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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1473-1480
RED CELLS
Tropomyosin isoform 5b is expressed in human erythrocytes:
implications of tropomodulin-TM5 or tropomodulin-TM5b complexes in the
protofilament and hexagonal organization of membrane skeletons
Lanping Amy Sung,
Ke-Ming Gao,
Leland J. Yee,
Constance J. Temm-Grove,
David M. Helfman,
Jim
J.-C. Lin, and
Majid Mehrpouryan
From the Department of Bioengineering and Center for Molecular
Genetics, University of California, San Diego, La Jolla, CA;
the Nutritional Science Department, University of Arizona,
Tucson, AZ; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and
the Department of Biological Sciences, University of Iowa, Iowa City,
IA.
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Abstract |
The human erythrocyte membrane skeleton consists of hexagonal
lattices with junctional complexes containing F-actin protofilaments of
approximately 33-37 nm in length. We hypothesize that complexes formed
by tropomodulin, a globular capping protein at the pointed end of actin
filaments, and tropomyosin (TM), a rod-like molecule of approximately
33-35 nm, may contribute to the formation of protofilaments. We have
previously cloned the human tropomodulin complementary DNA and
identified human TM isoform 5 (hTM5), a product of the
 -TM gene, as one of the major TM isoforms in erythrocytes. We now identify TM5b, a product of the  -TM gene, to be the
second major TM isoform. TM5a, the alternatively spliced isoform of the -TM gene, which differs by 1 exon and has a weaker
actin-binding affinity, however, is not present. TM4, encoded by
the  -TM gene, is not present either. In sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, hTM5 comigrated with the
slower TM major species in erythrocyte membranes, and hTM5b comigrated
with the faster TM major species. TM5b, like TM5, binds strongly to
tropomodulin, more so than other TM isoforms. The 2 major TM isoforms,
therefore, share several common features: They have 248 residues, are
approximately 33-35 nm long, and have high affinities toward F-actin
and tropomodulin. These common features may be the key to the mechanism
by which protofilaments are formed. Tropomodulin-TM5 or
tropomodulin-TM5b complexes may stabilize F-actin in segments of
approximately 33-37 nm during erythroid terminal differentiation and
may, therefore, function as a molecular ruler. TM5 and TM5b further
define the hexagonal geometry of the skeletal network and allow
actin-regulatory functions of TMs to be modulated by tropomodulin.
(Blood. 2000;95:1473-1480)
© 2000 by The American Society of Hematology.
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Introduction |
The erythrocyte membrane skeletal network is a
spectrin-actin-based 2-dimensional protein network anchored to the
endoface of the membrane. It provides the mechanical
stability and elastic deformability for the lipid bilayer. The protein
network is organized into hexagonal lattices (Figure
1A), with 6 long spring-like spectrin tetramers (approximately 200 nm) binding to 1 short actin filament (approximately 33-37 nm) at the junctional complex (indicated by
arrows).1-3 Several other actin-binding proteins, such as myosin, tropomyosin4 (TM), and
tropomodulin,5,6 exist in erythrocytes. The properties,
kinetics, and expression of these actin-associated proteins may play
important roles in regulating the length of actin filaments and thus
the spectrin-actin organization and the mechanical properties of the
erythrocyte membrane.
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| Fig 1.
Hexagonal lattices, protofilament, and tropomodulin-TM
complex of the erythrocyte membrane skeletal network.
(A) The top view of the hexagonal network based on the
electron micrographs.1-3 As indicated by an arrow, 6 spectrin tetramers are associated with 1 junctional complex. The
junctional complexes are approximately 33-37 nm in length. Each
spectrin tetramer is approximately 200 nm long and comprises 2  
spectrin dimers associated with a head-to-head fashion. The 2 tail ends
of the spectrin tetramer join the junctional complexes, and the 2 head
ends meet with each other in the middle. The pair of smaller complexes
in the mid regions of the spectrin tetramers are protein 4.2/band
3/ankyrin complexes, which hang the membrane skeletal network to the
lipid bilayer. (B) The molecular model of an actin protofilament in the
erythrocyte membrane skeleton. The filament's length is approximately
6-7 G-actin and is associated with only 2 TM molecules, 1 in each
groove of the actin filament, and 1 tropomodulin molecule at the
pointed end. The barbed end may either be uncapped or capped by
adducin,7 gelsolin,8 or another barbed-end
capping protein. The number of G-actin limits the number of spectrin
binding to the protofilament and defines the hexagonal geometry of the
membrane network. (C) The model of the tropomodulin-TM complex that
stabilizes the actin protofilament. Tropomodulin binds near the
N-terminal of TM5 or TM5b (homodimer or heterodimer
approximately 33-35 nm in length, 6 actin-binding sites), at the
pointed end of the actin filament. The complex functions as a measuring
device, determining the number of G-actin to be protected. N and C
stand for the N-terminal and C-terminal of the TM molecule,
respectively.
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While the actin filaments in nonmuscle cells are generally long and
vary in length, the actin filaments in erythrocyte membranes, as
revealed by electron microscopy, were relatively short and uniform,
approximately 33-37 nm in length. The mechanism by which such short
actin protofilaments is generated is not clear. Since 33-37 nm
approximates the length of a TM molecule, it was thought that the
protofilament is the result of actin-TM complexing.2 It was
not clear, however, why the actin-TM complex should stop with only 1 TM molecule.
TM is a family of actin-binding proteins present in the thin filaments
of muscle cells and in the microfilaments of nonmuscle cells. TM
molecules comprise 2 monomers and polymerize in a head-to-tail fashion
along the grooves of the actin double helix. TM molecules function to
stiffen the filament, stabilize its polymerization, and regulate its
interaction with other actin-binding proteins. There are several
TM isoforms encoded by at least 4 distinct genes in humans: , ,
, and .9-11 The diversity of TM is further generated
by alternative promoters and alternative splicing of primary RNA.
Tissue-restriction or specific expression of TM suggests that different
TM isoforms may perform distinct functions in different tissues,
especially in regulating cell motility and in organizing actin
filaments.9-14
In human erythrocytes, TMs of Mr 29 000 and
Mr 27 000 (molar ratio of 3:1) have been
reported.4 There are approximately 70 000 copies of TM
molecules per ghost. We have previously identified 1 major TM isoform
to be human TM isoform 5 (hTM5), which comigrated with the slower
migrating TM species.15 It is a gene product of the human
-TM gene, and it is approximately 33-35 nm in length. It was
not clear what is the other major faster migrating TM isoform(s) expressed in human erythrocyte membranes.
Tropomodulin is a 359-residue16 TM-binding protein first
discovered in human erythrocyte membranes.5 There are
approximately 30 000 copies of tropomodulin per ghost. Tropomodulin
binds to the N-terminal end of the TM molecule6,15 and has
the ability to inhibit the cooperativity of TM and the TM binding
to actin.6 Together with TM, tropomodulin inhibits the
elongation and depolymerization of the actin filaments at the pointed
end. It is, therefore, a capping protein17 that binds
to the pointed end of the actin filaments18 and is involved
in the regulation of actin filament length.19,20
We have cloned the first tropomodulin complementary DNA (cDNA) from
human fetal liver and reticulocytes16 and mapped the human
tropomodulin gene to chromosome 9, long arm, band 22 (9q22).21 Northern blot analysis of human tissues revealed that in
addition to erythrocytes, tropomodulin is highly expressed in the heart and skeletal muscle, with moderate or very little expression in some
other issues.21 The function of tropomodulin, therefore, is
important not only in erythrocytes but also in several other cell
types. For example, myofibril degeneration caused by tropomodulin overexpression has been shown to lead to dilated cardiomyopathy in
juvenile mice.22
We have previously demonstrated that tropomodulin binds to hTM5 more
strongly than other TM isoforms (eg, hTM2, hTM3, chicken skeletal
muscle isoforms, and chicken smooth muscle isoforms).15 It
is possible that tropomodulin may play an important role in regulating
the organization of actin filaments by preferentially binding to
specific TM isoforms at the pointed end of the actin filaments. Based
on our findings that tropomodulin binds to the N-terminal of hTM5 and
that hTM5 is one of the major TM isoforms in human erythrocytes, we
have previously proposed that tropomodulin complexed with hTM5 may
contribute to the protofilament formation in erythrocyte
membranes.15
Here we report that TM5b, one of the alternatively spliced gene
products of the -TM gene, is the second major TM isoform in
human erythrocytes. The fact that TM5 and TM5b are the 2 TM isoforms
that have not only the highest tropomodulin affinities but also the
highest F-actin affinities (among known low-molecular weight [LMW] TM
isoforms) suggests that the tight association of
tropomodulin/TM/F-actin may be the key to the mechanism of protofilament formation. Since both TM5 and TM5b are LMW TM isoforms with 6 actin-binding sites, they also favor the association of 6 spectrin molecules to 1 protofilament and, thus, the formation of
hexagonal lattices in the erythrocyte membrane skeletal network. TM5,
TM5b, and tropomodulin, therefore, are important molecules in the
development and organization of the membrane skeletal network.
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Materials and methods |
Antibodies
Mouse monoclonal antibodies (mAbs) CG3 (immunoglobulin M
[IgM]),23 LC1 (IgG),11,24 Pep3-43
(IgG),25 and Tmod-20415 have been described.
The rabbit antiserum R-41 was raised by using a combination of
recombinant hTM5 and chimeric hTM5/4 isoforms as the
immunogen. The peptide-specific antibody against tropomodulin was
raised in rabbits using a peptide corresponding to residues 70-84 of
human erythrocyte tropomodulin,16 conjugated to a protein carrier, keyhole limpet hemocyanin.
Recombinant TM isoforms
The expression and purification of rTM5,25 rTM5a,
rTM5b,26 hTM5,14 and chimeric TM
molecules14 in the bacterial expression systems have
been described.
Western blot analysis
Human and rat erythrocyte ghost membranes were
prepared27 and dissolved in equal volumes of sodium dodecyl
sulfate-solubilization (SDS-solubilization) solution containing 0.2%
bromophenol blue, 20% glycerol, 4% SDS, 100 mmol/L Tris-HCl
(tris(hydroxymethyl) aminomethane hydrochloride) (pH 6.8),
and 200 mmol/L dithiothreitol. Using 10% SDS-PAGE28
(polyacrylamide gel electrophoresis), 30 µg host membranes and 2 µg
recombinant TMs were electrophoresed and transblotted onto a
nitrocellulose membrane.29 The transblot was incubated with
either mouse mAbs CG3, LC1, Pep3-43, or LC24 or rabbit antiserum R-41
followed by a horse radish peroxidase (HRP)-conjugated secondary
antibody to mouse IgG or IgM or rabbit IgG. The transblot was then
developed enzymatically.
Sequential Western blot analysis
Human erythrocyte ghost membranes (30 µg) were electrophoresed
using 12% SDS-PAGE and transblotted onto a nitrocellulose membrane. The same strip was subjected to 2 distinct rounds of Western blotting with 2 different primary antibodies. Specifically, mAb
Pep3-431:100 was used, followed by an HRP-conjugated
secondary antibody specific for mouse IgG.1:1000 The strip
was developed enzymatically. After development, the same strip
was incubated with mAb LC1,1:1000 followed by an
HRP-conjugated secondary antibody1:1000 specific for mouse
IgG and enzymatic development.
Recombinant human tropomodulin
Human tropomodulin cDNA clone 1016 was subcloned into an
expression vector, pMal-c230,31 (New England
BioLabs, Beverly, MA). The constructed plasmid pMBP/Tmod-10
was used to transform Escherichia
coli (E coli) DH5. The induction of expression by isopropyl -D-thiogalactopyranoside (IPTG) and purification of the
fusion protein, MBP/Tmod-10, were performed according to the manufacturer's protocol, with a modification so that glucose was not
added in the culture media.
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| Fig 2.
Specificity of mAbs CG3, LC1, and Pep3-43
and the detection of TM5b in human erythrocyte ghost membranes.
(A) Using SDS-PAGE, 2 µg each of rTM5, rTM5a, and rTM5b and 30 µg
human erythrocyte ghost membranes were separated on a 10% gel. Three
identical sets of proteins were transblotted onto nitrocellulose
membranes and probed either with mAbs CG31:1000 (panel
labeled "mAb CG3"), LC11:500 (panel labeled "mAb
LC1"), or Pep3-431:100 (panel labeled mAb
"Pep3-43"). Secondary antibodies conjugated with HRP were used,
and the signals were developed enzymatically. Molecular weight
standards (lane 1) are phosphorylase b (97 kd), bovine serum
albumin (66 kd), ovalbumin (45 kd), carbonic anhydrase (31 kd), and
soybean trypsin inhibitor (21 kd). (B) This panel demonstrates the
presence of TM5b, but not TM5a, in human erythrocyte ghost membranes.
Using SDS-PAGE, we separated 2 µg each of purified rTM5a (lanes 1 and
5) and rTM5b (lanes 2 and 6) and 30 µg ghost membranes prepared from
rat (lanes 3 and 7) and human (lanes 4 and 8) erythrocytes on an 10%
gel in the absence (panel labeled "No Urea") and presence (panel
labeled "20% Urea") of 20% urea. Both transblots were probed
with mAb Pep3-431:100 and an HRP-conjugated secondary
antibody against mouse IgG; the color was developed enzymatically. The
arrow points to the band that comigrated with rTM5b in human and rat
ghost membranes. (C) Amino acid (1 letter code) sequences of
hTM5,35 rTM5,25 rTM5a, and rTM5b36
are shown; numbers on the top refer to the amino acid residues. The  indicates residue identical with those in hTM5, with human
(h) and rat (r) identified. rTM5a is identical to rTM5b
except for the boxed region (residues 152-177) encoded by an
alternative exon.
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Solid-phase binding assay
Recombinant TMs (2 µg) were electrophoresed in 10%
SDS-PAGE28 and transblotted onto a nitrocellulose
membrane.29 The binding assay was identical to an
established procedure for Western blot analysis (Bio-Rad, Hercules, CA)
except that the transblot was incubated with 26 µg/mL recombinant
tropomodulin in Tris-buffered saline (pH 7.4) for 1.5 hours before
immunological analyses. For detection, a rabbit peptide-specific
antibody to tropomodulin was used, followed by an HRP-conjugated
secondary antibody to rabbit IgG and enzymatic development.
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Results |
Detection of TM5a and/or TM5b isoforms in human erythrocyte
membranes
To search for additional unknown TM isoforms in human erythrocytes,
we first investigated the specificity of a group of mAbs (ie, CG3, LC1,
and Pep3-43) and then used them to detect specific TM isoforms in human
erythrocyte membranes (Figure 2). The mAbs CG3 and LC1
were previously used to identify hTM5, a gene product of the
-TM gene, in human erythrocytes.15 In search for
a TM isoform, which may migrate faster than hTM5 in SDS-PAGE, mAb
Pep3-43 was chosen to identify hTM5a and/or TM5b, the 2 alternatively spliced gene products of the -TM gene, in human erythrocyte
membranes.
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| Fig 3.
TM5 and TM5b are the 2 major TM isoforms in human
erythrocyte membranes.
(A) A Coomassie blue stain of several purified TM isoforms and their
chimeric molecules separated on a 12% gel using SDS-PAGE. (B) The
Western blot analysis of the above, with antibody R-41. Samples are 30 µg human ghost membrane (lane 1), chicken leg TM (lane 2), chicken
gizzard TM (lane 3), hTM5/2 (lane 4), hTM2 (lane 5), hTM3
(lane 6), hTM5 (lane 7), hTM5/3 (lane 8), and hTM3/5 (lane 9).
Approximately 10 µg TM isoforms (except hTM3/5, which has less) are
applied. (C) The following lanes depict the Western blot analysis of 30 µg human ghost membrane by mAb Pep3-431:100 (lane 3),
antibody R-411:100 (lane 4), and mAb LC11:500
(lane 5). The panel also depicts the Coomassie blue stains of human
ghost membrane (lane 2) and the molecular weight standards
(lane 1). (D) A sequential Western blot analysis demonstrates the
distinction between hTM5 and hTM5b. Lane 1 presents the first round
of Western blot with mAb Pep3-43,1:100 while lane 2 shows
the same strip after the second round of Western blot with mAb
LC1.1:1000
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Throughout the Western blot analyses (Figure 2A and 2B), samples
labeled 5, 5a, and 5b are purified bacterial recombinant proteins of TM
isoforms derived from the corresponding rat cDNA clones, and samples
labeled h and r are total proteins of ghost membranes
purified from human and rat erythrocytes, respectively. To understand
the specificity of these mAbs and the results, 4 published amino acid
sequences, hTM535, rat TM525
(rTM525), rTM5a, and rTM5b36 are shown and
aligned in Figure 2C.
The middle panel of Figure 2A, labeled mAb CG3,
demonstrates that mAb CG3, which recognizes an epitope within residues
29-44 of the hTM5 amino acid sequence, was able to cross-react with the
purified rTM5 but not with rTM5a or rTM5b. Thus, the mAb CG3 is
specific for hTM5 and rTM5 and can distinguish between TM5 and TM5a/5b.
In human erythrocyte ghost membranes (lane 8), the mAb CG3 was capable
of detecting and confirming the presence of hTM5.15 Such
specificity can be explained by the amino acid sequences of these TM
isoforms (Figure 2C): In residues 29-44, rTM5 is identical to hTM5,
whereas rTM5a and rTM5b share only a 50% sequence identity with hTM5.
The mAb CG3 is, therefore, TM5 specific.
The left panel of Figure 2A, labeled mAb LC1, demonstrates
the specificity of mAb LC1; mAb LC1 was able to detect hTM5 in human
erythrocyte ghosts (lane 4), as previously reported.15 However, mAb LC1 did not cross-react with purified rTM5 (lane 1) as did
mAb CG3. The entire sequences of rTM5 and hTM5 are absolutely identical
except for 1 residue: at the fourth position, rTM5 has a
serine4 (Ser) and hTM5 has an isoleucine4 (Ile)
(Figure 2C). We (L. A. Sung and J. J.-C. Lin, unpublished result) have previously discovered that mAb LC1 did not recognize mouse TM5 (mTM5).
The entire sequences of mTM537 and hTM5 are also absolutely identical except for 1 residue, also at the fourth position, where mTM5
has a threonine4 (Thr) and hTM5 has an Ile.4
The findings that the mAb LC1 did not recognize rTM5 or mTM5 indicates
the absolute requirement of Ile4 for the recognition of the
mAb LC1. In fact, by using TM5 molecules naturally existing in
different species, we mapped the epitope of mAb LC1 to include
Ile4 of hTM5. The mAb LC1 panel also showed that mAb LC1
did not cross-react with purified recombinant rTM5a (lane 2) or rTM5b
(lane 3). This finding is reasonable since rTM5a and rTM5b not only
have a Ser4 but also have 3 additional different residues
immediately downstream from Ser4 (Figure 2C). The mAb LC1
is, therefore, not only TM5-specific but also human-specific.
Finally, the right panel of Figure 2A, labeled mAb Pep3-43,
demonstrates the specificity of mAb Pep3-43 and shows that it detected
a specific band in human erythrocyte membranes. ThemAb Pep3-43
recognized both rTM5a (lane 10) and rTM5b (lane 11) purified from
recombinant bacteria. This finding is expected because the 2 alternatively spliced TM isoforms have an identical N-terminal sequence
(Figure 2C), which contains the peptide sequence n-AGSSSLEAVERRKIRSLC-c (residues 2-19) used in generating mAb Pep3-43.25 It is
important to note that mAb Pep3-43 did not cross-react with rTM5 (lane
9). In a separate experiment, mAb Pep3-43 was also shown not to
cross-react with purified recombinant hTM5 (data not shown). The mAb
Pep3-43 is, therefore, TM5a/5b specific. In the human erythrocyte ghost membrane (lane 12), mAb Pep3-43 was found to recognize a specific band
of ~Mr 34 000 that comigrated with
the purified recombinant rTM5a and rTM5b.
This series of experiments concludes that: (1) these 3 mAbs (LC1, Pep3-43, and GC3) are capable of distinguishing between TM5
and TM5a/5b, and mAb LC1 can even distinguish between hTM5 and TM5 of
other species (eg, mouse and rat); (2) mAb Pep3-43, which was made
against an N-terminal peptide of rTM5a/5b, is capable of cross-reacting
with hTM5a/5b; and (3) human erythrocyte membranes contain hTM5a,
hTM5b, or both.
It is not surprising that mAb Pep3-43 can recognize hTM5a/5b. hTM5a/5b
differs from rTM5a/5b in only 2 conserved amino acid changes at
position 30 (tyrosine [Thr or T] versus Ser or S) and position 37 (histidine [His or H] versus glutamine [Gln or Q]).12 In addition, the rTM5a/5b peptide used as the immunogen is located at
the conserved N-terminal region, which is shared by rTM5a/5b and
hTM5a/5b.
TM5a and TM5b are 2 alternatively spliced gene products from the -TM
gene. TM5a has a weaker actin-binding affinity than TM5b and
differs from TM5b only in the alternatively spliced exon 6, which
encodes residues 152-177 (boxed in Figure 2C).9 Their relative molecular weights, therefore, are indistinguishable under the
above experimental condition. The -TM gene also
encodes skeletal muscle -TM, which is a high molecular weight (HMW)
TM and has a different N-terminal.
TM5b, not TM5a, present in human erythrocyte membranes
To determine whether TM5a, TM5b, or both are present in human
erythrocyte membranes, we carried out a second set of Western blot
analyses (Figure 2B). To be complete, we included rat erythrocyte membranes here as a control, since mAb Pep3-43 was made against TM5a/5b
from rats. The experiments were carried out in both the presence and
absence of urea. The panel labeled "No Urea" showed a single
polypeptide recognized by mAb Pep3-43 in rat (lane 3) and human (lane
4) ghost membranes. Both rat and human polypeptides comigrated with the
purified recombinant rTM5a (lane 1) and rTM5b (lane 2). Since rTM5a and
rTM5b comigrated with each other under this experimental condition (as
in Figure 2A), 20% urea was included in the other gel (panel labeled
"20% Urea"). Under this condition, the migration of the
denatured rTM5a and rTM5b can be distinguished from each other: rTM5a
(lane 5) migrated slightly slower than rTM5b (lane 6). Under this
condition, a single mAb Pep3-43-reactive polypeptide with an apparent
Mr of about 38 000 was found in the human ghost
membrane (lane 8). This polypeptide comigrated exactly with rTM5b (lane
6) and not with rTM5a (lane 5). The results clearly demonstrated that
TM5b, not TM5a, is present in the human membrane skeletal network.
Differential migration of hTM5 and hTM5b in SDS-PAGE
In human erythrocyte membranes, there are 2 closely migrating TM
bands revealed by SDS-PAGE in the absence of urea.4 Because we previously reported that hTM5 is the isoform that comigrates with
the major slower migrating species of the TM bands,15 it is
of interest to know whether hTM5b is the isoform that comigrates with
the faster migrating species.
R-41 is a polyclonal antibody that cross-reacts with several nonmuscle
TM isoforms. TM2, TM3, TM5, and several chimeric TM molecules were used
in a Western blot analysis to demonstrate its wide range of recognition
among TM isoforms (Figure 3). R-41 was then
used to demonstrate the existence of 2 major TM bands in human
erythrocyte ghost membranes.
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| Fig 4.
Construction, expression, and purification of human
recombinant tropomodulin and its binding to rTM5 and rTM5b.
(A) Human tropomodulin cDNA clone 10 was subcloned at the EcoR1
site downstream from the MalE gene, which encodes MBP. (B) The
induction of the MBP/tropomodulin fusion protein. Total proteins of
E coli were separated on a 7% gel using SDS-PAGE and stained
by Coomassie blue, before the addition of IPTG (lane 2)
and 1 hour (lane 3), 2 hours (lane 4), and 3 hours (lane 5) after the
addition of IPTG. Affinity-purified tropomodulin fusion protein
(Mr 79 000) was either stained by Coomassie blue
(lane 6, 2 µg) or transferred onto a nitrocellulose membrane and
detected with tropomodulin-specific antibody using Western blot
analysis (lane 7, 0.1 µg). Molecular weight standards (lane 1) are
the same as in Figure 2. (C) A solid-phase binding assay demonstrates
the binding of human tropomodulin to rTM5 and rTM5b. Using SDS-PAGE, 2 µg of rTM5 (lanes 1 and 3) and rTM5b (lanes 2 and 4) were separated
on a 10% gel, transblotted onto a nitrocellulose membrane (panel
labeled "Protein Profile"), and overlaid with 26 µg/mL
recombinant human tropomodulin (panel labeled "Tmod Overlay").
The presence of bound tropomodulin was then analyzed by a rabbit
antibody against tropomodulin followed by HRP-antirabbit IgG, enzymatic
color development, and densitometry. (D) The panel shows the partial
sequence alignments of human,16 mouse,32
rat,33 and chicken38 tropomodulin in the region
between residues 94 and 138. One letter code for amino acids is used.
*Indicates identical residues among all 4 species. Numbers on the top
indicate residue numbers in human tropomodulin.
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In Figure 3, the 2 major TM bands of the erythrocyte membrane in the
band 7 area (indicated by *) can be clearly detected by R-41 in both
Figure 3B (lane 1) and Figure 3C (lane 4) in the absence of urea. To
demonstrate the positions of hTM5 and hTM5b, relative to the 2 R-41
reactive TM bands, 2 additional strips from the same transblot were
processed (Figure 3C): one was incubated with mAb Pep3-43 and the other
with mAb LC1. The results show that hTM5b (lane 3), as detected by mAb
Pep3-43, comigrated with the faster migrating species, while hTM5 (lane
5), as detected by mAb LC1, comigrated with the slower migrating species.
To confirm the differential migration pattern of TM5 and TM5b, we did a
sequential Western blot analysis (Figure 3D). In this experiment, a
transblot of erythrocyte membrane proteins was first incubated with mAb
Pep3-43 to reveal a single band of hTM5b. The same strip was then
further incubated with mAb LC1 to reveal a specific band of hTM5, which
appeared slightly above the band of hTM5b. The 2 bands were clearly
distinguishable. All of the above experiments, plus the finding that
hTM5 and hTM5b each comigrated with 1 of the 2 major TM bands, suggest
that TM5 and TM5b are indeed the 2 major TM isoforms present in human
erythrocyte membranes.
Human tropomodulin is capable of binding to rTM5 and rTM5b
Human recombinant tropomodulin was synthesized in E coli in
quantity and used to study the binding of tropomodulin to various TM
isoforms. Figure 4 shows the construction (panel A), expression (panel
B), and purification (panel B, lane 6) of human recombinant tropomodulin clone 10. The fusion protein contains residues 39-359 of
human tropomodulin (Mr 36 000), which includes the
TM-binding domain (residues 39-138)16 and the
maltose-binding protein (MBP, Mr 43 000). Total
bacterial proteins before and after the addition of
isopropylthiogalactoside (IPTG) were separated on a 7% gel by
SDS-PAGE. The correct reading frame of the affinity-purified tropomodulin fusion protein (Mr 79 000) was
verified with a tropomodulin-specific antibody by Western blot analysis
(panel B, lane 7).
A solid-phase binding assay was used to assess whether human
recombinant tropomodulin binds to purified recombinant rTM5 and rTM5b
and their relative binding affinities. The left panel of Figure 4C
shows the SDS-PAGE protein profiles of rTM5 (lane 1) and rTM5b (lane 2)
stained with Coomassie blue. The right panel, labeled "Tmod
Overlay," shows the presence of human tropomodulin that remained
bound to rTM5 and rTM5b on the transblot, as detected by a rabbit
peptide-specific antibody against human tropomodulin. This result
indicates that human tropomodulin is capable of binding to both rTM5
and rTM5b. The intensity of tropomodulin bound to rTM5b, however, was
about 80% of that bound to rTM5, which suggests that human
tropomodulin has a slightly higher affinity toward rTM5 than rTM5b.
Under the identical experimental condition, human tropomodulin had no
detectable binding or very little binding to other TM isoforms
including hTM2, hTM3, chicken skeletal muscle isoforms, and chicken
smooth muscle isoforms.15 The above finding indicates that
TM5 and TM5b are the 2 TM isoforms that bind to tropomodulin with a
higher affinity than other TM isoforms.
It is not surprising that tropomodulin derived from humans is capable
of binding to TM5 and TM5b derived from rats because both TM isoforms
and tropomodulin are highly conserved between humans and rats. On the
one hand, rTM5 differs from hTM5 only at the fourth position, and
rTM5a/5b differs from hTM5a/5b in only 2 conserved amino acid changes
at positions 30 and 3712 outside of the
tropomodulin-binding domain.34 On the other hand, the
TM-binding domain in tropomodulin (residues
94-13816,38) is located in a region where a
high degree of sequence identity is found among several species
including human,16 mouse,32 and
rat33 (Figure 4D). The tropomodulin-binding domain has been mapped to within the N-terminal 18 residues of
hTM5,34 and it is now known that human tropomodulin is
capable of binding to both rTM5 and rTM5b. These facts suggest that (1)
residue 4 (ie, Ile4 in hTM5, Ser4 in rTM5, and
Ser4 in rTM5b) is either not involved in the binding and/or
is located outside of the tropomodulin-binding domain and (2) the
N-terminal sequence variations between TM5 and TM5b (Figure 2C)
contribute to the minor difference of their affinities toward human tropomodulin.
Protofilaments in erythrocyte membrane skeleton
Using electron microscopy, the actin filaments revealed in a
negatively stained membrane skeletal network were approximately 33-37 nm in length.1,2 This length is equivalent to 6-7 G-actin in each strand of the actin filament (approximately 13 G-actin in
total) or 1 TM molecule (2 strands) bound in each groove of the actin
double helix. It was suggested that this is the result of actin-TM
complexing.2 It is not clear, however, why the TM molecule
should stop with 1 molecule.
TM, which binds to the side of F-actin, does not prevent actin
polymerization from the pointed end. TM stabilizes the pointed end of
actin filaments, in fact, by slowing depolymerization from the pointed
end.39 In vitro, in the presence of TM, both elongation and
depolymerization of the actin filaments can be inhibited by low
concentrations of tropomodulin. Higher concentrations of tropomodulin alone can also inhibit the elongation, even more effectively than depolymerization.17
In the model we previously proposed for nonmuscle actin filaments,
tropomodulin was shown to bind to the N-terminus of a terminal TM5
molecule positioned at the pointed end of a long actin
filament.15 Based on our new findings, we now propose a
molecular model of a short actin protofilament in erythrocytes (Figure
1B). In this model, tropomodulin is associated near the N-terminal end
of 1 TM molecule, which comprises either TM5 or TM5b (both are
approximately 33-35 nm in length) in the form of either a homodimer or
heterodimer. The molecule is at the pointed end of the short actin
filament, which is only approximately 33-37 nm in length. Since both
TM5 and TM5b are LMW TM isoforms with 6 actin-binding sites, they would
stabilize 6 G-actin in 1 strand and favor the binding of 6 spectrin
tetramers to 1 protofilament. TM5 and TM5b are the 2 TM isoforms that
have the strongest binding affinities (among all TM isoforms we tested,
including muscle and nonmuscle TM isoforms) toward tropomodulin. TM5
and TM5b are also the 2 isoforms (among LMW TM isoforms) that have the
highest affinities toward F-actin. These facts suggest that a very
tight association between the tropomodulin-TM complex and the actin
filament may be the key to the mechanism by which protofilaments are
formed and maintained in erythrocyte membranes. The protofilament may,
therefore, be viewed as a tightly associated basic unit of the actin
filaments, consisting of a tropomodulin, 2 TM dimeric molecules (1 in
each groove of the actin filament), and a double helical actin filament with a uniform length of approximately 33-37 nm. The complex formed by
a globular tropomodulin and a rod-like TM molecule of approximately 33-35 nm in length may, therefore, function as a measuring device or a
molecular ruler (Figure 1C) to reduce long actin filaments to short
protofilaments with a uniform length.
TM4 is not present in human erythrocyte membranes
There are several LMW TM isoforms expressed in nonmuscle cells
including TM5, TM5a, TM5b, TM4, and a few neuron-specific TM isoforms.9-11 TM4, a gene product of the -TM
gene, also consists of 248 residues. To see whether
human erythrocyte membranes also contain TM4, a Western blot analysis
using a TM4-specific mAb LC24 was conducted. A recombinant chimeric
human TM molecule (hTM5-g/4), which contains the epitope for mAb LC24,
was included as a positive control. The results showed that mAb LC24
(1:1000 dilution) detected a strong signal for the positive control,
but no detectable signal for TM4 was found in human ghost membranes
(data not shown). The studies on the specificity of mAbs Pep3-34, LC1,
and CG3, which demonstrated that they do not cross-react with TM4, have
been reported previously.11,23-25 There remains a small
possibility that there may be some minor or new TM isoforms in human
erythrocyte membranes that have not yet been identified.
| |
Discussion |
In this report, we identified TM5 and TM5b to be the 2 major TM
isoforms in human erythrocytes. Although they are products of 2 different genes (-TM and -TM genes,
respectively), they share several common features. Both are LMW
isoforms, have 248 residues, and are approximately 33-35 nm in
length.40,41 In addition, both have a high actin-binding
affinity10,12 and a high tropomodulin-binding
affinity.15 These properties are in agreement with previous
studies of the Mr in the TM mixtures purified from
erythrocytes in SDS-PAGE,4 the length of actin protofilaments (approximately 33-37 nm) in erythrocyte membrane skeletal network,1-3 and the high F-actin affinity found in
TM mixtures purified from erythrocytes.4
The common features shared by TM5 and TM5b have significant functional
implications, especially in the stability of protofilaments, the
hexagonal organization of the membrane skeletal network, and the
remodeling of the actin cytoskeleton during terminal differentiation. The following details the F-actin affinity of TM and why it is important for the stability of protofilaments; the length of TM and how
it defines the hexagonal geometry of the membrane skeleton; and the
tropomodulin affinity of TM and how that modulates the function of TM
isoforms and the length of the actin filaments.
High F-actin affinity allows TM5 and TM5b to form more stable
protofilaments. Erythrocytes are constantly subjected to the flow
dynamics of the cardiovascular system and are frequently deformed by
negotiating their way through narrow capillaries. Protofilaments
located in the center of the junctional complexes must be strong enough
to resist the pulling of spectrin in response to stresses in order to
maintain the integrity of the membrane skeletal network (Figure 1A and
1B). The main function of TM is to coat and stiffen the actin
filaments,42 thereby making them more resistant to
depolymerization and fragmentation.43,44 TM5 and TM5b, the
2 TM isoforms that have a high affinity toward F-actin, should function
better than other LMW TM isoforms in stabilizing protofilaments.
The length of TM molecules or the number of actin-binding sites along
the TM molecules defines the geometry of the membrane skeletal network.
In general, there are 7 actin-binding sites along HMW TM isoforms (284 residues, approximately 40-43 nm in length) and 6 sites along LMW TM
isoforms (248 residues, approximately 33-35 nm in length). As LMW
isoforms, TM5 and TM5b stabilize 6 G-actin (in 1 strand) and allow 6 spectrin dimers to bind to 1 protofilament (presumably 1
spectrin dimer binds to 1 pair of G-actin in the double helix). As a
result, LMW isoforms, rather than HMW isoforms, favor the organization
of hexagonal lattices observed in the membrane skeletal network (Figure
1A and 1B). The hexagonal arrangement allows a seamless continuation of
the spectrin-actin-based skeletal network throughout the entire cell membrane. This two-dimensional (2-D) membrane skeletal
network is essential for the mechanical stability of a circulating
erythrocyte, as the enucleated, biconcave mature erythrocyte no longer
has a supporting 3-D actin cytoskeleton in the cytoplasm.
The high tropomodulin affinity of TM5 and TM5b makes the
TM/tropomodulin complex an effective measuring device or a molecular ruler capable of metering off long actin filaments to short
protofilaments. This is because TM complexed with tropomodulin is able
to bind and block an actin filament at its pointed end, but it is not able to overlap with other TM isoforms in a head-to-tail
fashion along the actin filaments or at the barbed end (Figure
5). As a result, only the first 6 G-actin
in 1 strand (or 12 G-actin in the double helix) located at the pointed
end of the actin filaments are protected by TM. Given the stress and
strain undergone in the erythrocyte membrane during circulation,
segments of actin filaments not coated by TM are likely to be
fragmented or depolymerized. Thus, only short segments of the actin
filaments, which are protected by 1 TM, can survive. The complex,
therefore, contributes to the formation and maintenance of
protofilaments in erythrocytes. The fact that TM5 has high initial
binding affinity and low cooperativity to actin filaments12
should also favor the formation of protofilaments the length of only 1 TM.
View larger version (18K):
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| Fig 5.
A switch of other TM isoforms to TM5 or TM5b and/or an
increase of tropomodulin would favor the formation of actin
protofilaments.
The tropomodulin-TM complex is able to bind to the pointed end of a
long actin filament but not along it. The more tropomodulin bind to the
N-terminal of homodimer or heterodimer of TM5 and TM5b (approximately
33-35 nm in length), the less the head-to-tail association of TM
molecules can occur along the actin filaments. Increasing TM5, TM5b,
and/or tropomodulin, therefore, would favor the stabilization of
shorter actin filaments that are of the same size as an LMW TM
molecule.
|
|
Although tropomodulin is a capping protein at the pointed end, it has
more effects on the actin filaments than simply capping the pointed
end. Unlike the barbed-end capping proteins that bind directly and
block the G-actin at the barbed end, tropomodulin functions mainly
through the mechanism of complexing with the TM molecule. TM is a
regulatory rod-like molecule that overlaps with adjacent TM and
stabilizes the actin filament from one end to the other. Therefore, by
binding to one end (the N-terminal end) of TM, tropomodulin affects the
stability of the entire actin filament and, under certain conditions,
the growth from both ends. The degree to which tropomodulin
destabilizes the actin filaments (except for the 1 TM-protected segment
at the pointed end) depends on the proportion of TM molecules
that are complexed with tropomodulin. If all TMs are complexed with
tropomodulin (Figure 5) and if the actin filaments are under
stress, the actin filaments can only be 1 TM in length, without
growth from either end.
The erythrocyte membrane skeleton contains approximately 130 000
copies of TM (corresponding to approximately 75 000 dimeric TM
molecules), approximately 400 000 copies of G-actin (corresponding to
approximately 30 770 protofilaments), and approximately 30 000 copies
of tropomodulin.5 The number of TM molecules is enough to
stabilize all protofilaments, 1 in each groove of the actin filament.
The number of tropomodulin is enough to bind to half of the TM dimeric
molecules or every protofilament in the membrane skeleton. The
stoichiometry of these 3 molecules, therefore, favors the formation of
protofilaments that are only 1 TM molecule in length. The functional TM
molecules in protofilaments are expected to be homodimers and
heterodimers of TM5 and TM5b because dimers are the functional forms of
the molecules and TM5 is uniquely capable of dimerizing with
TM5b.25,45 TM5 and TM5b share several common features that
are important for the development and organization of the membrane
skeleton, and the 2 are expected to have similar functions in this
respect. It is likely, however, that each may have unique functions in
other aspects.
The actin cytoskeleton is remodeled during erythroid terminal
differentiation. The long actin filaments are shortened into short
protofilaments, and the 3-D cytoskeleton is transformed into a 2-D
membrane skeleton. It is not clear how these changes are achieved,
although they may be caused by an increase of complexes formed by TM5
and/or TM5b with tropomodulin (Figure 5). An increase in these
complexes may be achieved by 2 mechanisms1: TM isoform switching, ie, switching from other isoforms to TM5 and/or
TM5b, or by up-regulation of tropomodulin.2 Finding
either or both of these mechanisms would support the functional role of
tropomodulin -TM5 or -TM5b complexes in the formation of
protofilaments. Examination of the levels of TM5, TM5b, and
tropomodulin during erythroid terminal differentiation using a 2-phase
liquid culture system would shed new light on the regulatory
mechanism(s) involved in the development of erythrocyte membrane
skeletons. Experiments are also in progress to determine the definitive
roles of tropomodulin in the differentiation and biomechanics of
erythrocytes by targeted disruption of the tropomodulin gene in the
mouse embryonic stem cells.46
| |
Acknowledgments |
We thank Carlos Vera and Edgar Gutierrez for repeating some of the
experiments. We also thank Matt Levitt, Ellvin Mar, and Stacy Hwang for
assistance in manuscript preparation.
| |
Footnotes |
Submitted August 11, 1999; accepted October 5, 1999.
Supported in part by grant HL-43026 (L.A.S.) and grants DK47673 and
HD18577 (J.J.-C.L.) from the National Institutes of Health, Bethesda,
MD, grant MCB-9874492 (D.H.) from the National Science Foundation, and
fellowship 950216 (C.J.T.-G.) from the American Heart Association,
Dallas, TX. D.M.H. is an Established Investigator of the American Heart Association.
Reprints: Lanping Amy Sung, Department of Bioengineering and
Center for Molecular Genetics, EBU1 6406, Mail Code 0412, University of
California, San Diego, La Jolla, CA 92093-0412; e-mail:
amysung{at}bioeng.ucsd.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction