Download A nuclear matrix-associated high molecular mass nuclear antigen

3031
Journal of Cell Science 110, 3031-3041 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS4482
A nuclear matrix-associated high molecular mass nuclear antigen, HMNA, of
chicken and marked decrease of its immunoreactivity during the progression
of S phase
Kenji Shimada, Masahiko Harata and Shigeki Mizuno*
Laboratory of Molecular Biology, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1
Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981 Japan
*Author for correspondence
SUMMARY
A hnRNP-free nuclear matrix prepared from chicken
MSB-1 cells was used to raise monoclonal antibodies. The
monoclonal antibodies 2H3 and 3B7 showed identical nonhomogeneous immunofluorescence staining patterns of
nuclei in MSB-1 cells and chicken embryonic fibroblasts.
In a synchronized culture of MSB-1 cells, the immunoreactivity of nuclei with 2H3, but not with 3B7, antibody
decreased markedly during the progression of S phase, but
returned to the normal level at the next G1 phase. When
cells were treated with Triton X-100 prior to fixation with
paraformaldehyde or cells were fixed in methanol, nuclei
were reactive with 2H3 antibody throughout the S phase.
Both 2H3 and 3B7 antibodies recognized a high molecular
mass nuclear antigen (HMNA) of approximately 550 kDa,
which was associated with the nuclear matrix. HMNA was
resistant to extraction with 0.5 M NaCl from the nuclei at
the G1/S boundary but became extractable by the end of S
phase. A cDNA clone, pBHB36, containing a partial
sequence for HMNA was isolated by immunoscreening as
a double positive clone with 2H3 and 3B7 antibodies. The
deduced 1,150 residue-long sequence of pBHB36 shows no
homology with any molecules in the nucleotide and protein
sequence databases, and contains different epitope regions
for 2H3 and 3B7 antibodies. A possibility of hydrophobic
association of HMNA with nuclear protein(s) during the
progression of S phase is discussed.
INTRODUCTION
matrix. Interestingly, the reactivity of 2H3, but not 3B7,
antibody to HMNA markedly decreased during the progression
of S-phase in the cell cycle of MSB-1 cells. Different epitope
regions in HMNA for 2H3 and 3B7 antibodies were characterized by isolating a cDNA clone, pBHB36, which contained
a partial cDNA sequence for HMNA but was double-positive
to these antibodies in immunoscreening. The nucleotide and
deduced amino acid sequences of pBHB36 suggest that
HMNA is a novel protein species.
The nuclear matrix has been implicated to be involved in
various nuclear functions such as DNA replication, pre-mRNA
splicing, formation of chromatin domains, binding sites of
transcriptional factors and other regulatory proteins. It has been
suggested that the basic structure of nuclear matrix is corefilaments, which are made up of proteins belonging to the intermediate filament protein family (Jackson and Cook, 1988; He
et al., 1990) and that the core filaments are converted to thick
filaments by association with various nuclear components such
as lamins, heterogeneous nuclear ribonucleoproteins
(hnRNPs), DNA topoisomerase II and other proteins. Protein
compositions of nuclear matrices are variable depending upon
the procedure of fractionation and the source of nuclei from
which they were prepared (for reviews see Berezney et al.,
1995; Nickerson et al., 1995).
In this study, we first prepared a nuclear matrix fraction from
which hnRNPs were virtually completely removed from
chicken MSB-1 cells (Akiyama and Kato, 1974). The fraction
obtained was used as antigens to prepare monoclonal antibodies. Among those antibodies, 2H3 and 3B7 recognized a
high molecular mass nuclear protein antigen (HMNA) of
approximately 550 kDa, which was associated with the nuclear
Key words: Chicken, High molecular mass nuclear antigen, Nuclear
matrix, S phase
MATERIALS AND METHODS
Production of monoclonal antibodies against a nuclear
matrix fraction of MSB-1 cells
Nuclei were prepared from a 2 litre culture of chicken MSB-1 cells
(Akiyama and Kato, 1974) as described by Suka et al. (1993). The
nuclear pellet was suspended in 10 ml of CSK buffer (10 mM Pipes,
pH 6.8, 50 mM NaCl, 3 mM MgCl2, 0.3 M sucrose, 0.5 µg each/ml
of protease inhibitors antipain, chymostatin, elastatinal, leupeptin and
pepstatin A), incubated with 180 units/ml of DNaseI (Takara Biomedicals, Kusatsu, Japan) at 25°C for 20 minutes, followed by extraction with an equal volume of CSK buffer containing 0.5 M (NH4)2SO4
according to the method of Fey et al. (1986). The treated nuclei were
resuspended in 10 ml of CSK buffer and incubated with 25 µg/ml of
RNase A (Sigma, St Louis, MO, USA) at 25°C for 10 minutes. The
3032 K. Shimada, M. Harata and S. Mizuno
RNase digestion was repeated once again. The final pellet (nuclear
matrix fraction) was resuspended in 5 ml of buffer A (0.05 mM Pipes,
pH 6.5, 50 mM NaCl, 50 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.5
M hexyleneglycol, 0.15 M sucrose, 0.5 µg each/ml of protease
inhibitors).
A 70 µl portion (about 150 µg protein) of the nuclear matrix
fraction was mixed with 30 µl of 5 M NaCl and 100 µl of complete
Freund’s adjuvant, or incomplete Freund’s adjuvant (Difco Lab.,
Detroit, MI, USA) in the following two injections, and injected subcutaneously into a 4-week-old female Balb/c mouse. The high concentration of NaCl was noted to enhance antigenicity of the nuclear
matrix fraction. The final injection was made into the peritoneal cavity
with 100 µl (about 200 µg protein) of the nuclear matrix fraction
mixed with 30 µl of 5 M NaCl and 7 µl of 10% Triton X-100. Three
days after the final injection, the splenic cells were isolated and
hybridomas were raised using the standard procedures. A selected
hybridoma clone (1-2×107 cells) was further propagated in ascites of
a 6-week-old female Balb/c mouse, and the monoclonal antibody was
recovered by E-Z-SEP (Pharmacia Biotech, Uppsala, Sweden). A
monoclonal antibody was biotinylated using Biotinylation kit
(Amersham International plc, Amersham, UK).
Cell culture and immunofluorescence reactions
Chicken embryonic fibroblasts (CEFs) were cultured in DMEM
(Sigma) containing 8% FBS (Irvine Scientific, Santa Ana, CA, USA)
and 2% chicken serum (Sigma) at 37°C in 5% CO2/95% air. MSB-1
cells were cultured in suspension in RPMI 1640 containing 8% FBS
and 2% chicken serum at 42°C in 5% CO2/95% air. A synchronous
culture of MSB-1 cells was started after release from the aphidicolin
block at the G1/S boundary as described (Suka et al., 1993). MSB-1
cells spread on a slide glass in Cytospin 2 (Shandon, Runcorn, UK)
or CEFs grown on a slide glass were fixed in PLP solution containing 2% paraformaldehyde (McLean and Nakane, 1974) at 25°C for
15 minutes, washed in PBS (1.5 mM KH2PO4, 6.4 mM
Na2HPO4.12H2O, 137 mM NaCl, 2.7 mM KCl, pH 7.2), treated in
ice-cold acetone for 10 minutes, washed again in PBS, and subjected
to reaction with a monoclonal antibody at 25°C for 1 hour, followed
by reaction with fluorescein isothiocyanate (FITC)-labelled goat antimouse IgG (Cappel/ICN Pharmaceuticals, Costa Mesa, CA, USA) in
PBS containing 1% bovine serum albumin (BSA) and 3% goat serum
(Rockland Immunochemicals, Gilbertsville, PA, USA). The cells were
counterstained with 1 µg/ml propidium iodide (PI) and observed
under a fluorescence microscope (Olympus BH2-RF, Olympus,
Tokyo, Japan; or Leica DMRB, Wetzlar, Germany). When reactions
with two monoclonal antibodies were performed, the PLP-fixed and
acetone-treated cells were subjected to reaction with one monoclonal
antibody, followed by reaction with Cy5-labelled donkey anti-mouse
IgG (Amersham). Cells were then subjected to reaction with a biotinylated second monoclonal antibody, followed by reaction with
avidin-FITC (Vector Lab, Burlingame, CA, USA). The fluorescence
was observed under a confocal laser scanning fluorescence microscope (MRC-1024; Bio-Rad, Hemel Hempstead, UK).
Flow cytometric analysis of DNA contents
MSB-1 cells (about 106 cells) from a synchronous culture were resuspended in 300 µl of PBS, mixed with 700 µl of 99.5% ethanol (prechilled at −20°C), and stored at −20°C. The ethanol-fixed cells were
collected by centrifugation at 1,500 g for 5 minutes, resuspended in
200 µl of PBS containing 20 µg/ml PI and 100 µg/ml RNase A, and
subjected to flow cytometry with Coulter Epics Elite system (Coulter,
Miami, FL, USA) as described by Rapi et al. (1996).
In situ preparation of the nuclear matrix
CEFs cultured on a slide glass were washed in IS buffer (3.75 mM
Tris-HCl, pH 7.4, 0.05 mM spermine, 0.125 mM spermidine, 0.5 mM
EDTA, 1% β-thiodiglycol, 20 mM KCl, 300 mM sucrose), placed on
ice and covered with IS buffer containing 0.1% digitonin and 0.5 mM
CuSO4 for 10 minutes. The following steps were carried out at 25°C.
Cells were treated with IS-extraction buffer (5 mM Hepes, pH 7.4,
0.25 mM spermidine, 2 mM KCl, 2 mM EDTA, 0.1% digitonin, 25
mM 3,5-diiodosalicylic acid lithium salt (LIS), 300 mM sucrose) for
5 minutes, washed in IS-digestion buffer (20 mM Tris-HCl, pH 7.4,
0.05 mM spermine, 0.125 mM spermidine, 20 mM KCl, 70 mM NaCl,
10 mM MgCl2, 300 mM sucrose), incubated in IS-digestion buffer
containing 350 units/ml DNaseI (Takara Biomedicals) and 100 µg/ml
RNase A (Sigma) for 10 minutes and then washed in IS-fixation buffer
(10 mM Hepes, pH 7.5, 100 mM NaCl, 3 mM MgCl2, 300 mM
sucrose). This preparation (in situ nuclear matrix) was fixed in ISfixation buffer containing 2% paraformaldehyde.
Fractionation of nuclei, salt extraction, and western
blotting
Nuclear matrix
Nuclear matrix (nuclear scaffold) was prepared from the nuclei of
Fig. 1. Immunofluorescence staining of MSB-1 cells, fixed under different conditions, with 2H3 or 3B7 antibody. MSB-1 cells fixed with PLP
(A,B), first treated with 0.5% Triton X-100 then fixed with 2% paraformaldehyde (C,D) or fixed with 100% methanol (E,F) were first reacted
with 3B7 antibody, followed by Cy5-conjugated anti-mouse IgG (red) (A,C,E). After washing with PBS, samples were subjected to reaction
with biotinylated 2H3 antibody, followed by FITC-conjugated avidin (green) (B,D,F). Fluorescence images were observed under the confocal
laser scanning fluorescence microscope. It is noted that not all nuclei were reactive to 2H3 antibody in B. Bar, 50 µm.
Behavior of a nuclear matrix antigen in S phase 3033
5×107 non-synchronized MSB-1 cells, according to the method of
Mirkovitch et al. (1984), except that the nuclei were pretreated in 150
µl of the EDTA-free isolation buffer containing 0.5 mM CuSO4 on ice
for 10 minutes, and that the LIS-extracted nuclei were digested with
100 units/ml DNaseI, instead of restriction enzymes, in the digestion
buffer, from which Trasyol and phenylmethylsulfonyl fluoride
(PMSF) were omitted but 1 µg each/ml of protease inhibitors were
added, at 37°C for 6 minutes.
Salt extraction
Nuclei isolated from about 1×107 synchronized MSB-1 cells at 0 time
or 3 hours after release from the aphidicolin block, were digested with
35 units/ml of DNaseI in 200 µl of buffer A at 25°C for 10 minutes. A
portion of the DNaseI-treated nuclei (derived from about 3×106 cells)
was pelleted by centrifugation at 900 g for 5 minutes and resuspended
in 100 µl of 50 mM Tris-HCl, pH 7.5, 10 mM EDTA containing 0.15,
0.3 or 0.5 M NaCl in the absence or presence of 1% NP-40. After
placing on ice for 15 minutes, the suspension was centrifuged at 2,000
g for 10 minutes at 4°C, and supernatant and pellet were separated.
Western blotting
Nuclear subfractions were subjected to SDS-6% or 7.5% polyacrylamide gel electrophoresis (PAGE), and proteins electroblotted onto a
nitrocellulose membrane (BA85, Schleicher & Schuell, Dassel,
Fig. 2. Decreasing reactivity of MSB-1 cell nuclei to
2H3 antibody during the progression of S phase.
(A) Flow cytometric analysis of the relative contents
of PI-stained DNA in MSB-1 cells at the time as
indicated after release from the aphidicolin block at
the G1/S boundary. (B) FITC-fluorescence staining of
MSB-1 cell nuclei which reacted with 2H3 antibody at
the time points correspondoing to those in A.
(C) Percentage of nuclei reactive with 2H3 antibody
(stippled bars) and viable cell count (filled circles) at
the time points corresponding to those in A and B.
3034 K. Shimada, M. Harata and S. Mizuno
Germany) was subjected to reaction with 2H3, 3B7 or anti-chicken
lamin B1 monoclonal antibody in TBST buffer (10 mM Tris-HCl, pH
8.0, 150 mM NaCl, 0.05% Tween-20) containing 5% skimmed milk
(Snow Brand Milk Products, Tokyo, Japan), followed by reaction with
alkaline phosphatase-conjugated sheep anti-mouse IgG (Promega,
Madison, WI, USA).
Isolation of HMNA
Nuclei prepared from a 4 litre culture of MSB-1 cells were digested
with 45 units/ml of DNaseI in 20 ml of buffer A at 20°C for 15 minutes
and centrifuged at 400 g for 5 minutes. The pellet was resuspended in
30 ml of lysis buffer (25 mM Tris-HCl, pH 8.0, 0.35 M NaCl, 1% NP40, 1 µg each/ml of protease inhibitors), stirred for 1 hour at 4°C,
followed by sonication for 20 seconds twice with Sonifier (Branson,
Danbury, CT, USA). After centrifugation at 9,000 g for 10 minutes,
the supernatant was diluted with 30 ml of 25 mM Tris-HCl, pH 8.0,
1% NP-40, 1 µg each/ml of protease inhibitors, centrifuged again and
the supernatant was applied to a 1 ml column of HiTrapQ (Pharmacia
Biotech) which had been equilibrated with a column buffer (25 mM
Tris-HCl, pH 8.0, 0.15 M NaCl, 0.1% NP-40, 1 µg each/ml of protease
inhibitors). A flow-through fraction from the HiTrapQ column was
then applied repeatedly with a flow rate of 0.5 ml/minute (total 3 to 6
hours) to a 3 ml column of 3B7 antibody bound to HiTrap NHSActivated (Pharmacia Biotech) which had been equilibrated with the
column buffer. After washing with the column buffer containing 0.5
M NaCl, bound proteins were eluted with 0.1 M glycine, pH 3.0 (1
ml/fraction) into tubes each containing 100 µl of 1 M Tris-HCl, pH
8.0, 2.5 µg each/ml of protease inhibitors. The protein-containing
fraction was concentrated under vacuum and subjected to SDS-5%
PAGE, followed by western blotting with 3B7-antibody or staining
with 0.1% Coomassie Brilliant Blue R250. The high molecular mass
protein, which was proved to be reactive with 3B7-antibody, was electroeluted in 25 mM Tris, 192 mM glycine, pH 8.3, 0.1% SDS, at 250
V, 80 mA, passed through a Columngard-LCR4 (pore size 0.5 µm;
Millipore, Bedford, MA, USA) and concentrated as above.
2026-2415, were amplified by PCR, subcloned into pQE30 (Qiagen,
Chatsworth, CA, USA), and transformed into E. coli XL1 bluepREP4. The polypeptide with a (His)6-tag at its N terminus was
produced after induction with isopropyl β-D-thiogalactopyranoside
(IPTG). Cells were lysed in PBST buffer (50 mM Na-phosphate, pH
8.0, 0.3 M NaCl, 1% Tween-20), and the (His)6-tagged polypeptide
was purified using a Ni2+-NTA-agarose column (QIAGEN) according
to the manufacturer’s protocol. Polyclonal antibodies against each
(His)6-tagged polypeptide were raised in 4-week-old Balb/c female
mice and affinity-purified on the antigen polypeptide fixed on a PVDF
membrane (PE Appled Biosystems).
cDNA cloning, DNA sequencing and northern blot
hybridization
A λgt11 cDNA library of MSB-1 cells (prepared by H. Tsuda of the
authors’ laboratory) was immunoscreened with 2H3 and/or 3B7
antibody, and inserts of positive clones were recloned using pBluescript SK+ vector (Stratagene, La Jolla, CA, USA). The cDNA
sequence was determined on a series of deletion constructs prepared
by exonuclease III and Mung bean nuclease using PRISM Sequenase
Kit (PE Applied Biosystems, Foster City, CA, USA), Taq Dye Primer
Cycle Sequencing Kit (PE Applied Biosystems), ∆Tth DNApolymerase Auto Sequencer Core Kit (Toyobo, Osaka, Japan) or Bca Best
Dideoxy Sequencing Kit (Takara Biomedicals), and 373A DNA
sequencer (PE Applied Biosystems). Poly(A)+ RNA, prepared from
MSB-1, CEF, Japanese quail QT-6, mouse Balb3T3, green monkey
COS-7 or human HeLa cells, was electrophoresed on a 1% agarose
gel (1 µg/lane), transferred onto Hybond-N+ (Amersham) and
subjected to northern blot hybridization in 5× SSPE (1× SSPE: 10 mM
Na phosphate, pH 7.7, 0.18 M NaCl, 1 mM EDTA) containing 1%
SDS, 200 µg/ml of sheared, denatured salmon sperm DNA and 50%
formamide with the 32P-labelled 3.5 kb insert DNA of pBHB36 at
42°C for 20 hours. The membrane was washed in 1× SSPE, 0.1% SDS
at 60°C for 20 minutes and radioactivities were visualized by
BAS2000 bio-image analyzer (Fuji Photo Film, Tokyo, Japan). After
removing the probe, the same blot was rehybridized with the chicken
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe
(Dugaiczyk et al., 1983).
Production of polypeptides in Escherichia coli and
preparation of antisera
Two cDNA fragments of pBHB36; nucleotide positions 421-792 and
Fig. 3. Detection of a high molecular mass nuclear antigen (HMNA)
which is reactive with both 2H3 and 3B7 antibodies. (A) Proteins in
the nuclei, from 8×105 cells (left lane) or 4×105 cells (right lane), and
cytoplasmic fraction (from 8×105 cells) of MSB-1 cells were
separated by SDS-PAGE and stained with Coomassie Brilliant Blue
(CBB) or subjected to western blotting with 2H3 or 3B7 antibody.
Human low density lipoprotein (LDL), in which apolipoprotein B100 (approximately 510 kDa) is the major component, was coelectrophoresed as a molecular mass marker. Other molecular mass
markers were given by the broad range protein molecular weight
standard (Bio-Rad; Hercules, CA, USA). (B) Extract of MSB-1
nuclei (NE) and a fraction eluted from the 3B7-antibody column
(Ab-C) were subjected to SDS-PAGE and CBB staining. (C) HMNA
in B (Ab-C) was electroeluted and subjected to SDS-PAGE, followed
by silver staining or western blotting with 2H3 or 3B7 antibody.
HMNA is shown by an arrow in B and C.
Behavior of a nuclear matrix antigen in S phase 3035
RESULTS
Nuclear immunofluorescence staining with
monoclonal antibodies 2H3 and 3B7
Among monoclonal antibodies against the hnRNP-depleted
nuclear matrix fraction of chicken MSB-1 cells, 2H3 and 3B7
were noted by confocal laser scanning fluorescence
microscopy to show identical immunofluorescence staining
patterns in the nuclei of CEFs or MSB-1 cells, which had been
fixed with PLP containing 2% paraformaldehyde. However, it
was noted that not all nuclei were reactive with 2H3 antibody
(Fig. 1B). On the other hand, when MSB-1 cells were treated
with 0.5% Triton X-100 prior to the above fixation (Fig. 1D)
or fixed in 100% methanol at −20°C (Fig. 1F), all the nuclei
were reactive with 2H3 antibody. All the nuclei were reactive
with 3B7 antibody irrespective of the method of fixation (Fig.
1A,C and E).
Decreased reactivity of nuclei with 2H3 antibodty
during the progression of S phase
When synchronously cultured MSB-1 cells were subjected to
reaction with 2H3 antibody, numbers of immunostained nuclei
decreased substantially at 1.5 and 2.5 hours after release from
the aphidicolin block at the G1/S boundary, but then increased
again and reached a high level at 4.5 hours (Fig. 2B). Flow
cytometry on PI-stained cells indicated that the 1.5- and 2.5
hour points corresponded to mid S and late S/G2 phases,
respectively (Fig. 2A). When percentages of immunostained
nuclei and viable cell numbers are compared, it is evident that
the number of immunostainable nuclei with 2H3 antibody
decreased about 5-fold by the late S/G2 phase but it returned
to a high level again by the next G1 phase (Fig. 2C).
Fig. 4. Existence of HMNA in the nuclear matrix fraction. MSB-1
cell nuclei treated with 0.5 mM CuSO4 were fractionated into 25 mM
LIS-soluble and insoluble fractions. The insoluble fraction was
digested with DNaseI and centrifuged to obtain supernatant (S) and
pellet (P). The pellet was regarded as the nuclear matrix fraction.
Unfractionated nuclei and the nuclear subfractions were subjected to
SDS-PAGE, followed by CBB staining or western blotting with 3B7
antibody. Protein molecular mass markers are as in Fig. 3.
Fig. 5. Existence of HMNA in the in situ prepared nuclear matrix.
CEFs cultured on a slide glass were treated with 0.5 mM CuSO4 and
0.1% digitonin (A,B), and then nuclear matrix was prepared in situ
by extraction with LIS and digestion with DNase I and RNase A
(C,D). Preparations were fixed with 2% paraformaldehyde and
subjected to reaction with anti-chicken p120 monoclonal antibody,
followed by Cy5-conjugated anti-mouse IgG (A,C). The same
preparations were then subjected to reaction with biotinylated 2H3
antibody, followed by FITC-conjugated avidin (B,D). Bar, 50 µm.
Fig. 6. Susceptibility of HMNA to the high salt extraction at the late
S/G2 phase. Nuclei were prepared from MSB-1 cells at 0 hour or 3
hours after release from the aphidicolin block, treated with DNase I,
and extracted with the buffer containing different concentrations of
NaCl, in the absence (A) or presence (B) of 1% NP-40. Supernatant
(S) and residual nuclear pellet (P) were separated by centrifugation,
and each fraction was subjected to SDS-PAGE and western blotting
with 2H3 + 3B7 antibodies (A), 3B7 antibody (B) or anti-chicken
lamin B1 monoclonal antibody (A,B).
3036 K. Shimada, M. Harata and S. Mizuno
Behavior of a nuclear matrix antigen in S phase 3037
patterns with these antibodies (Fig. 1) suggested that 2H3 and
3B7 antibodies recognized different epitopes in the HMNA
molecule.
When the nuclei of MSB-1 cells were fractionated into LISsoluble, LIS-insoluble but solubilized after DNaseI digestion,
and residual ‘nuclear matrix’ fractions, according to the
method of Mirkovitch et al. (1984), and each fraction subjected
to western blotting with 3B7 antibody, HMNA was detected
exclusively in the nuclear matrix fraction (Fig. 4).
Association of HMNA with the nuclear matrix was also
demonstrated by in situ preparation of nuclear matrix, followed
by immunofluorescence staining. CEFs grown on a slide glass
were treated with 0.5 mM CuSO4 and 0.1% digitonin, and then
the nuclear matrix was prepared in situ. The nuclear matrix
thus prepared, or control cells after treatment with CuSO4 and
digitonin, were subjected to a series of immunofluorescence
reactions with anti-p120 antibody, which recognized hnRNP U
protein (Kiledjian and Dreyfuss, 1992) of chicken (K.
Shimada, unpublished), and with 2H3 antibody. The immunofluorescence of p120 in the nuclear matrix was hardly
detectable (Fig. 5A versus C), whereas intensities of the
immunofluorescence of HMNA in the control nuclei and in the
in situ prepared nuclear matrix were similar (Fig. 5B versus
D). We conclude from these results that HMNA is associated
with the hnRNP-depleted nuclear matrix.
Fig. 7. Nucleotide and deduced amino acid sequences of a 2H3/3B7
double-positive cDNA clone, pBHB36. (A) Nucleotide and deduced
amino acid sequences of the entire cDNA insert of pBHB36, which
will appear in DDBJ, EMBL and GenBank nucleotide sequence
databases with the accession number D88440. A region showing
homology to histone H1.10 (underlined), a region consisting of
repeated elements (enclosed), a cluster of serine residues (broken
underline), a stretch of basic amino acid residues (wavy underline)
and potential sites of phosphorylation with casein kinase II (ck2),
protein kinase C (pkc), or protein kinase A (pka) are indicated. The
sequence for the insert of pBH18, which was selected by
immnoreaction with 2H3 antibody only, is shown between two
arrows. (B) Twenty tandemly repeated sequence elements, except for
the middle one, in the region enclosed in A and their predicted
secondary structure. A small letter in the consensus sequence means
a residue which appears in less than 50% of the repeats.
A high molecular mass nuclear antigen (HMNA)
recognized by 2H3/3B7 antibodies and its
association with the nuclear matrix
When cytoplasmic and nuclear fractions of MSB-1 cells were
subjected to western blotting with 2H3 or 3B7 antibody, an
extremely high molecular mass protein (HMNA) was recognized by both antibodies in the nuclear fraction (Fig. 3A). The
molecular mass of HMNA was estimated to be about 550 kDa
from the comparison of its mobility in the SDS-PAGE with that
of apolipoprotein B-100 (approximately 510 kDa; Cladaras et
al., 1986). As minor protein bands were detected in the western
blotting of nuclear fraction, we aimed to isolate HMNA, as
described in Materials and Methods. The fraction eluted from
a 3B7 antibody column contained multiple protein components
as shown by SDS-PAGE (Fig. 3B), suggesting the presence of
both degradation products and proteins associated with
HMNA. The HMNA recovered by electroelution was reactive
with both 2H3 and 3B7 antibodies (Fig. 3C). These results
together with the identical nuclear immunofluorescence
Changes in the extractability of HMNA from the
nuclei during S phase
The observation that the reactivity of HMNA with 2H3
antibody decreased markedly during the progression of S phase
when MSB-1 cells were first fixed with paraformaldehyde but
that such a decrease was not observed when the cells were
treated with Triton X-100 prior to fixation or were fixed in
100% methanol suggests that HMNA becomes associated with
other nuclear protein components and/or HMNA undergoes
conformational change during the S-phase. In relation to these
notions, extractability of HMNA from the nuclei of synchronized MSB-1 cells was examined by western blotting immediately after release from the G1/S boundary (0 hour) and 3 hours
after the release, when the reactivity with 2H3 antibody
became very low (Fig. 2). HMNA remained in the nuclei at
both 0 and 3 hour points even when the treatment with 0.5%
Triton X-100 was included during the isolation of nuclei (Suka
et al. 1993) (Fig. 6A). HMNA was resistant to the extraction
with 0.3 M NaCl and only slightly extracted with 0.5 M NaCl
from the nuclei at 0 hour. On the other hand, at 3 hours, it was
extracted partially with 0.3 M NaCl and nearly completely with
0.5 M NaCl. About half of lamin B1 remained with the nuclear
pellet after the extraction with 0.5 M NaCl at 3 hours (Fig. 6A).
When the nuclei were extracted with 0.5 M NaCl in the
presence of 1% NP-40, a substantial fraction of HMNA
remained with the nuclear pellet at 0 hour, but it was nearly
completely extracted at 3 hours (Fig. 6B). Lamin B1 was
extracted from the nuclei nearly completely with 0.5 M NaCl
in the presence of 1% NP-40 at both the 0 and 3 hour points,
reflecting its association with the inner nuclear membrane
(Hennekes and Nigg, 1994; Martin et al., 1995) (Fig. 6B).
These results suggest that HMNA is associated with the nuclear
matrix but not with the nuclear membrane, but that its mode of
association with the nuclear matrix changes to a state which is
susceptible to the high salt extraction by the late S/G2 phase.
3038 K. Shimada, M. Harata and S. Mizuno
Fig. 8. Subregions of HMNA produced as (His)6-tagged polypeptides in E. coli, containing an epitope for 2H3 or 3H7 antibody. (A) The (His)6tagged polypeptide corresponding to amino acid residues 676 to 805, containing the repeated elements (Fig. 8B), was detected by western
blotting with 2H3 but not with 3B7 antibody in the IPTG-induced E. coli extract or after purification using a Ni2+-NTA agarose column.
(B) The (His)6-tagged polypeptide corresponding to amino acid residues 141 to 264, containing the H1-homology region (Fig. 8A), was
detected as in A with 3B7 but not with 2H3 antibody. The extracts or purified polypeptides in A and B were also stained with Coomassie
Brilliant Blue (CBB) for comparison with the band detected by western blotting.
Nucleotide and deduced amino acid sequences of a
2H3/3B7 double-positive cDNA clone
A cDNA expression library constructed from MSB-1 cells was
screened by an immunoreaction with 2H3 or 3B7 antibody, and
a clone pBHB36, which contained a partial sequence of HMNA
but was reactive with both antibodies, was obtained. Nucleotide
sequence of the 3.5 kb insert of pBHB36 and its deduced amino
acid sequence are shown in Fig. 7A. These sequences do not
show significant similarities to any sequences in the nucleotide
(DDBJ, EMBL and GenBank) and protein (PIR and Swiss-Plot)
sequence databases. However, the deduced sequence contains
several characteristc motifs; a region (amino acid residues 201
to 240) showing 42% identitiy with a part (residues 160 to 202)
of the C-terminal tail domain of a subtype (.10 H1) of chicken
histone H1 (Coles et al., 1987) (underline), a region (residues
691 to 909) consisting of twenty repeats of approx. ten-residueslong element (boxed), which was noted by the Harr plot analysis
(Harr et al., 1982), clusters of serine residues (residues 1,037 to
1,060 and 1,131 to 1,150; broken underline) and a stretch of
basic residues (1,124 to 1,128; wavy underlined). The deduced
sequence of 1,150 residues is slightly hydrophlic (the average
hydrophobicity value of Kyte and Doolittle (1982) is −0.34), and
has relatively high contents of Ala (22.5%), Pro (20.5%), Ser
(11.4%) and Thr (11.7%). Potential phosphorylation sites by
protein kinase A (2 sites), protein kinase C (4 sites) and casein
kinase II (7 sites) are present. The above mentioned twenty
repeats with their predicted secondary structure are shown in Fig.
7B. It is evident that the ten-residues-long element is tandemly
repeated except for the middle region.
Epitope regions for 2H3 and 3B7 antibodies
From the cDNA expression library of MSB-1 cells a clone,
pBH18, which was reactive only with 2H3 antibody, was
obtained. This clone contained an approx. 1.6 kb insert and its
sequence was included in that of pBHB36 (the region between
two arrows in Fig. 7A). As pBH18 contained the sequence
encoding the above twenty repeats, this region was predicted
to be an epitope for 2H3 antibody. In order to prove this prediction, the cDNA sequence corresponding to about a half of
the repeats (amino acid residues 676-805) was obtained by
PCR and expressed in E. coli to produce a (His)6-tagged
polypeptide. The polypeptide, either in the E. coli extract or
after the purification, was reactive with 2H3 but not with 3B7
antibody (Fig. 8A).
The cDNA sequence corresponding to amino acid residues
141 to 264, which contained the H1-homology region (Fig.
7A), was also obtained by PCR and expressed in E. coli to
produce a (His)6-tagged polypeptide. This polypeptide, either
in E. coli extract or after the purification, was reactive with 3B7
but not with 2H3 antibody (Fig. 8B).
A polyclonal antibody was raised in mice against the (His)6tagged polypeptide containing half of the twenty repeats
(residues 676-805) and was affinity-purified. The purified
antibody reacted specifically with HMNA in the nuclear extract
of MSB-1 cells, as did 2H3 antibody (Fig. 9A). When CEFs
and MSB-1 cells were subjected to reaction with the affinitypurified antibody, similar nuclear immunofluorescence patterns
and the presence of poorly reactive nuclei were observed (Fig.
9B) as observed with 2H3 antibody (Fig. 1B).
Detection of mRNA for HMNA or its homologue
Northern blot hybridization of poly(A)+ RNAs from CEF,
MSB-1, Japanese quail QT-6, mouse Balb3T3, green monkey
COS-7 and human HeLa cells with the insert of pBHB36 as a
probe detected an approx. 15 kb mRNA species in the chicken
cells (CEF and MSB-1). A high molecular mass transcript of
approximately 12 kb was detected in the Japanese quail cells,
but no signals of hybridization were detected for the RNA
samples from the mammalian cells (Fig. 10). The mRNA
molecular size of about 15 kb seems to be consistent with the
molecular mass of 550 kDa for HMNA which was estimated
from the electrophoretic mobility in SDS-PAGE (Fig. 3).
Behavior of a nuclear matrix antigen in S phase 3039
Fig. 9. Similar immunological properties exhibited by the antiserum
against the (His)6-tagged polypeptide 676-805 as those by 2H3
antibody. (A) Western blotting of nuclear extracts of MSB-1 cells
with 2H3 antibody (lane 1), pre-immune serum (lane 2), or the
affinity purified antiserum against the (His)6-tagged polypeptide 676805 (lane 3). HMNA is shown by an arrow. (B) CEFs or MSB-1 cells
were subjected to reaction with the antiserum against the (His)6tagged polypeptide 676-805 and the immunoreaction was detected
with FITC-labelled anti-mouse IgG. Nuclei were counterstained with
PI. Presence of unstained or weakly immunostained nuclei is noted
as in Fig. 1B. Bar, 50 µm.
DISCUSSION
Comparison of the structure of HMNA with that of
other nuclear matrix proteins
In the present study, 1,150 amino acid residues of HMNA were
deduced from the cDNA sequence of pBHB36, which is likely
to represent about one fifth of the huge molecule of HMNA.
The homology plot analysis against nucleotide and protein
sequence databases indicates that the deduced partial sequence
of HMNA is unrelated to the sequences of the following
hnRNP proteins: human hnRNP proteins A1, A2, B1, C, C2,
F, K, L, M, M4, 87F and Drosophila hnRNP protein 48. There
are no RNA-binding motifs such as RNP motif, arginine-rich
motif, RGG box (Burd and Dreyfuss, 1994) nor the
serine/arginine-rich repeat (RS domain) which is conserved
among pre-mRNA splicing factors (Screaton et al., 1995). The
deduced partial sequence of HMNA does not show homology
with those of nuclear matrix-associated DNA-binding proteins;
DNA topoiosmerase II (Tsai-Pflugfelder et al., 1988; Jenkins
et al., 1992), SATB1 (Dickinson et al., 1992), SAF-A/hnRNPU (Kiledjian and Dreyfuss, 1992; Fackelmayer et al., 1994),
SAF-B (Renz and Fackelmayer, 1996) and matrin F/G (Hakes
and Berezney, 1991), nor does it show homology with high
molecular-mass proteins found in nuclear matrix fractions;
NuMA (238 kDa) (Yang et al., 1992), Ki-67 (320 and 359 kDa)
(Schluter et al., 1993) and NP220 (221 kDa) (Inagaki et al.,
1996). HMNA is clearly unrelated to lamins in that the
molecular mass of HMNA is much higher than that of lamins,
HMNA and lamins are not immunologically cross-reactable,
and HMNA but lamin B1 is totally extractable from the nuclei
with 0.5 M NaCl at the late S/G2 phase (Fig. 6). All these com-
Fig. 10. Northern blot hybridization of poly(A)+ RNA samples with
32P-labelled 3.5 kb insert of pBHB36 or chicken GAPDH cDNA
probe. RNA samples were from chicken MSB-1 cells (lane 1),
chicken embryonic fibroblasts (lane 2), Japanese quail QT-6 cells
(lane 3), mouse Balb3T3 cells (lane 4), green monkey COS-7 cells
(lane 5), and human HeLa cells (lane 6). Size markers are RNA
Ladder (Gibco BRL).
parisons suggest that HMNA is a novel protein associated with
the hnRNP-depleted nuclear matrix.
The 2H3 epitope in HMNA and its decreasing
immunoreactivity during the progression of S phase
The most intriguing feature of HMNA is that its immunoreactivity to 2H3 monoclonal antibody changes remarkably during
the cell cycle. The reactivity starts to decrease after release
from the aphidicolin block, reaches to the level where only
about 15% of cells are reactive by the late S/G2 phase and
returns to a highly reactive state at the next G1 phase. Considering that synchronization of MSB-1 cells is incomplete
judging from the flow cytometric pattern at 0 time (Fig. 2A),
the immunoreactivity should reach essentially zero level by the
end of S phase. This cell cycle-dependent behavior is unique
to HMNA among the known nuclear matrix-associated
proteins. Although a precise mechanism for this change
remains to be investigated, we like to point out those results
which seem to be pertinent to this phenomenon. The fact that
HMNA becomes reactive to 2H3 antibody when cells are
treated with Triton X-100 prior to fixation with paraformaldehyde or when cells are fixed in methanol, suggests most simply
that some protein component(s) forms a hydrophobic association with the 2H3 epitope during the progression of S phase
and the association breaks up upon entry to G1 phase. Covalent
modification, like phosphorylation, or its disappearance, like
dephosphorylation, of the epitope region per se is unlikely to
cause prevention or reappearance of immunoreaction with 2H3
antibody, but it is more likely that such a modification or
demodification affects the association or dissociation of other
protein component(s). Another possibility may be that some
other protein component(s) associates with a region outside the
2H3 epitope, which then causes conformational change of the
epitope region to become unreactable with the antibody.
3040 K. Shimada, M. Harata and S. Mizuno
The 2H3 epitope contains most likely the repeat of a tenresidues-long sequence (SPMGAAtTtq; Fig. 7B), because the
polypeptide corresponding to residues 676-805 and containing
half of the twenty repeats, which was produced in E. coli as a
form fused with a (His)6-tag, reacted specifically with 2H3
antibody, and the affinity-purified polyclonal antibody raised
against this polypeptide showed the same characteristics as did
2H3 antibody. Although there are no authentic phosphorylation sites within the repeats (Fig. 7A), the (S/T)P motifs in this
region may be phosphorylated by some species of nuclear
protein kinase. It is predicted that this region forms repeated
β-sheets (Fig. 7B), which may be involved in the hydrophobic
protein/protein association as discussed above. The above
repeat in HMNA is different from other repeats found in
nuclear proteins: WD-repeat (Neer et al., 1994), ankyrin repeat
(Michaely and Bennett, 1992) and Ki-67 repeat (Schluter et al.,
1993) consist of much longer repeating units having lesser
degrees of sequence homogeneity. The repeat of a thirteenresidues-long motif in NP220 (Inagaki et al., 1996) has a
higher level of sequence homogeneity but is acidic in nature.
MCM proteins, the yeast minichromosome maintenance
protein family, consisting of six members, are candidates of the
replication licensing factors in eukaryotes. They form a multimeric complex of 400-600 kDa and become a part of the prereplication complex at the origin of replication during late M
phase to G1 phase (Romanowski et al., 1996; Thommes et al.,
1997). In S phase, when DNA replication starts, MCM proteins
disappear from the nucleus in budding yeast (Yan et al., 1993),
but they stay in the nucleus throughout interphase in fission
yeast (Okishio et al., 1996) and higher eukaryotes (Kimura et
al., 1994; Todorov et al., 1995). In the latter cases, localization
and molecular states of the released MCM proteins within the
nucleus are unknown. However, it is particularly intriguing that
the released MCM proteins are extractable from the nuclei with
0.5% Triton X-100 (Kimura et al., 1994; Todorov et al., 1995),
because it is tempting to speculate that the released MCM
proteins associate with HMNA through hydrophobic interactions and decrease immunoreactivity of the 2H3 epitope during
the progression of S phase.
We thank H. Kimura for valuable discussion, E. Nigg for the
supply of anti-chicken lamin B1 antibody, and K. Magoori and T.
Yamamoto for human LDL. This work was supported by a Grant-inAid for Scientific Research on Priority Areas No. 07282102 from the
Ministry of Education, Science, Sports, and Culture of Japan.
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(Accepted 17 October 1997)