescence results obtained on permeabilized
N-RBC (Fig 2) showed that in this experiment about 44%
of N-RBC showed a bright fluorescent rim. A-RBC (Fig 2)
showed, at best, a marginal and dim fluorescence.
To be certain that nonspecific binding or variations in
permeabilization did not account for these findings, these
NEONATAL RBC
Fig 2. Immunofluorescence using an antimyosin antibody followed by an FITC-labeled swine antirabbit IgG on permeabilized RBC. A single
representative experiment is shown. Forty-four percent of N-RBC showed bright fluorescence, while there was only a dim fluorescent staining on
A-RBC. Original magnification x 1,000. These micro photographs were taken under and printed under the identical conditions and exposure times
as the results with preimmune serum shown in Fig 3.
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COLIN AND SCHRIER
3054
A-RBC and N-RBC were, in parallel, incubated with
preimmune rabbit serum and also with our affinity columnpurified rabbit polyclonal antispectrin antibody. There was
uniform permeabilization and minimal nonspecific staining
(Fig 3).
To more precisely determine the amount of myosin that
was membrane-associated compared with the whole RBC
content in neonates and adults, ELISA was performed on
ghosts and sonically disrupted RBC. Before any measurement, platelets and WBCs were removed. ELISA measurements performed on four sets of adult and neonatal RBC
allowed us to determine that A-RBC contained 2,760
myosin copies per cell, 800 copies (29%) of which are
bound to the membrane. N-RBC contain 7,600 myosin
copies per cell, of which 2,960 (39%) are membraneassociated. If one corrects these actual values for the
recoveries observed for myosin standards added to sonically
lysed RBC, the values are shown in Fig 4 with 4,315 myosin
copies per A-RBC, of which 1,250 are in A-ghosts, while
N-RBC contained 10,855 copies per RBC, of which 4,230
are in N-ghosts. Therefore, there are 2.5 times more myosin
in N-RBC than in A-RBC and 3.4 times more myosin in
N-ghosts than in A-ghosts.
DISCUSSION
In 1985, Fowler et all" and Wong et all' identified and
purified RBC myosin. Fowler et all" found it both in
cytoplasm"' and in the membrane, with about one third
present in the membrane. It was possible that some of the
differences between N-RBC and A-RBC noted previously
could be caused in part by differences in myosin content
and distribution. Matovcik et all3 previously showed that
N-ghosts contained more myosin than A-ghosts, but the
Western blotting methods used were not quantitative.
The extensive cross-reactivity between platelet and RBC
myosin has been well documented.""" Therefore, we prepared a monospecific polyclonal antiplatelet myosin antibody against highly purified human platelet myosin, extracted as previously described.14
ADULT RBC
Fig 4. Myosin content of N-ghosts and RBC and of A-ghosts and
RBC measured by ELISA. Values are expressed as copies of myosin
per RBC or per ghost, corrected for recovery as described in the text.
Immunofluorescence showed that approximately half
(44%) of N-RBC showed bright staining, while only a dim
fluorescence could be seen in A-RBC (Fig 2). The glutaraldehyde fixation used can cause a degree of heterogeneity in
immunoreactivity, as seen with the antispectrin immunofluorescence in Fig 3. However, we propose that this modest
variation does not account for the differences seen in
antimyosin reactivity in neonatal versus adult RBC in Fig 2.
Neonatal blood contains only 3% to 5% reticulocytes, much
less than 44% of N-RBC, which were quite positive for
myosin by immunofluorescence. Therefore, the myosin
increase seen in N-RBC is not the consequence of a small
subpopulation of unusual RBC containing very large
amounts of myosin.
Our ELISA data were in general agreement with that
Fowler et al,"' who identified 6,000 myosin copies per adult
RBC, and with Wong et a]," who reported 2,400. We
detected 4,315 copies per adult RBC and showed that there
NEONATAL RBC
PR EIMMUNE
ANTISPECTRIN
Fig 3. The A-RBC and N-RBC
shown in Fig 2 were also incubated with preimmune rabbit serum to control for specificity and
also with an antispectrin antibody to control for variations in
permeability.There was minimal
nonspecific fluorescence, not different for A-RBC and N-RBC, and
spectrin fluorescence was quite
uniform in both populations,indicating minimal variation in permeabilization(original magnification x 1,000).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3055
NEONATAL RBC MYOSIN
were 2.5 times more myosin in N-RBC (10,855 copies) than
in A-RBC. A-ghosts contained 1,250 myosin copies on a
per-cell basis or 29% of the total cellular myosin, while
N-ghosts contained 4,230 copies on a per-cell basis or 39%
of N-RBC myosin. Therefore, there are 3.4 times more
membrane-associated myosin copies in N-RBC compared
with A-RBC, partly because the membrane-associated
fraction in N-RBC is disproportionately increased. Immunofluorescence is probably dependent on levels of myosin
between 4,315 and 10,855 copies per RBC, explaining why
immunofluorescence was not readily detectable in A-RBC.
The ELISA and immunofluorescence data are supported
by myosin Western blotting of RBC and ghosts (Fig l),
showing increased immunoreactivity of myosin heavy chains
when equal numbers of RBC and equal amounts of ghost
protein are analyzed.
Myosin has been shown to be involved in cell motility and
shape alteration through attachment to the plasma membrane in human intestinal epithelial cells” or platelets.’8.”
The increased amount of myosin in N-RBC and especially
in the membrane-associated fraction could account for
some of the mechanical differences noted previously, particularly if myosin binds to protein 4.1 in vivo as it does in
vitro.”
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1991 78: 3052-3055
Myosin content and distribution in human neonatal erythrocytes are
different from adult erythrocytes
FC Colin and SL Schrier
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