Báo cáo Y học: Structural and biochemical characterization of calhepatin, an S100-like calcium-binding protein from the liver of lungfish (Lepidosiren paradoxa) docx

Structural and biochemical characterization of calhepatin,
an S100-like calcium-binding protein from the liver of lungfish
(
Lepidosiren paradoxa
)
Santiago M. Di Pietro and Jose
´
A. Santome
´
Instituto de Quı
´
mica y Fisicoquı
´
mica Biolo
´
gicas (IQUIFIB), Facultad de Farmacia y Bioquı
´
mica, Universidad de Buenos Aires,
Argentina
We report the biochemical characterization of calhepatin, a
calcium-binding protein of the S100 family, isolated from
lungfish (Lepidosiren paradoxa) liver. The primary struc-
ture, determined by Edman degradation and MS/MS,
shows that the sequence identities with the other members
of the family are lower than those between S100 proteins
from different species. Calhepatin is composed of 75
residuesandhasamolecularmassof8670Da.Itissmaller
than calbindin D
9k
(78 residues), the smallest S100 des-
cribed so far. Sequence analysis and molecular modelling

predict the two EF-hand motifs characteristic of the
S100 family. Metal-binding properties were studied by a
direct
45
Ca
2+
-binding assay and by fluorescence titration.
Calhepatin binds Ca
2+
and Cu
2+
but not Zn
2+
.Cu
2+
binding does not change the affinity of calhepatin for
Ca
2+
. Calhepatin undergoes a conformational change
upon Ca
2+
binding as shown by the increase in its intrinsic
fluorescence intensity and k
max
, the decrease in the
apo-calhepatin hydrodynamic volume, and the Ca
2+
-
dependent binding of the protein to phenyl-Superose. Like
most S100 proteins, calhepatin tends to form noncova-

lently associated dimers. These data suggest that calhepatin
is probably involved in Ca
2+
-signal transduction.
Keywords: calcium-binding protein; EF-hand; liver; lungfish;
S100.
Cytoplasmic Ca
2+
is a ubiquitous second messenger. The
rise in intracellular Ca
2+
is a widely established signal
controlling a variety of processes in eukaryotic cells, such as
cell growth and differentiation, cell motility, muscle con-
traction, gene expression, secretion, nerve impulse trans-
mission, and apoptosis. The signal is partly transduced into
metabolic or mechanical responses by calcium-binding
proteins (CaBPs) which interact with cellular effectors in a
Ca
2+
-dependent fashion [1].
S100 is a multigenic family of small dimeric CaBPs
(78–119 amino acid residues) of the EF-hand superfamily,
comprising 16 known members from mammalian species
(S100 A1 to S100 A13, S100 B, Calbindin D
9k
,S100P)and
two putative additional members identified in chicken and
channel catfish (MRP126 and ictacalcin, respectively).
They have two EF-hand Ca

2+
-binding motifs, the
N-terminal one having an extended loop characteristic of
the S100 family [1–5]. S100 proteins show tissue-specific
and cell-specific expression [4]. Some members of the
family also bind Zn
2+
and/or Cu
2+
[5]. Most S100
proteins can form noncovalent dimers by a symmetric
homodimeric fold mediated by hydrophobic contacts not
found in other CaBPs [5–7]. Some S100 proteins form
disulfide cross-linked homodimers [5]. The location of
target protein-binding sites on opposite sides of the S100
homodimers could allow an S100 dimer to cross-bridge
two homologous or heterologous S100 target proteins [2].
Calbindin D
9k
is the only S100 protein identified so far that
does not form dimers [2,5–8].
We previously identified S100 A8 and S100 A9 from pig
granulocytes [9] and discovered another member of the S100
family, the S100 A12 [10]. Hofmann et al. [11] proved that
S100 A12, and possibly other members of the S100 family,
mediates the activation of a novel proinflammatory axis by
binding to RAGE (receptor for advanced glycation end
products), a cell surface receptor. In this work, we focus on
the characterization of a CaBP from lungfish (Lepidosiren
paradoxa) liver, an S100 protein that apparently does not

belong to any known S100 member. Its isolation, primary
structure, metal-binding properties, and tissue expression
pattern are described. As it is expressed mainly in hepatic cells
andnootherS100memberhasbeenreportedinliver,this
protein will be referred to as calhepatin.
MATERIALS AND METHODS
Materials
45
CaCl
2
(12.89 CiÆg
)1
) was from Dupont NEN. Endopro-
teinases Glu-C and Lys-C were obtained from Promega.
All other reagents were purchased from Sigma, Baker,
Bio-Rad, Amersham Pharmacia Biotech and/or Applied
Biosystems.
Correspondence to J. A. Santome
´
,IQUIFIB,Facultadde
Farmacia y Bioquı
´
mica, UBA, Junı
´
n 956, Buenos Aires (1113),
Argentina. Fax: + 54 11 4508 3652, Tel.: + 54 11 4508 3651,
E-mail: santome@qb.ffyb.uba.ar
Abbreviations: CaBP, calcium-binding protein; MALDI-TOF,
matrix-assisted laser desorption ionization time-of-flight.
Note: The amino-acid sequence reported in this work has been

deposited in the SWISS-PROT databank under accession number
P82978.
(Received 21 March 2002, accepted 24 May 2002)
Eur. J. Biochem. 269, 3433–3441 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03023.x
Preparation of the lungfish liver cytosolic fraction
and purification of calhepatin
Livers were excised from L. paradoxa weighing 600–700 g,
cut into small pieces, suspended in homogenization buffer
(40 m
M
sodium phosphate, 150 m
M
KCl, 4 m
M
EDTA,
pH 7.4), and disrupted in a glass/Teflon homogenizer. The
homogenate was then centrifuged at 20 000 g for 15 min,
and the resulting supernatant further centrifuged at
105 000 g for 90 min in a Beckman XL-90 ultracentrifuge.
The entire procedure was carried out at 4 °C. The superna-
tant (4 mL) was loaded on a Sephadex G-75 column
(2.5 · 40 cm) equilibrated with 15 m
M
Tris/HCl (pH 9.0)/
1m
M
EDTA. Elution was performed at 4 °Cwiththesame
buffer at a flow rate of 16 mLÆh
)1
. The 6–18-kDa fraction

was applied to a DEAE-cellulose column (1.1 · 10 cm)
equilibrated with 15 m
M
Tris/HCl(pH9.0).Thematerial
bound to the column was subsequently eluted with 10, 20,
30, 40, 50 and 100 m
M
NaCl in the same buffer. A portion of
each fraction was concentrated and changed into 50 m
M
Tris/HCl buffer (pH 7.4) by using a CentriprepÒ concen-
trator (Amicon) and assayed for
45
Ca
2+
-binding activity as
described below. The 10 m
M
fraction containing the calhep-
atin was concentrated by using the above concentrator and
loaded on a Mono Q HR 5/5 column (Pharmacia LKB)
previously equilibrated with 15 m
M
Tris/HCl (pH 9.0). The
column was developed on an FPLC system (Pharmacia
LKB), at a flow rate of 0.8 mLÆmin
)1
, with a 0–100 m
M
linear gradient of NaCl concentration over 60 min. Calhep-

atin was eluted at % 40 m
M
NaCl. Protein purity was
checked by SDS/PAGE (16% gel), isoelectric focusing, and
RP-HPLC in a Vydac C
4
column (4.6 · 250 mm).
Preparation of the cytosolic fraction from lungfish
and rat tissues
Tissues were cut into small pieces, suspended in 40 m
M
sodium phosphate (pH 7.4) containing 150 m
M
KCl, 4 m
M
EDTA and 4 m
M
dithiothreitol, and homogenized in a
Teflon Potter homogenizer. Homogenates were then cen-
trifuged at 20 000 g for 15 min, and the resulting super-
natants further centrifuged at 105 000 g for 90 min in a
Beckman XL-90 ultracentrifuge.
Electrophoresis
SDS/PAGE (16% gel) was carried out as described by
Scha
¨
gger & von Jagow [12]. Isoelectric focusing was
performed in a Phast System (Pharmacia).
Antiserum production
Calhepatin (500 lg) was mixed with Freund’s adjuvant and

injected subcutaneously into a rabbit (first immunization),
followed by a 250-lg boost 3 weeks later (second immun-
ization). After 3 weeks, the animal was bled from the
marginal ear vein and the serum was obtained. The
antibodies were purified using conventional methods invol-
ving ammonium sulfate precipitation, DEAE-cellulose
chromatography, and gel filtration on a Superdex 200
column. They were then concentrated with the Centriprep
concentrator up to a concentration of 10 mgÆmL
)1
and
stored at )40 °C.
Western blotting and immunoprecipitation experiments
Western blotting was carried out as described by Harlow &
Lane [13]. Immunoprecipitation was performed at 4 °C
using protein A–Sepharose beads [13].
Chromatographic analysis
Gel-filtration analysis of pure calhepatin was performed as
described by Drohat et al. [7] by FPLC on a Superose 12
HR 10/30 column (Pharmacia) calibrated with standard
proteins. The column was equilibrated and eluted with
50 m
M
Tris/HCl/120 m
M
KCl/0.1 m
M
EDTA (pH 7.4) for
the apo-calhepatin or with 50 m
M

Tris/HCl/120 m
M
KCl/2 m
M
CaCl
2
(pH 7.4) for the holo-protein, at a flow
rate of 0.5 mLÆmin
)1
. Both protein forms (7 l
M
monomer
concentration) were incubated in the corresponding equili-
bration buffer for 30 min before being loaded. The mono-
meric and dimeric fractions obtained from the Superose
column were incubated at room temperature for 12 h and
applied again to the gel-filtration column under the same
buffer and flow rate conditions.
Hydrophobic interaction chromatography was carried
out on a phenyl-Superose HR 5/5 column (Pharmacia)
equilibrated with 50 m
M
Tris/HCl/120 m
M
KCl/1 m
M
CaCl
2
(pH 7.4). After injection of 30 lgpureproteinon
the equilibration buffer, the column was eluted with

4 column vol. of the same buffer and then with 4 column
vol. of 50 m
M
Tris/HCl/120 m
M
KCl/5 m
M
EDTA
(pH 7.4).
Enzymatic digestion and peptide purification
For Glu-C protease digestion, 250 lg calhepatin was
incubated in 0.1
M
Tris/HCl (pH 7.9)/2
M
guanidine
hydrochloride with 4 lg enzyme, at 20 °C for 24 h. For
Lys-C protease digestion, 250 lg calhepatin was incubated
in 0.1
M
Tris/HCl (pH 8.5)/2
M
guanidine hydrochloride
with 5 lg enzyme, at 20 °C for 24 h. Peptides were
separated by RP-HPLC (Pharmacia LKB) on a Vydac
C
18
column (4.6 · 250 mm) equilibrated with solvent A
[0.1% (v/v) trifluoroacetic acid in water]. Elution was
performed at a flow rate of 0.8 mLÆmin

)1
with a 0–50%
linear gradient of solvent B [80% (v/v) acetonitrile, 0.08%
(v/v) trifluoroacetic acid] over 80 min.
Amino-acid analysis and sequencing
Peptide amino-acid analyses and automatic amino-acid
sequence determination by Edman degradation were carried
out in an Applied Biosystems 420A Amino Acid Analyzer
and an Applied Biosystems 477A Protein Sequencer,
respectively, at the LANAIS-PRO (National Protein Sequen-
cing Facility, UBA-CONICET), Buenos Aires, Argentina.
Amino-acid sequence determination of the N-terminal
peptide by MS/MS was performed in an Electrospray
Ionization Ion Trap (Finnigan LCQ) at the Harvard
University Microchemistry Facility, Cambridge, MA, USA.
In-gel digestion and peptide purification
Isolated calhepatin from lungfish intestine was digested
with sequencing-grade trypsin by the in-gel procedure of
3434 S. M. Di Pietro and J. A. Santome
´
(Eur. J. Biochem. 269) Ó FEBS 2002
Rosenfeld et al. [14], as modified by Hellman et al.[15].The
resulting peptides were recovered by passive elution and
then separated by HPLC on a Brownlee
TM
Aquapore RP
300 C
18
column (2.1 · 220 mm) by using a combination of
linear gradients of acetonitrile in 0.1% aqueous trifluoro-

acetic acid.
Sequence alignment
Multiple sequence alignment was performed by using
MUL-
TALIN
( />/NPSA/npsa_multalin.html) with a BLOSUM 62 matrix
and parameter default values.
Evolutionary tree
It was constructed with
CLUSTAL W
(http:/www.ebi.ac.uk/
clustalw/) by the Neighbour Joining method and using
Kimura’s distance correction procedure.
Mass spectrometry
The molecular mass of lungfish calhepatin was deter-
mined with a Bruker Biflex III matrix-assisted laser
desorption ionization time-of-flight (MALDI-TOF) mass
spectrometer.
Molecular modelling
The three-dimensional structure of lungfish calhepatin was
modelled using
SWISS
-
MODEL
, an automated modelling
package of the ExPASy Molecular Biology Server, available
through the internet ( />SWISS-MODEL.html) [16]. Calhepatin was modelled with
the
PROMOD
program on the basis of its similarity to

homologous structures existing in the Brookhaven Protein
Data Bank. After primary modelling, the structure was
energy-minimized using
GROMOS
. The model was checked
with the
WHAT
-
IF
program which utilizes
WHAT
-
CHECK
verification routines.
45
Ca
2+
-Binding assay
Apo-(lungfish calhepatin) was prepared by incubation of
freshly purified protein with 2 m
M
EGTA and 2 m
M
EDTA
and subsequent dialysis against 50 m
M
Tris/HCl (pH 7.4).
45
Ca
2+

binding was determined by the method of Mani and
Kay[77]using15l
M
apo-(lungfish calhepatin) as described
by Dell’Angelica et al. [10]. Binding data were analyzed by
nonlinear regression curve fitting using the following
equation, where v is the number of moles of Ca
2+
bound
per mol of monomer, x is the free Ca
2+
concentration, n is
the number of binding sites per monomer, and K
a1
and K
a2
are macroscopic binding constants:
 ¼½ðn=2ÞK
a1
x þ nK
a1
K
a2
x
2
=ð1 þ K
a1
x þ K
a1
K

a2
x
2
Þ
Fluorescence measurements
The intrinsic fluorescence of 2 l
M
calhepatin was recorded
at 20 °C on a Jasco FP-770 spectrofluorimeter (Japan
Spectroscopic Co., Hachioji City, Japan). The excitation
wavelength was set to 278 ± 5 nm. Each spectrum (285–
420 nm) represents an average of three scans. The fluores-
cence intensity was corrected for sample dilution, the latter
never exceeding 4%. Curve fitting was performed as
described by Dell’Angelica et al.[10].Datawereanalyzed
by the following equation where F
0
is the fluorescence at
zero ligand concentration, F
m
is the maximum fluorescence
change, T is the total ligand concentration, P is the protein
monomer concentration, and K
a
is the apparent association
constant:
F ¼ F
0
þ F
m

ð2PÞ
À1
 T þ P þ K
À1
a
À½ðT þ P þ K
À1
a
Þ
2
À 4PT 
1=2
no
The Cu
2+
-binding-induced fluorescence change was
corrected for nonspecific quenching by subtracting the
values of a linear term obtained from the final portion of the
Cu
2+
-binding curve, corresponding to fluorescence quench-
ing before binding saturation. Uncorrected Cu
2+
fluores-
cence quenching data were also analyzed by direct fitting to
an equation containing an additional linear term, but the
value obtained for the association constant was indistin-
guishable from that obtained with corrected data.
RESULTS
Purification of lungfish calhepatin

The 105 000 g supernatant from lungfish liver was frac-
tionated on a Sephadex G-75 column. The 6- to 18-kDa
fraction containing the calhepatin was applied to a DEAE-
cellulose column, and the 10 m
M
NaCl fraction containing
Ca
2+
-binding activity was further purified by anion-
exchange chromatography on a Mono Q column. The
protein was eluted at 40 m
M
NaCl from the last column in a
symmetric peak that was homogeneous as confirmed by
SDS/PAGE (Fig. 1), isoelectric focusing and RP-HPLC
(not shown).
Fig. 1. SDS/PAGE analysis of calhepatin-containing samples at
different stages of purification. Lane 1, 105 000 g supernatant of
lungfish liver homogenate; lane 2, after Sephadex G-75 gel filtration;
lane 3, after DEAE-cellulose chromatography; lane 4, after Mono Q
chromatography.
Ó FEBS 2002 Hepatic S100 calcium-binding protein (Eur. J. Biochem. 269) 3435
Biochemical properties of lungfish calhepatin
The protein migrates on SDS/PAGE as a 6-kDa polypep-
tide (Fig. 1) showing an aberrant mobility, in the same way
as other CaBPs [10,18–20]. The molecular mass as deter-
mined by MALDI-TOF MS is 8672 Da.
Analysis of the apo-calhepatin, at 7 l
M
monomer

concentration, by Superose 12 gel filtration showed two
peaks of apparent molecular mass 6.1 ± 0.1 and
16.8 ± 0.1 kDa (mean ± SD), respectively, showing that
the protein exists as both a monomer (15%) and a dimer
(85%). Taking into account the chromatography time scale
(% 25 min) the existence of the two well-separated forms
suggests a very slow monomer–dimer equilibrium. To
confirm that there is an equilibrium between the two forms,
the monomeric (0.3 l
M
monomer concentration) and
dimeric (1.1 l
M
monomer concentration) fractions obtained
from the Superose column were incubated for 12 h and
applied again to the gel-filtration column. In both cases, the
two forms were obtained again and the ratio of monomer to
dimer fraction was % 60 : 40 and 40 : 60, respectively,
indicating that the dissociation constant is in the micro-
molar order. When the same determination was performed
with the holo-calhepatin, at 7 l
M
monomer concentration,
almost 100% of the protein was recovered as a dimer of
apparent molecular mass 14.6 ± 0.2 kDa. The holodimeric
fraction obtained from the Superose column (0.9 l
M
monomer concentration) was incubated for 12 h and
applied again to the gel-filtration column. Almost 100%
of the protein was again recovered as a dimer, indicating

that the monomer-dimer dissociation constant for the
holoprotein is in the submicromolar range. On the other
hand, differences between the apo-calhepatin and holo-
calhepatin hydrodynamic volume (16.8 ± 0.1 kDa and
14.6 ± 0.2 kDa, respectively) suggest that calhepatin
undergoes a conformational change on Ca
2+
binding.
Ca
2+
binding affected the chromatographic behaviour of
calhepatin on a phenyl-Superose column. The protein was
completely bound to the column in the presence of 1 m
M
CaCl
2
but could be eluted from the column with 5 m
M
EDTA (not shown).
Primary structure of lungfish calhepatin
Purified protein (250 pmol) was subjected to four cycles of
Edman degradation. No phenylthiohydantoin derivative
could be identified, indicating that its N-terminal amino
acid is blocked. Calhepatin fragments were generated by
digestion with proteases Glu-C and Lys-C, fractionated by
RP-HPLC (Fig. 2A,B) and submitted to Edman degra-
dation and amino-acid analysis. Information on the
complete amino-acid sequence except for the four -
N-terminal residues was obtained. According to the
amino-acid determination and the blocked N-terminal

residue of the peptide, peak 1 from Lys-C digestion
corresponds to the N-terminal portion of lungfish calhep-
atin. The material corresponding to this peak was
submitted to sequencing by MS/MS. A summary of the
sequence analyses and the resulting primary structure of
lungfish calhepatin is shown in Fig. 3.
The protein is composed of 75 residues. From its amino-
acid sequence, assuming that the N-terminus is an acetyl
group, the molecular mass was calculated to be 8670 Da.
This value is close to that obtained by MALDI-TOF MS
(8672 Da). The calculated isoelectric point [21] (pI ¼ 5.12)
agrees with the experimental value (pI ¼ 5.15 for both the
holo and apo protein).
Sequence comparison and evolutionary relationship
The alignment of the amino-acid sequence of lungfish
calhepatin with other members of the S100 family indicates
that the number of amino-acid identities between calhepatin
and other S100 proteins ranges from 12 to 21 (Fig. 3). These
values are far lower than those between S100 proteins from
different species. This is strong evidence that calhepatin is a
novel S100 protein, as shown in the evolutionary tree of the
S100 family in Fig. 4.
Despite the above evolutionary relationships, according
to the
BLASTP
program [22], the higher similarity when the
calhepatin amino-acid sequence is compared with all
database proteins corresponds to one or more segments of
CaBPswithahighermolecularmassthanthatofS100
proteins. Identity can reach 41%.

Structural modelling of lungfish calhepatin
The three-dimensional structure of the lungfish calhepatin
monomer was predicted by using the
SWISS
-
MODEL
model-
ling package [16] (Fig. 5). Molecular modelling of the
Fig. 2. RP-HPLC separation of calhepatin peptides generated by
enzymatic digestion. (A) The peptide mixture obtained by Glu-C
digestion was fractionated on a Vydac C
18
column (4.6 · 250 mm)
equilibrated with solvent A [0.1% (v/v) trifluoroacetic acid in water].
The column was eluted with a 0–50% linear gradient (dashed line) of
solvent B [80% (v/v) acetonitrile, 0.08% (v/v) trifluoroacetic acid].
(B) The products of Lys-C digestion were separated as described
for the Glu-C peptide mixture. Numbered peaks represent peptides
submitted to sequencing and/or amino-acid analysis.
3436 S. M. Di Pietro and J. A. Santome
´
(Eur. J. Biochem. 269) Ó FEBS 2002
sequence was conducted with the
PROMOD
program using
the known three-dimensional structures of other members
of the S100 family as templates. The lungfish calhepatin
monomer model has the same overall conformation as that
of other members of the S100 family containing four a helix
segments (Fig. 5A). Calhepatin residues present in sequence

positions equivalent to residues crucial for dimerization and
monomer stability, in Sl00 A4 and other S100 proteins [23]
(Fig. 3), are clustered between helix I and IV (Fig. 5B), in
the same way as in Sl00 A4 and other S100 proteins [23].
This agrees with biochemical data showing that calhepatin
is able to dimerize.
Fluorescence titration
The intrinsic emission spectrum of calhepatin and those
of the protein with increasing amounts of Ca
2+
are shown
in Fig. 6A. Figure 6B displays the corrected maximum
of fluorescence intensity for each Ca
2+
concentration
and allows detection of one binding site with K
a(app)
¼
(3.6 ± 0.5) · 10
5
M
(mean ± SD, n ¼ 3).
Intrinsic fluorescence determinations were also applied to
study the binding of Zn
2+
,Mg
2+
and Cu
2+
. Neither Zn

2+
nor Mg
2+
changes calhepatin fluorescence, suggesting that
they have no binding sites in the protein. In addition, they
have no effect on Ca
2+
binding (not shown). Calhepatin
fluorescence intensity decreased, and k
max
changed with
Cu
2+
additions (Fig. 7A). The analysis of the corrected
maximum of fluorescence (Fig. 7B) provides evidence of the
presence of a single site with K
a(app)
¼ (1.5 ± 0.2) · 10
7
M
(mean ± SD, n ¼ 3).
Direct Ca
2+
-binding studies
The
45
Ca
2+
-binding isotherm of calhepatin at 20 °Cin
25 m

M
Tris/HCl (pH 7.4) is shown in Fig. 8. The binding
constants determined are K
a1
¼ (2.9 ± 0.3) · 10
5
M
and
K
a2
¼ (6.0 ± 0.7) · 10
3
M
(n ¼ 2.1 ± 0.05). In the presence
of 1 m
M
Cu
2+
, binding constants are K
a1
¼ (2.0 ±
0.3) · 10
5
M
and K
a2
¼ (4.6 ± 0.6) · 10
3
M
(n ¼ 2.2 ±

0.2), thus Cu
2+
binding does not significantly change
the affinity of calhepatin for Ca
2+
(values are all mean ±
SD; n ¼ 3).
Tissue expression of calhepatin
To investigate the pattern of calhepatin expression in
lungfish tissues, cytosolic fractions from liver, skeletal
muscle, intestine, lung, brain, adipose tissue, heart and
skin were submitted to electrophoresis and immunoblot-
ting. Rabbit antibodies to calhepatin only detected the
protein in liver and at a much lower level in intestine
(Fig. 9). Furthermore, the antibodies did not cross-react
with cytosolic proteins from rat liver or intestinal tissues
(Fig. 9), suggesting that calhepatin-like proteins are not
expressed in rat and that the antibodies do not
cross-react with calbindin D
9k
. Consistently with the
immunoblotting results, when cytosolic fractions from
lungfish and rat tissues were submitted to immunopre-
cipitation experiments with the calhepatin antibodies
followed by SDS/PAGE analysis, only lungfish liver
and intestine showed the calhepatin band (data not
shown).
Fig. 3. Primary structure of calhepatin and its sequence alignment with S100 family members. Peptide fragments are indicated by solid arrows when
determined by sequencing, and by a dotted line arrow for those inferred on the basis of their amino-acid analysis. Each peptide is labelled with a
letter (E for peptides derived by Glu-C digestion and K for peptides obtained by Lys-C digestion) and a number that agrees with that of Fig. 2.

Numbers above the sequence indicate residue positions in the protein. aaaaa correspond to predicted a helices. Underlined residues are equivalent
to the residues of S100 A4 critical for S100 A4 dimerization [23]. The amino-acid sequence of calhepatin was aligned with those of the human form
of each S100 protein, except for MRP126 and ictacalcin, which have only been isolated from chicken and catfish, respectively. The number
of identities between each S100 protein and calhepatin is indicated after each amino-acid sequence. Both canonical (C) and noncanonical (NC)
EF-hands are also indicated.
Ó FEBS 2002 Hepatic S100 calcium-binding protein (Eur. J. Biochem. 269) 3437
To confirm that the lungfish intestinal protein recognized
by the antibodies is calhepatin, it was submitted to in-gel
tryptic digestion by the procedure of Rosenfeld et al.[14].
The peptide mixture was fractionated by RP-HPLC, and
the two peptides sequenced (SGTLSVDELY and IIEK)
were found to be identical with those corresponding to
calhepatin fragments 19–28 and 46–49, respectively.
DISCUSSION
The Ca
2+
signal is transduced by a variety of CaBPs.
Whereas a number of Ca
2+
-dependent responses are
mediated by calmodulin, a ubiquitous CaBP universally
present in cells, the S100 proteins are cell-type-specific
mediators of the Ca
2+
signal [3]. Kligman & Hilt [3]
described the structural features that determine whether a
protein is a member of the S100 family. They have two
EF-hands per monomer. One of them, located in the
C-terminal region, comprises 12 amino-acid residues and is
similar to those found in calmodulin. The other differs from

the calmodulin-related protein EF-hand as it contains 14
residues. The N-terminal and C-terminal regions contain
conserved hydrophobic amino-acid domains. The CaBP
reported here, calhepatin, shares all these structural
characteristics.
Members of the S100 family are acidic CaBPs comprising
between 78 (calbindin D
9k
) and 119 (MRP126) residues,
whereas calhepatin consists of 75 residues, this being the
smallest S100 protein reported. As far as we know, this is the
first time that an S100 has been described in liver.
Calhepatin probably has specific functions in this organ
taking into account that most S100 proteins are often
expressed in a tissue-specific manner [3–5,24] and calhepatin
is expressed almost exclusively in liver.
In the evolutionary tree of the S100 family (Fig. 4),
calhepatin appears as a new member, calbindin D
9k
being
the most closely related to it. The two S100 proteins share
the characteristic of having a low number of residues,
although divergence between their genes seems to have
occurred long ago. The lack of cross-reaction between the
calhepatin antibodies and rat intestinal calbindin D
9k
agrees
with their gene divergence. Interestingly, calbindin D
9k
is the

Fig. 4. Evolutionary tree of the S100 protein family. Unrooted evolu-
tionary tree based on the multiple sequence alignment of 59 S100
protein primary structures constructed with
CLUSTAL W
(http:/
www.ebi.ac.uk/clustalw/) by the Neighbour Joining method and using
the Kimura correction of distances. hu, human (Homo sapiens);
ca, catfish (Ictalurus punctatus); ra, rat (Rattus norvegicus); bo, bovine
(Bos taurus); mo, mouse (Mus musculus); rb, rabitt (Oryctolagus
cuniculus); ch, chiken (Gallus gallus); ho, horse (Equus caballus);
lf, lungfish (Lepidosiren paradoxa); pi, pig (Sus scrofa).
Fig. 5. Structural modelling of calhepatin
monomer. (A) Three-dimensional structure of
lungfish calhepatin predicted using the Swiss-
Model automated modelling package based
on the crystal and/or NMR structures of other
members of the family. The molecule is
depicted in strand representation, and a helices
are numbered from I to IV. (B) Residues Leu7,
Arg8 and Phe11 from a-helix I, and Trp60,
Phe63, Ala66 and Phe67 from a helix IV
(located at equivalent positions to those cru-
cial for S100 A4 dimerization and monomer
stability [23]) are shown in dark and light grey
representation, respectively. The figure was
generated using the
RASMOL
program.
3438 S. M. Di Pietro and J. A. Santome
´

(Eur. J. Biochem. 269) Ó FEBS 2002
only family member identified so far that does not form
dimers, acting as a Ca
2+
modulator, rather than as a Ca
2+
sensor [25]. Analysis of apo-calhepatin and holo-calhepatin
by Superose 12 gel filtration showed that the protein is in a
monomer–dimer equilibrium and that the dissociation
constant is in the micromolar range for the apoprotein
and in the submicromolar range for the holoprotein, as
reported for other S100 family members [7]. Tarabykina
et al. [23] studied crucial residues for dimerization in Sl00A4
and found that three residues in helix I and four in helix IV
are critical for S100A4 dimerization. Figure 3 shows these
residues and those present in equivalent positions in the
other S100 proteins. Most of the critical residues are present
in calhepatin and calbindin D
9k
. However, the latter has a
shorter helix IV, a characteristic that could explain its
inability to dimerize.
Kligman & Hilt [3] proposed that the interaction of a
particular S100 protein with an effector protein occurs
after Ca
2+
binding induces a conformational change,
exposing hydrophobic domains which then interact
with corresponding hydrophobic domains in the effector.
According to this currently accepted mechanism [2,26],

calhepatin should undergo the Ca
2+
-dependent con-
formational changes responsible for the transmission of
information to effector proteins. Our fluorescence experi-
ments indicate a Ca
2+
-induced change in the environment
of at least the tryptophan residue located in one of the
Fig. 6. Ca
2+
fluorescence titration. (A) Fluorescence spectra of 2 l
M
calhepatin in 25 m
M
Tris/HCl, pH 7.4, with 0–810 l
M
Ca
2+
.(B)Ca
2+
titration curve showing corrected maximum fluorescence at each Ca
2+
concentration. Curve fitting was performed as indicated in Materials
and methods.
Fig. 7. Cu
2+
fluorescence titration. (A) Fluorescence spectra of
2 l
M

calhepatin in 25 m
M
Tris/HCl, pH 7.4, with 0–100 l
M
Cu
2+
.
(B) Cu
2+
titration curve showing corrected maximum fluorescence
at each Cu
2+
concentration. Curve fitting was performed as indicated
in Materials and methods.
Fig. 8.
45
Ca
2+
-Binding isotherms.
45
Ca
2+
binding to 15 l
M
calhepatin
in 25 m
M
Tris/HCl, pH 7.4, was determined by the method of Mani &
Kay [17] following the procedure of Dell’Angelica et al.[10].Curve
fitting was performed as indicated in Materials and Methods.

Ó FEBS 2002 Hepatic S100 calcium-binding protein (Eur. J. Biochem. 269) 3439
four positions critical for dimerization in helix IV (Figs 3
and 5). The binding of Ca
2+
to calhepatin increases its
intrinsic fluorescence intensity and k
max
. This result, the
decrease in the protein hydrodynamic volume, and the fact
that Ca
2+
-loaded calhepatin is retained on a phenyl-
Superose column and can be eluted with EDTA suggests
that calhepatin undergoes a conformational change on
Ca
2+
binding that exposes hydrophobic regions. This
probably involves residues shown in spacefill representa-
tion in Fig. 5B. Unfortunately, although preliminary
immunoprecipitation experiments do precipitate calhepa-
tin, they fail to coprecipitate calhepatin effector protein
partners.
The metal-binding properties of calhepatin were studied
by a direct
45
Ca
2+
-binding assay and fluorescence titration.
The binding of 2 Ca
2+

/monomer is consistent with the
presence of two EF-hand motifs. The affinity constants
determined agree with the fact that S100 protein affinity for
Ca
2+
is low, the affinity of the C-terminal EF-hand being
greater than that of the N-terminal EF-hand [3]. Such
characteristics suggest that S100 proteins may be activated
only in subcellular compartments where the Ca
2+
concen-
tration reaches a relatively high level [3]. Additional modes
of affinity control may involve other factors such as other
cations. The affinity of S100 proteins for Ca
2+
can be
modulated by Zn
2+
binding in some subfamilies such as
S100B [27], S100A5 [28], S100A6 [20] and S100A12 [10].
Our results indicate that Cu
2+
, unlike Zn
2+
and Mg
2+
,
binds to calhepatin. Copper binding does not change
calhepatin affinity for Ca
2+

, but it is not unlikely that in
some cases calhepatin biological activity could be regulated
by Cu
2+
instead of Ca
2+
[5]. Preliminary cross-linking
experiments show that both Ca
2+
and Cu
2+
increased the
dimer/monomer ratio, suggesting that, like Ca
2+
,Cu
2+
also enhance calhepatin dimerization.
Surprisingly, according to the
BLASTP
program [22], the
higher scores of similarity when the calhepatin amino-acid
sequence is compared with all database proteins correspond
to one or more segments of CaBPs of higher molecular mass
than S100 proteins. This contains several EF-hands such as
Ca
2+
-dependent protein kinases from Arabidopsis thaliana,
Zea mays, Glycine max, Dunaliella tertiolecta, Picea mari-
ana, Solanum tuberosum, Plasmodium falciparum and other
species. In addition, calmodulin-like proteins from A. thali-

ana, Z. mays, Mus musculus, Homo sapiens skin and
Suberites domuncula have two fragments with similar
characteristics. As some CaBPs appear to have evolved
from a single ur-domain by two cycles of gene duplication
and fusion [8], calhepatin may be related to that ancient
domain.
ACKNOWLEDGEMENTS
We thank Dr Ulf Hellman and the Ludwig Institute for Cancer
Research (Uppsala, Sweden) for MALDI-TOF MS analysis. We
acknowledge S. B. Linskens and E. V. Dacci for amino-acid analysis
and sequence determination by Edman degradation. We also thank R.
Davis for language supervision. This work was supported by Consejo
Nacional de Investigaciones Cientı
´
ficas y Te
´
cnicas de la Repu´ blica
Argentina Grant 4115 and Universidad de Buenos Aires Grant TB75.
REFERENCES
1. Heizmann, C.W. & Hunziker, W. (1991) Intracellular calcium-
binding proteins: more sites than insights. Trends Biochem. Sci. 16,
98–103.
2. Donato, R. (1999) Functional roles of S100 proteins, calcium-
binding proteins of the EF-hand type. Biochim. Biophys. Acta
1450, 191–231.
3. Kligman, D. & Hilt, D.C. (1988) The S100 protein family. Trends
Biochem. Sci. 13, 437–443.
4. Scha
¨
fer, B.W. & Heizmann, C.W. (1996) The S100 family of

EF-hand calcium-binding proteins: functions and pathology.
Trends Biochem. Sci. 21, 134–140.
5. Heizmann, C.W. & Cox, J.A. (1998) New perspectives on S100
proteins: a multi-functional Ca
2+
-, Zn
2+
-andCu
2+
-binding
protein family. Biometals 11, 383–397.
6. Potts, B.C.M., Smith, J., Akke, M., Macke, T.J., Okazaki, K.,
Hidaka,H.,Case,D.A.&Chazin,W.J.(1995)Thestructureof
calcyclin reveals a novel homodimeric fold for S100 Ca
2+
-binding
proteins. Nat. Struct. Biol. 2, 790–796.
7. Drohat, A.C., Nenortas, E., Beckett, D. & Weber, D.J. (1997)
Oligomerization state of S100B at nanomolar concentration
determined by large-zone analytical gel filtration chromatography.
Protein Sci. 6, 1577–1582.
8. Kawasaki, H., Nakayama, S. & Kretsinger, R.H. (1998) Clas-
sification and evolution of EF-hand proteins. Biometals 11,
277–295.
9. Dell’Angelica, E.C., Schleicher, C.H., Simpson, R.J. & Santome
´
,
J.A. (1996) Complex assembly of calgranulins A and B, two
S100-like calcium-binding proteins from pig granulocytes. Int. J.
Biochem. Cell Biol. 28, 53–62.

10. Dell’Angelica, E.C., Schleicher, C.H. & Santome
´
, J.A. (1994)
Primary structure and binding properties of calgranulin C, a novel
S100-like calcium-binding protein from pig granulocytes. J. Biol.
Chem. 269, 28929–28936.
11. Hoffmann, M.A., Drury, S., Fu, C., Qu, W., Taguchi, A., Lu, Y.,
Avila, C., Kambham, N., Bierhaus, A., Nawroth, P., Neurath, M.,
Merser, J., Stern, D. & Schmidt, A.M. (1999) RAGE mediates a
novel proinflammatory axis: a central cell surface receptor for
S100/calgranulin polypeptides. Cell 97, 889–907.
12. Scha
¨
gger, H. & von Jagow, G. (1987) Tricine-sodium dodecyl
sulfate-polyacrylamide gel electrophoresis for the separation
of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166,
368–379.
13. Harlow, E. & Lane, D. (1988) Antibodies. A Laboratory
Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
14. Rosenfeld, J., Capdevielle, J., Guillemot, J.C. & Ferrara, P. (1992)
In-gel digestion of proteins for internal sequence analysis after
one- or two-dimensional gel electrophoresis. Anal. Biochem. 203,
173–179.
15. Hellman, U., Wernstedt, C., Go
´
n
˜
ez,J.&Heldin,C.H.(1995)
Improvement of an in-gel digestion procedure for the

Fig. 9. Western-blot analysis. The 105 000 g supernatant of lungfish
liver (lane 1), skeletal muscle (lane 2), intestine (lane 3), lung (lane 4),
brain (lane 5), adipose tissue (lane 6), heart (lane 7) and skin (lane 8)
homogenates and those of rat liver (lane 9) and intestine (lane 10) were
subjected to SDS/PAGE and transferred to nitrocellulose membranes.
Immunodetection was carried out with polyclonal rabbit anti-calhep-
atin IgG.
3440 S. M. Di Pietro and J. A. Santome
´
(Eur. J. Biochem. 269) Ó FEBS 2002
micropreparation of internal protein fragments for amino acid
sequencing. Anal. Biochem. 224, 451–455.
16. Peitsch, M.C. (1996) ProMod and Swiss-Model: internet-based
tools for automated comparative protein modelling. Biochem. Soc.
Trans. 24, 274–279.
17. Mani, R.S. & Kay, C.M. (1990) Isolation and characterization of a
novel molecular weight 11000 Ca
2+
-binding protein from smooth
muscle. Biochemistry 29, 1398–1404.
18. Boyhan, A., Casimir, C.M., French, J.K., Teahan, C.G. & Segal,
A.W. (1992) Molecular cloning and characterization of grancalcin,
a novel EF-hand calcium-binding protein abundant in neutrophils
and monocytes. J. Biol. Chem. 267, 2928–2933.
19. Dianoux, A.C., Stasia, M.J., Garin, J. & Vignais, P.V. (1992) A
23-kilodalton protein, a substrate of protein kinase C, in bovine
neutrophil cytosol is a member of the S100 family. Biochemistry
31, 5898–5905.
20. Pedrocchi, M., Scha
¨

fer,B.W.,Durussel,I.,Cox,J.A.&Heiz-
mann, C.W. (1994) Purification and characterization of the
recombinant human calcium-binding S100 proteins CAPL and
CACY. Biochemistry 33, 6732–6738.
21. Moore, D.S. (1985) Amino acid and peptide net charges: a simple
calculation procedure. Biochem. Ed. 13, 10–11.
22. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J.
(1990) Basic local alignment tool. J. Mol. Biol. 215, 403–410.
23. Tarabykina, S., Scott, D.J., Herzyk, P., Hill, T.J., Tame, J.R.H.,
Kriajevska, M., Lafitte, D., Derrick, P.J., Dodson, G.G.,
Maitland, N.J., Lukanidin, E.M. & Bronstein, I.B. (2001) The
dimerization interface of the Metastasis-associated protein
S100A4 (Mts1). J. Biol. Chem. 276, 24212–24222.
24. Mandinova, A., Atar, D., Scha
¨
fer, B.W., Spiess, M., Aebi, U. &
Heizmann, C.W. (1998) Distinct subcellular localization of cal-
cium binding S100 proteins in human smooth muscle cells and
their relocation in response to rises in intracellular calcium. J. Cell
Sci. 111, 2043–2054.
25. Skelton, N.J., Ko
¨
rdel, J., Akke, M., Forse
´
n, S. & Chazin, W.
(1994) Signal transduction versus buffering activity in Ca
2+
-
binding proteins. Nat. Struct. Biol. 1, 239–245.
26. da Silva, A.C.R. & Reinach, F.C. (1991) Calcium binding induces

conformational changes in muscle regulatory proteins. Trends
Biochem. Sci. 16, 53–57.
27. Baudier, J., Glasser, N. & Gerard, D. (1986) Ions binding to S100
proteins I. Calcium- and zinc-binding properties of bovine brain
S100aa, S100a (ab), and S100b (bb)protein:Zn
2+
regulates Ca
2+
binding on S100b protein. J. Biol. Chem. 261, 8192–8203.
28. Scha
¨
fer, B.W., Fritschy, J.M., Murmann, P., Troxler, H.,
Durussel, I., Heizmann, C.W. & Cox, J.A. (2000) Brain S100A5 is
a novel calcium-, zinc-, and copper ion-binding protein of the
EF-hand superfamily. J. Biol. Chem. 275, 30623–30630.
Ó FEBS 2002 Hepatic S100 calcium-binding protein (Eur. J. Biochem. 269) 3441