From the Institute of Medical Chemistry and Biochemistry,
University of Innsbruck, Fritz
Preglstrasse 3, A-6020 Innsbruck, Austria
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INTRODUCTION |
DNA in eukaryotes is organized and compacted in chromatin. The
fundamental subunit of chromatin is the nucleosomal core, which consists of 146 base pairs of DNA wrapped 1.75 times around an octamer
of core histones (reviewed in Ref. 1). The linker histones H1, H5, and
H10 are associated with the core histone-DNA complex and
with the linker DNA between adjacent nucleosomes and are thought to
modulate the condensation/decondensation of the chromatin fiber, thus
influencing many nuclear activities such as transcription, replication,
recombination, and DNA repair (2). H10 was first described
in 1969 by Panyim and Chalkley (3, 4) as an H1-like protein present in
mammalian tissues with little or no cellular proliferation and was
later shown to increase at a terminal stage of differentiation (5-10).
Some cells, however, accumulate significant amounts of the protein
while still actively proliferating (11, 12) or accumulate it upon
proliferation arrest without concomitant differentiation (12). In
addition, H10 seems to be the only histone undergoing
changes during malignant transformation (13). Recently, it was found
that transformation of NIH 3T3 fibroblasts by
c-Ha-rasVal12 oncogene causes chromatin
decondensation accompanied by alterations in the content of histone
H10 (14). All these findings suggest a role for
H10 in the regulation of either cell proliferation or
cellular differentiation.
In every tissue in which H10 has been detected, two
subfractions were present (15-18). It appears that these two
H10 proteins, up to now named H10a and
H10b, have specific individual functions in chromatin (15).
The relative proportions of the two H10 forms seem to
differ from tissue to tissue (15) and exhibit age-dependent
changes in rat brain cortical neurons (17). The two H10s
are resolvable by ion-exchange chromatography on Bio-Rex 70 (16, 19) or
acetic acid-urea gel electrophoresis (15-18). Most recently, Lindner
et al. (20) developed a high performance capillary electrophoresis method allowing separation of H10 and its
subfractions from other histone H1 subtypes. The two H10
proteins run coincidentally on sodium dodecyl sulfate-polyacrylamide gels, suggesting that the difference between them is one of charge and
not of size (15). Since neither treatment with alkaline phosphatase nor
exposure to alkaline conditions changed the separation of the
H10 peak into two subfractions, phosphorylation and
ADP-ribosylation have been ruled out as possible post-translational
modifications responsible for the different forms (16, 20, 21).
Although some investigators speculated that the two forms of
H10 might be coded by different genes (15, 17), Doenecke
et al. (22) found that the mammalian genomes contain only
one H10 gene.
To gain insight into the nature of the two H10
subfractions, we took advantage of a combined reversed phase high
performance liquid chromatography
(RP-HPLC)1/hydrophilic
interaction liquid chromatography (HILIC) technique recently developed
in our laboratory for separating acetylated core and phosphorylated H1
histones (23, 24). By applying this two-step HPLC method human placenta
histone H10 was resolved into four components, which were
treated with cyanogen bromide and chymotrypsin. HILIC analysis of the
peptide fragments obtained indicated that the four H10
protein subfractions differ in their N-terminal end consisting of 30 residues. The subsequent Edman degradation of the N-terminal peptides
and mass spectra analysis of the four untreated H10
subfractions demonstrated that human placenta histone H10
consists of a mixture of intact (H10 Asn-3) and deamidated
forms (H10 Asp-3) both blocked (H10a Asn-3,
H10a Asp-3) and unblocked (H10b Asn-3,
H10b Asp-3) on their N terminus. Deamidation occurs at
Asn-3 in the sequence Thr-Glu-Asn-Ser... . Applying the procedure
described for human placenta histone H10, we also analyzed
histone H10 from rat and mouse liver and brain. We also
found four H10 subfractions consisting of all the possible
combinations involving either acetylated or unacetylated N-terminal
residues and/or Asn or Asp at position 3. We thus show for the first
time the occurrence of N-terminally unblocked H10 histones
and of in vivo deamidated forms of linker histones. Furthermore, we found an accumulation of both the N-terminally acetylated and the deamidated forms of H10 with aging and
that the relative proportions of all four forms of H10
histones differ from tissue to tissue.
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EXPERIMENTAL PROCEDURES |
Materials--
Sodium perchlorate (NaClO4) and
triethylamine (TEA) were purchased from Fluka (Buchs, Switzerland), and
hydroxypropylmethylcellulose (4000 centipoises) and trifluoroacetic
acid were obtained from Sigma (Munich, Germany). All other chemicals
were purchased from Merck (Darmstadt, Germany).
Preparation of H1 Histones--
H1 histones were extracted from
human placenta and from rat liver and brain with perchloric acid (5%,
w/v) according to the procedure of Lindner et al. (25) with
slight modifications. The organs were homogenized in 2 volumes of
buffer A (250 mM sucrose, 50 mM Tris-HCl, pH
7.5, 25 mM KCl, 5 mM MgCl2, 0.2 mM phenylmethylsulfonyl fluoride, 50 mM
NaHSO3, 45 mM sodium butyrate, and 10 mM 2-mercaptoethanol) in a Potter homogenizer. After
centrifugation for 10 min at 3,500 rpm in the SS-34 rotor of a Sorvall
centrifuge, the pellet was washed twice with buffer A and then
homogenized in buffer A containing 0.2% Triton X-100 in a Dounce
homogenizer. Nuclei thus obtained were collected by centrifugation (10 min at 3,500 rpm), and the nuclear pellet was washed once with buffer A
without Triton X-100. The pellet was centrifuged and then extracted
with 1 volume of 10% (w/v) HClO4 and 2 volumes of 5%
(w/v) HClO4 for 1 h with occasional vortex mixing.
HClO4-insoluble material was removed by centrifugation at
10,000 rpm for 10 min, and soluble proteins were precipitated by adding
trichloroacetic acid to a 20% (w/v) final concentration in the
presence of protamine sulfate (20 µg/ml). The precipitated H1
histones were left on ice for 60 min and then centrifuged at 10,000 rpm
for 10 min, washed twice with cold acidified acetone, and three times
with pure acetone, dissolved in 1 ml of water containing 0.1%
2-mercaptoethanol, lyophilized, and stored at
20 °C until used for
HPLC.
For comparative purposes, human placenta H1 histones were also isolated
by extraction with 0.5 M NaCl as described previously (26).
To remove the low mobility group proteins, NaCl extract was treated
with trichloroacetic acid (2%, w/v). After centrifugation at 10,000 rpm for 15 min, the linker histones were precipitated with 20% (w/v)
trichloroacetic acid and isolated as described above.
High Performance Liquid Chromatography--
The equipment used
consisted of two 114M pumps, a 421A system controller, and a model 165 variable-wavelength UV-visible detector (Beckman Instruments, Palo
Alto, CA). The effluent was monitored at 210 nm, and the peaks were
recorded using Beckman System Gold software. Triethylammonium phosphate
buffers were prepared by adding appropriate amounts of a 1 M stock solution prepared by adding TEA to phosphoric acid
until a pH of 3.0 was reached. Buffer compositions are expressed as
(v/v) throughout this text.
Reversed-phase HPLC--
The separation of whole linker histones
was performed on a Nucleosil 300-5 C4 column (250 × 8 mm inner diameter; 5-µm particle pore size; 30-nm pore size;
end-capped; Machery-Nagel, Düren, Germany). The lyophilized
proteins were dissolved in water containing 200 mM
2-mercaptoethanol, and samples of ~700 µg were injected onto the
column. The histone H1 sample was chromatographed within 40 min at a
constant flow of 1.5 ml/min with a two-step acetonitrile gradient
starting at solvent A/solvent B (63:37) (solvent A, water containing
0.1% trifluoroacetic acid; solvent B, 70% acetonitrile and 0.1%
trifluoroacetic acid). The concentration of solvent B was increased
from 37 to 45% solvent B within 10 min and from 45 to 54% solvent B
within 30 min. The histone H10 fraction was collected, and,
after adding protamine sulfate (20 µg/ml) and 50 µl of
2-mercaptoethanol (0.2 M), lyophilized, and stored at
20 °C.
The peptides obtained by digestion of human placenta H10
histones by chymotrypsin were separated using a Nucleosil 300-5 C18 column (250 × 3 mm inner diameter; 5-µm
particle pore size; end-capped; Macherey-Nagel, Düren, Germany).
Samples of ~100 µg were injected onto the column. Chromatography
was performed within 70 min at a constant flow of 0.35 ml/min with a
two-step acetonitrile gradient starting at solvent A/solvent B (90:10)
(solvent A, water containing 0.1% trifluoroacetic acid; solvent B,
85% acetonitrile and 0.1% trifluoroacetic acid). The concentration of
solvent B was increased linearly from 10 to 45% within 60 min and from
45 to 100% within 10 min. Fractions obtained in this way were
collected and, after adding 20 µl of 2-mercaptoethanol (0.2 M), lyophilized, and stored at
20 °C.
To separate the peptide fractions obtained by cyanogen bromide cleavage
of human placenta H10 histones, samples of 50 µg were
injected onto the column. Samples were chromatographed within 85 min at
a constant flow of 0.35 ml/min with a multi-step acetonitrile gradient
starting at solvent A/solvent B (80:20) (solvent A, water containing
0.1% trifluoroacetic acid; solvent B, 85% acetonitrile and 0.1%
trifluoroacetic acid). The concentration of solvent B was increased
linearly from 20 to 38% within 15 min, from 38 to 52% within 60 min,
and from 52 to 100% within 10 min. Fractions were collected and, after
adding 20 µl of 2-mercaptoethanol (0.2 M), lyophilized,
and stored at
20 °C.
Hydrophilic Interaction Liquid Chromatography--
The histone
fraction H10 (150 µg) isolated by RP-HPLC was analyzed on
a PolyCAT A column (250 × 4.6 mm inner diameter; 5-µm particle
size; 100-nm pore size; ICT, Vienna, Austria) at 23 °C and at a
constant flow of 1.0 ml/min using a multi-step gradient starting at
solvent A/solvent B (100:0) (solvent A, 70% acetonitrile, 0.015 M TEA/H3PO4, pH 3.0; solvent B,
70% acetonitrile, 0.015 M
TEA/H3PO4, pH 3.0, and 0.68 M
NaClO4). The concentration of solvent B was increased from
0 to 65% B within 5 min, from 65 to 100% within 45 min, and then
maintained at 100% within 30 min. The isolated protein fractions were
desalted using RP-HPLC. Histone fractions obtained in this way were
collected and, after adding 20 µl of 2-mercaptoethanol (0.2 M), lyophilized and stored at
20 °C.
The peptide fraction I (~120 µg) obtained by RP-HPLC of
chymotrypsin-digested human H10 histones (Fig. 3) was
analyzed using a two-step gradient starting at solvent A/solvent B
(100:0) (solvent A, 70% acetonitrile, 0.015 M
TEA/H3PO4, pH 3.0; solvent B, 70%
acetonitrile, 0.015 M TEA/H3PO4, pH
3.0, and 0.68 M NaClO4). The concentration of
solvent B was increased from 0 to 65% B within 5 min and from 65 to
100% B within 45 min. The individual HILIC fractions were desalted
using RP-HPLC. The peptide fractions obtained in this way were
collected and, after adding 20 µl of 2-mercaptoethanol (0.2 M), lyophilized and stored at
20 °C.
Chymotrypsin Digestion--
Whole histone H10
(~100 µg) obtained from human placenta by RP-HPLC fractionation
(Fig. 1) was digested with
-chymotrypsin (EC 3.4.21.1) (Sigma type
I-S, 1:150 w/w) in 100 µl of 100 mM sodium acetate
buffer, pH 5.0, for 3-4 h at room temperature. The digest was
subjected to RP-HPLC.
Cyanogen Bromide Cleavage--
Whole histone H10
(~100 µg) obtained from human placenta by RP-HPLC fractionation
(Fig. 1) was dissolved in 50 µl of 70% formic acid containing 10 mg/ml cyanogen bromide (21, 27). The tubes were sealed with Parafilm
and left in the dark at room temperature, with occasional shaking, for
20 h. The solution thus obtained was then immediately analyzed by
RP-HPLC.
Amino Acid Sequence Analysis--
Peptide sequencing was
performed on an Applied Biosystems Inc. (ABI) model 492 Procise protein
sequenator. Sequencer grade solvents were purchased from ABI. To
partially deblock the acetylated N terminus, the histones were
incubated in the cartridge for 3 h at 48 °C using gaseous
trifluoroacetic acid.
Mass Spectrometric Analysis--
Determination of the molecular
masses of the four histone H10 subfractions obtained by the
HILIC run (Fig. 1B) was carried out by electrospray ion-mass
spectrometry technique using a MAT 900 instrument (Finnigan/MAT GmbH,
Bremen, Germany). Samples (5-10 µg) were dissolved in 50% aqueous
methanol containing 0.1% formic acid and injected into ion source.
Matrix-assisted laser desorption ionization coupled with time-of-flight
mass spectrometry (MALDI-TOF-MS) was used for determination of the
molecular masses of the seven peptide fractions obtained by RP-HPLC
separation (Fig. 3) of chymotrypsin-digested human placenta
H10. MALDI-TOF-MS was performed on a KOMPACT MALDI III
(Kratos Analytical, Manchester, UK) linear type mass spectrometer
operating in the positive ion mode of detection. The matrix solution
was prepared by making a saturated solution of
4-hydroxy-
-cyanocinnamic acid with water/acetonitrile (1:2). Sample
preparations were performed as in the following: typically 2 µl of
the matrix stock solution was placed in an Eppendorf tube, and 1 µl
of each sample peptide solution (10 pM/µl) and 1 µl of
ubiquitin solution (10 pM/µl) were added as internal mass
standard. The solution was briefly mixed using vortex stirring. 0.8 µl of the matrix/peptide/ubiquitin mixture was applied onto the
sample slide.
Acid-Urea Gel Electrophoresis--
Polyacrylamide (15%) gel
electrophoresis (16 cm × 18 cm × 0.75 mm) was carried out
in acetic acid-urea, essentially as described by Lennox et
al. (28). The gels were stained for 1 h with 0.1% Coomassie
Blue in 40% (v/v) ethanol, 5% (v/v) acetic acid and destained in 20%
(v/v) ethanol, 5% (v/v) acetic acid.
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RESULTS AND DISCUSSION |
HILIC Separation of Human Placenta Histone H10 into
Four Subfractions--
Perchloric acid-extracted linker histones from
human placenta were fractionated using RP-HPLC with a semi-preparative
column filled with Nucleosil 300-5 C4 and a two-step
water/acetonitrile gradient. The three fractions obtained (Fig.
1A) were characterized by SDS-
and AU-PAGE (data not shown). The histone H10 fraction
eluted at about 19 min as a single peak. By applying a new high
performance capillary electrophoresis method (20, 29), this fraction
was further separated into two major peaks (data not shown). This
result was not further surprising since it is known (15-18) that long
acid-urea-polyacrylamide gels also resolve histone H10 into
two subfractions (designated H10a and
H10b).

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Fig. 1.
HILIC separation of human placental histone
H10. A, perchloric acid-extracted linker
histones (700 µg) from human placenta were injected onto a Nucleosil
300-5 C4 column (250 × 8 mm). The histone H1 was
fractionated by RP-HPLC using a two-step acetonitrile gradient starting
at 63% A, 37% B (solvent A, water containing 0.1% trifluoroacetic
acid; solvent B, 70% acetonitrile and 0.1% trifluoroacetic acid). The
concentration of solvent B was increased from 37 to 45% B within 10 min and from 45 to 54% B within 30 min. The flow rate was 1.5 ml/min.
The protein was monitored at 210 nm. The histone H10
fraction was collected and, after adding protamine sulfate (20 µg/ml)
and 50 µl 2-mercaptoethanol (0.2 M), lyophilized and
stored at 20 °C; B, the histone H10
fraction (~150 µg) isolated by RP-HPLC (A) was analyzed
on a PolyCAT A column (250 × 4.6 mm) at 23 °C at a constant
flow of 1.0 ml/min using a two-step gradient starting at 100% A, 0% B
(solvent A, 70% acetonitrile, 0.015 M
TEA/H3P04, pH 3.0; solvent B, 70%
acetonitrile, 0.015 M TEA/H3P04, pH
3.0, and 0.68 M NaCl04). The concentration of
solvent B was increased from 0 to 65% B in 5 min, from 65 to 100% B
in 45 min, and then maintained at 100% for 30 min. The effluent was
monitored at 210 nm. The isolated protein fractions (designated
1-4) were desalted using RP-HPLC. Histone
fractions obtained were collected and, after adding 20 µl of
2-mercaptoethanol (0.2 M), lyophilized and stored at
20 °C. The samples were used for CE and ESI mass-spectrometric
analysis (data not shown) as well as for AU-PAGE (Fig. 2).
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Excellent separations of modified core and H1 histones, recently
achieved in our laboratory using the HILIC method (23, 24), prompted us
to test this HPLC technique for fractionating histone H10
from human placenta. Therefore, the histone H10 fraction
obtained in the RP-HPLC run (Fig. 1A) was subjected to HILIC
using a PolyCAT A column with a triethylammonium phosphate buffer
system, pH 3.0, in the presence of 70% acetonitrile. As shown in Fig.
1B, five major peaks were found. Since the peak eluting at
about 15 min consists of protamine sulfate, which was generally added
to the linker histone fractions isolated by RP-HPLC in order to
stabilize the proteins (24), peaks 1-4 are due to H10
subfractions. The results obtained with CE and HILIC clearly indicate
that the individual H10 subcomponents differ in both their
charge and hydrophilicity. Furthermore, it is evident that the proteins
of peaks 3 and 4 exhibit a more hydrophilic nature than do the proteins
of peaks 1 and 2.
Characterization of the Four H10 Subfractions Obtained
by HILIC--
To characterize the proteins in Fig. 1B, each
peak was first subjected to AU gel electrophoresis. It was found that
peaks 1 and 2 in Fig. 1B correspond to
the subfraction called H10a and peaks 3 and
4 to that of H10b (shown in Fig.
2). In order to localize the region
responsible for the diversity of the H10 proteins, whole
H10 histone obtained from the RP-HPLC run shown in Fig.
1A was treated with cyanogen bromide. Histone
H10 contains only 1 methionine residue at position 30 and,
therefore, cleavage with cyanogen bromide should produce two peptide
fragments, a larger peptide from residue 31 to the C terminus and a
smaller one originating from the N-terminal H10 domain
(residues 1-30; shown in Table I). In
our experiments, however, we were unable to detect the small peptide
when using RP-HPLC and CE. This finding that the small CNBr peptide is
not detectable agrees well with observations made by other
investigators using gel electrophoresis (16, 30). Both CE and HILIC
analysis of the large C-terminal peptide produced by treatment with
cyanogen bromide and characterized by Edman degradation revealed that
this fragment was homogeneous (data not shown). Based on this result we
assumed that the heterogeneity of H10 is due to differences
within the first 30 residues of the N-terminal H10 region.
In order to obtain peptides containing this N-terminal region we
digested whole H10 histone from human placenta with
chymotrypsin and separated the peptides by means of RP-HPLC. The
fragmentation yielded seven main peptide peaks, as shown in Fig.
3. The purity and homogeneity of the
fractions were assessed by CE (data not shown). Fraction I alone was
non-uniform and resolved into two components. To identify the seven
peptide fractions, amino acid sequencing of the first three amino acids
and MALDI-TOF analysis were performed. The result is shown in Table I.
Thus, it was established that fraction I consists of a peptide
containing the N-terminal 52 residues of histone H10. To
unambiguously confirm our assumption that the N-terminal domain is
responsible for the microheterogeneity of histone H10, we
analyzed fraction I under HILIC conditions. As expected, four
subfractions designated 1' to 4' were obtained (Fig.
4). The chromatogram closely resembled
that obtained by HILIC analysis of undigested H10 (shown in
Fig. 1B).

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Fig. 2.
AU-PAGE of the four histone H10
subfractions obtained by HILIC (Fig. 1B).
Polyacrylamide (15%) gel electrophoresis (16 × 18 × 0.075 cm) was carried out in acetic acid/urea (28). The gel was pre-run
overnight at 50 mA. Samples were electrophoresed at 10 mA (constant
current) for 28 h. Lanes 1 and 6, linker
histone markers (10 µg) from human placenta; lanes 2-5,
H10 histone subfractions 4, 3, 2, and 1, respectively,
obtained from the HILIC run (Fig. 1B). The protein load was
2-4 µg.
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Table I
Chymotryptic peptides of human placenta histone H10 and human
H10 primary structure
The peptide fractions of chymotrypsin-digested human placenta
H10 were separated by RP-HPLC (Fig. 3) and analyzed by both
amino acid sequencing of the first three amino acids and by
MALDI-TOF-MS. Peptide I, amino-terminal sequence (residues 1-52);
peptide II, IKS (53-69); peptide III, SIK (70-80); peptide IV, KQT
(81-92); peptide V, RLA (93-106); and peptide VI, KKT (107-193).
Histone H10 sequence data were taken from human H10
cDNA
(32).
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Fig. 3.
RP-HPLC of peptide fractions of
chymotrypsin-digested human placenta H10. Whole
histone H10 from human placenta was digested with
chymotrypsin as described under "Experimental Procedures." The
digest (containing ~100 µg of peptides) was injected onto a
Nucleosil 300-5 C18 column (250 × 3 mm). Analysis
was performed at a constant flow of 0.35 ml/min using a two-step
acetonitrile gradient starting at 90% A, 10% B (solvent A, water
containing 0.1% trifluoroacetic acid; solvent B, 85% acetonitrile and
0.1% trifluoroacetic acid). The concentration of solvent B was
increased linearly from 10 to 45% (60 min) and from 45 to 100%
(10 min). The effluent was monitored at 210 nm. Peptide fractions
I-VI were analyzed by CE, amino acid sequencing
of the first three amino acids, and by MALDI-TOF-MS (data not shown).
Fraction I was used for HILIC analysis (Fig. 4).
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Fig. 4.
HILIC separation of peptide fraction I
obtained by RP-HPLC of chymotrypsin-digested human H10
histone. The sample (~120 µg) was analyzed on a PolyCAT A
column (250 × 4.6 mm) at 23 °C at a constant flow of 1.0 ml/min using a two-step gradient starting at 100% solvent A, 0%
solvent B (solvent A, 70% acetonitrile, 0.015 M
TEA/H3PO4, pH 3.0; solvent B, 70%
acetonitrile, 0.015 M TEA/H3PO4, pH
3.0, and 0.68 M NaClO4). The concentration of
solvent B was increased from 0 to 65% B(5 min) and from 65 to 100% B
(45 min). The HILIC fractions (designated 1'-4')
were desalted using RP-HPLC. The peptide fractions obtained in this way
were collected and applied on an ABI protein sequencer.
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Sequence analysis performed with the intact H10 proteins
was problematic because of chemical side reactions in the course of Edman degradation, resulting in non-reliable sequence analyses data. We
consequently attempted to determine the amino acid sequence of the
individual HILIC peaks of Fig. 4. Previous reports indicated that the N
terminus of the H10 proteins is blocked, restricting the
determination of primary structure in that region (15, 31). However, as
shown in Table I, we did sequence all RP-HPLC fractions including the
N-terminal fraction I of Fig. 3. We therefore concluded that the
N-terminal residue is blocked in some, however not in all four, histone
H10 fractions obtained by HILIC. This assumption proved to
be true; although sequence determination of the H10
subfractions of HILIC peaks 3' and 4' was unproblematic, it was not
possible to sequence the fractions of peaks 1' and 2'. Thus, the
proteins designated H10a and H10b in Fig. 2 are
H10 histones with N-terminal blocked and N-terminal
unblocked residue, respectively.
Since HILIC resolves H10a and H10b into two
further subfractions each, as shown in Fig. 1B, at least one
additional difference had to exist within their N-terminal domains. In
fact, this difference was found when 33 steps of the Edman degradation
were performed on HILIC peaks 3' and 4' and the resulting amino acid
sequences compared with the primary sequence of human H10
as determined from cDNA cloning (32). The primary structure of
subfraction 3' was identical to that of human H10 having
asparagine in position 3 (henceforth called H10b Asn-3). In
HILIC subfraction 4', however, the third residue was found to be
aspartic acid. We named the corresponding deamidated histone
H10 protein H10b Asp-3. As for the HILIC
subfractions 1' and 2' containing the blocked N-terminal residue, we
assumed that they could also differ by deamidation at position 3. To
confirm this, we partially deblocked the HILIC fractions as described
under "Experimental Procedures" and determined the amino acid
sequences. In fact, subfractions 1' and 2' differed at position
3, subfraction 1' containing asparagine and subfraction 2' aspartic
acid. The corresponding proteins were designated H10a Asn-3
and H10a Asp-3, respectively.
To examine the nature of the blocking residue of the human placenta
H10 histones, we subjected the four H10
subfractions obtained by HILIC to ion-spray mass spectrometric analysis
(data not shown). Significant mass differences were not observed
between the H10a Asn-3 and H10a Asp-3 nor
between the H10b Asn-3 and H10b Asp-3 histones.
This result agrees well with our finding that the Asn-3 forms consist
of intact H10 and the Asp-3 forms of deamidated
H10 histones (mass difference is 1 Da). However, a mass
difference of 43 Da was found between the H10a Asn-3 and
H10b Asn-3 histones as well as between the H10a
Asp-3 and H10b Asp-3 histones. This mass difference
corresponds to the presence (H10a Asn-3 and
H10a Asp-3) and absence (H10b Asn-3 and
H10b Asp-3) of an acetyl group. We assume that the acetyl
group is bound to the N-terminal nitrogen of the H10
histone, thus blocking the Edman degradation of the H10a
Asn-3 and H10a Asp-3 histones. In this context it should be
noted that N-terminal acetylation is a characteristic of H1 histones
(33). However, the occurrence of both the blocked and the unblocked
protein forms appears unique to mammalian histones.
From the results obtained it was possible to compare the gel
electrophoretic behavior of the four H10 subcomponents with
that of H10a and H10b. AU gel electrophoresis
(shown in Fig. 2) revealed that H10a Asn-3 and
H10a Asp-3 (the less positively charged H10
histones; lanes 5 and 4, respectively) migrate
slower than H10b Asn-3 and H10b Asp-3 (the more
positively charged histone H10 proteins; lanes 3 and 2, respectively). It seems likely to us, therefore, that
the H10 subfractions, in the literature originally
designated H10a and H10b (15-17), actually
consist of a mixture of H10a Asn-3 and H10a
Asp-3 and of H10b Asn-3 and H10b Asp-3,
respectively.
Age-dependent Changes in the Proportions of the Four
Forms of H10 Histones--
A further important question
was whether the four histone H10 forms are present only in
human tissue. In order to answer this question we subjected histone
H10 isolated from various tissues (liver and brain) of
mouse and rat to HILIC. Just like the human histone H10,
both the mouse and rat histone H10 were separated into four
subfractions (shown in Fig. 5 for rat histone H10). To identify these subfractions we used the
same analytical procedures as described above for the identification of
human histone H10. We also found here that the HILIC
fractions designated 1 and 2 in Fig. 5 consist of N-terminally blocked
intact (H10a Asn-3) and deamidated (H10a Asp-3)
histone H10 forms, respectively, whereas fractions 3 and 4 are composed of N-terminally unblocked intact (H10b Asn-3)
and deamidated (H10b Asp-3) histones H10,
respectively. We thus detected N-terminally blocked and unblocked H10 histones in all three species. This result is in
contrast to the results of Smith et al. (31) who assumed
that its two H10 subfractions obtained from various tissues
of man, mouse, and rat are N-terminally blocked, just as all the H1
histones.

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Fig. 5.
HILIC analysis of histone H10
from liver and brain of rats of various ages. H10 was
from the livers of rats aged 10 days (A) and 15 months
(B) and the brains of rats aged 10 days (C) and
15 months (D). The H10 fractions (~150 µg)
isolated by RP-HPLC were analyzed on a PolyCAT A column (250 × 4.6 mm). Conditions were the same as for Fig. 1B. 1, H10a Asn-3; 2, H10a Asp-3; 3, H10b
Asn-3; 4, H10b Asp-3.
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An obvious question raised by the present results was whether
N-terminal acetylation and/or deamidation of histone H10 is
a physiologically important process. In this context it should be noted
that previous investigations have shown an age-related change in the
proportion of H10 "subtypes" a and b in rat brain
cortical neurons (17) and, moreover, differing ratios of histones
H10a and H10b according to the tissue examined
(15). We were interested, therefore, in examining the occurrence of the
individual H10 forms prepared from various tissues (liver
and brain) of rats aged 10 and 450 days using our HILIC technique. As
can be seen from Fig. 5, the H10 pattern differs not only
between rat liver and brain (Fig. 5, A and B
compared with C and D) but also between young and
old rat liver (Fig. 5A compared with B) and
between young and old rat brain (Fig. 5C compared with
D). In order to obtain more precise and, in addition,
quantitative data on the age-dependent changes of
N-terminally acetylated and deamidated H10 forms, we
analyzed liver and brain H10 histones from rats aged 10 days, 30 days, 6 months, and 15 months. Fig.
6A reveals that the proportion
of N-terminally acetylated H10 (sum of H10a
Asn-3 and H10a Asp-3) is about 30% higher in senescent (15 months old) rat livers and brains than in young ones (10 days old). It
is worth mentioning, however, that an increase of about 37% in
acetylated histone H10 was already observed in rat brains
of 30-day-old animals. The dramatic age-dependent increase
of the deamidated histone H10 forms (H10a Asp-3
plus H10b Asp-3) is shown in Fig. 6B. The
proportion of deamidated H10 forms was about 7.5-fold
higher in brain and about 3-fold higher in liver of rats 15 months of
age than that in 10-day-old animals.

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Fig. 6.
Age-dependent increase of both
the N-terminally acetylated and the deamidated histone H10
forms. H10 was from the livers and brains of rats aged
10 days, 30 days, 6 months, and 15 months. The H10
fractions (~150 µg) isolated by RP-HPLC were analyzed under the
same HILIC conditions used in Fig. 5. The amount of each
H10 form was quantified using a Beckman System Gold
software. A, age-dependent increase of the
N-terminally acetylated H10 forms (H10a Asn-3 + H10a Asp-3); B, age-dependent
increase of the deamidated H10 forms (H10a
Asp-3 + H10b Asp-3). Results represent means ± S.D.
for three to five independent experiments.
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N-terminal acetylation of proteins is a common modification among
eukaryotic proteins (34, 35). Although the structural or functional
significance of N-terminal acetylation is unknown, it seems likely that
the N terminus of a protein has a major effect on the regulation of
protein metabolism (36) or on the rate of degradation (34-36). The
N-terminal acetylation of a protein is usually complete (34). However,
we found the N-terminally acetylated and unacetylated forms to occur
simultaneously for the H10 histones. Regarding the linker
histones, this kind of uncompleted acetylation of N-terminal residues
seems to be limited to histone H10. For this reason, it is
conceivable that this modification serves as a histone
H10-specific molecular timer of histone turnover. It would
be of interest, therefore, to explore further the biological
consequences of a blocked and an unblocked N terminus of
H10 histones.
Protein deamidation is a well documented nonenzymatic process (37, 38).
Some studies have shown an age-dependent increase of
deamidation of proteins that turn over little or not at all during the
lifetime of the organism (39, 40). This accumulation of deamidated
forms of proteins possibly results from a decrease in the rate at which
deamidated proteins degrade with age (41). The fate of deamidated
asparaginyl residues in proteins is unknown. Robinson et al.
(42) suggested that deamidation of proteins could serve as a molecular
timer determining the lifetime of proteins. In a few cases the
deamidation of specific asparagine residues has been connected with
changes in protein function (43). Although deamidation caused by a
deamidase was also described most recently (44, 45), in this study we
did not find any evidence of such an enzymatic deamidation.
It should be noted that nonenzymatic deamidation is known to occur also
in vitro during the isolation procedure and the handling and
storage of peptides and proteins (46). It was important, therefore, to
make sure that the described alterations in histone H10
forms are indeed the result of in vivo aging in the tissues. Despite the fact that all procedures for isolation and handling of
proteins were carefully performed under identical conditions, the level
of deamidated histone H10 forms shows an
age-dependent increase in the tissues investigated. Furthermore, neither the gentle extraction of linker histones using 0.5 M NaCl instead of 5% perchloric acid nor prolonged
standing of the histones precipitated with 20% trichloroacetic acid
(overnight instead of only 60 min) resulted in detectable changes in
the amounts of deamidated forms (data not shown). It can thus be
concluded that the deamidation events observed are actually in
vivo processes in aging organs. This study, therefore, provides
the first evidence of in vivo deamidation in linker histone
proteins.
Whether the age-dependent increase of deamidated and/or
N-terminally acetylated histone H10 forms is responsible
for the age-related decrease in transcriptional activity found in
various mammalian tissues including the brain (47-49) is unknown at
present. It is reported that this decline of transcriptional activity
is primarily due to changes in the chromatin structures (49, 50). Both
the N-terminal acetylation and the deamidation of Asn-3 reduce the
positive charge of the N-terminal domain of histone H10. It
is conceivable, therefore, that the age-related changes in N-terminal
acetylation and deamidation may directly affect the interaction of
H10 proteins with DNA. This effect might be one of the
reasons for the structural changes in chromatin observed in aging
tissues.
We acknowledge Finnigan MAT GmbH
(Bremen, Germany) and Dr. Karl-Hans Ongania for performing
mass spectrometric measurements. We express our appreciation to
Shimadzu (Vienna) for lending a mass spectrometer KOMPACT MALDI III. We
thank R. Berberich and A. Devich for their excellent technical
assistance.