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
Trang 1Structural and biochemical characterization of calhepatin,
an S100-like calcium-binding protein from the liver of lungfish
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
residues and has a molecular mass of 8670 Da It is smaller
than calbindin D9k (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
direct45Ca2+-binding assay and by fluorescence titration
Calhepatin binds Ca2+ and Cu2+ but not Zn2+ Cu2+ binding does not change the affinity of calhepatin for
Ca2+ Calhepatin undergoes a conformational change upon Ca2+binding as shown by the increase in its intrinsic fluorescence intensity and kmax, the decrease in the apo-calhepatin hydrodynamic volume, and the Ca2+ -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 Ca2+-signal transduction Keywords: calcium-binding protein; EF-hand; liver; lungfish; S100
Cytoplasmic Ca2+is a ubiquitous second messenger The
rise in intracellular Ca2+ 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
Ca2+-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 D9k, S100 P) and
two putative additional members identified in chicken and
channel catfish (MRP126 and ictacalcin, respectively)
They have two EF-hand Ca2+-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 Zn2+ and/or Cu2+ [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 D9kis 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 and no other S100 member has been reported in liver, this protein will be referred to as calhepatin
M A T E R I A L S A N D M E T H O D S
Materials
45CaCl2(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, Facultad de
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-PROTdatabank under accession number
P82978.
(Received 21 March 2002, accepted 24 May 2002)
Trang 2Preparation 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 mM sodium phosphate, 150 mM KCl, 4 mM 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 4C The
superna-tant (4 mL) was loaded on a Sephadex G-75 column
(2.5· 40 cm) equilibrated with 15 mMTris/HCl (pH 9.0)/
1 mMEDTA Elution was performed at 4C with the same
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 mM T ris/HCl (pH 9.0) T he material
bound to the column was subsequently eluted with 10, 20,
30, 40, 50 and 100 mMNaCl in the same buffer A portion of
each fraction was concentrated and changed into 50 mM
Tris/HCl buffer (pH 7.4) by using a Centriprep
concen-trator (Amicon) and assayed for45Ca2+-binding activity as
described below The 10 mMfraction 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 mMTris/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 mM
linear gradient of NaCl concentration over 60 min
Calhep-atin was eluted at 40 mM NaCl Protein purity was
checked by SDS/PAGE (16% gel), isoelectric focusing, and
RP-HPLC in a Vydac C4column (4.6· 250 mm)
Preparation of the cytosolic fraction from lungfish
and rat tissues
Tissues were cut into small pieces, suspended in 40 mM
sodium phosphate (pH 7.4) containing 150 mMKCl, 4 mM
EDTA and 4 mM 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 4C 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 mMTris/HCl/120 mMKCl/0.1 mMEDTA (pH 7.4) for the apo-calhepatin or with 50 mM Tris/HCl/120 mM KCl/2 mM CaCl2(pH 7.4) for the holo-protein, at a flow rate of 0.5 mLÆmin)1 Both protein forms (7 lMmonomer concentration) were incubated in the corresponding equili-bration buffer for 30 min before being loaded The mono-meric and dimono-meric 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 mM Tris/HCl/120 mM KCl/1 mM CaCl2 (pH 7.4) After injection of 30 lg pure protein on the equilibration buffer, the column was eluted with
4 column vol of the same buffer and then with 4 column vol of 50 mM Tris/HCl/120 mM KCl/5 mM EDTA (pH 7.4)
Enzymatic digestion and peptide purification For Glu-C protease digestion, 250 lg calhepatin was incubated in 0.1M Tris/HCl (pH 7.9)/2M guanidine hydrochloride with 4 lg enzyme, at 20C for 24 h For Lys-C protease digestion, 250 lg calhepatin was incubated
in 0.1M Tris/HCl (pH 8.5)/2M guanidine hydrochloride with 5 lg enzyme, at 20C for 24 h Peptides were separated by RP-HPLC (Pharmacia LKB) on a Vydac
C18 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
Trang 3Rosenfeld et al [14], as modified by Hellman et al [15] T he
resulting peptides were recovered by passive elution and
then separated by HPLC on a BrownleeTMAquapore RP
300 C18column (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 (http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=
/NPSA/npsa_multalin.html) with a BLOSUM 62 matrix
and parameter default values
Evolutionary tree
It was constructed withCLUSTAL 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 (http://www.expasy.ch/swissmod/
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 usingGROMOS The model was checked
with the WHAT-IF program which utilizes WHAT-CHECK
verification routines
45
Ca2+-Binding assay
Apo-(lungfish calhepatin) was prepared by incubation of
freshly purified protein with 2 mMEGTA and 2 mMEDTA
and subsequent dialysis against 50 mMTris/HCl (pH 7.4)
45Ca2+binding was determined by the method of Mani and
Kay [77] using 15 lMapo-(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 Ca2+bound
per mol of monomer, x is the free Ca2+concentration, n is
the number of binding sites per monomer, and Ka1and Ka2
are macroscopic binding constants:
¼ ½ðn=2ÞKa1xþ nKa1Ka2x2=ð1 þ Ka1xþ Ka1Ka2x2Þ
Fluorescence measurements
The intrinsic fluorescence of 2 lMcalhepatin was recorded
at 20C 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] Data were analyzed
by the following equation where F0is the fluorescence at zero ligand concentration, Fmis the maximum fluorescence change, T is the total ligand concentration, P is the protein monomer concentration, and Kais the apparent association constant:
F¼ F0þ Fmð2PÞ1
T þ P þ Kn 1a ½ðT þ P þ K1a Þ2 4PT 1=2o The Cu2+-binding-induced fluorescence change was corrected for nonspecific quenching by subtracting the values of a linear term obtained from the final portion of the
Cu2+-binding curve, corresponding to fluorescence quench-ing before bindquench-ing saturation Uncorrected Cu2+ 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
R E S U L T S
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 mMNaCl fraction containing
Ca2+-binding activity was further purified by anion-exchange chromatography on a Mono Q column The protein was eluted at 40 mMNaCl 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.
Trang 4Biochemical 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 lM 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 lM monomer concentration) and
dimeric (1.1 lMmonomer 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 lMmonomer 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 lM
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 Ca2+binding
Ca2+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 mM
CaCl2 but could be eluted from the column with 5 mM
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 aminoacid 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] (pI¼ 5.12) agrees with the experimental value (pI¼ 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 theBLASTPprogram [22], the higher similarity when the calhepatin amino-acid sequence is compared with all database proteins corresponds to one or more segments of CaBPs with a higher molecular mass than that of S100 proteins Identity can reach 41%
Structural modelling of lungfish calhepatin The three-dimensional structure of the lungfish calhepatin monomer was predicted by using theSWISS-MODEL model-ling package [16] (Fig 5) Molecular modelmodel-ling 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.
Trang 5sequence was conducted with thePROMODprogram 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 Ca2+are shown
in Fig 6A Figure 6B displays the corrected maximum
of fluorescence intensity for each Ca2+ concentration
and allows detection of one binding site with Ka(app)¼
(3.6 ± 0.5)· 105M(mean ± SD, n¼ 3)
Intrinsic fluorescence determinations were also applied to
study the binding of Zn2+, Mg2+and Cu2+ Neither Zn2+
nor Mg2+changes calhepatin fluorescence, suggesting that
they have no binding sites in the protein In addition, they
have no effect on Ca2+binding (not shown) Calhepatin
fluorescence intensity decreased, and kmax changed with
Cu2+ additions (Fig 7A) The analysis of the corrected
maximum of fluorescence (Fig 7B) provides evidence of the
presence of a single site with Ka(app)¼ (1.5 ± 0.2) · 107
M (mean ± SD, n¼ 3)
Direct Ca2+-binding studies The 45Ca2+-binding isotherm of calhepatin at 20C in
25 mMTris/HCl (pH 7.4) is shown in Fig 8 The binding constants determined are Ka1¼ (2.9 ± 0.3) · 105M and
Ka2¼ (6.0 ± 0.7) · 103M(n¼ 2.1 ± 0.05) In the presence
of 1 mM Cu2+, binding constants are Ka1¼ (2.0 ± 0.3)· 105M and Ka2¼ (4.6 ± 0.6) · 103M (n¼ 2.2 ± 0.2), thus Cu2+ binding does not significantly change the affinity of calhepatin for Ca2+(values are all mean ± SD; n¼ 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 D9k 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.
Trang 6To 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
D I S C U S S I O N
The Ca2+ signal is transduced by a variety of CaBPs Whereas a number of Ca2+-dependent responses are mediated by calmodulin, a ubiquitous CaBP universally present in cells, the S100 proteins are cell-type-specific mediators of the Ca2+ 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 D9k) 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 D9kbeing 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 D9kagrees with their gene divergence Interestingly, calbindin D9kis 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 program.
Trang 7only family member identified so far that does not form
dimers, acting as a Ca2+modulator, rather than as a Ca2+
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 D9k 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 Ca2+ 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 Ca2+-dependent
con-formational changes responsible for the transmission of
information to effector proteins Our fluorescence experi-ments indicate a Ca2+-induced change in the environment
of at least the tryptophan residue located in one of the
Fig 6 Ca2+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 Ca2+
concentration Curve fitting was performed as indicated in Materials
and methods.
Fig 7 Cu2+ fluorescence titration (A) Fluorescence spectra of
2 l M calhepatin in 25 m M Tris/HCl, pH 7.4, with 0–100 l M Cu2+ (B) Cu2+ 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.45Ca2+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.
Trang 8four positions critical for dimerization in helix IV (Figs 3
and 5) The binding of Ca2+ to calhepatin increases its
intrinsic fluorescence intensity and kmax This result, the
decrease in the protein hydrodynamic volume, and the fact
that Ca2+-loaded calhepatin is retained on a
phenyl-Superose column and can be eluted with EDTA suggests
that calhepatin undergoes a conformational change on
Ca2+ 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 direct45Ca2+-binding assay and fluorescence titration
The binding of 2 Ca2+/monomer is consistent with the
presence of two EF-hand motifs The affinity constants
determined agree with the fact that S100 protein affinity for
Ca2+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 Ca2+
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 Ca2+ can be
modulated by Zn2+binding in some subfamilies such as
S100B [27], S100A5 [28], S100A6 [20] and S100A12 [10]
Our results indicate that Cu2+, unlike Zn2+and Mg2+,
binds to calhepatin Copper binding does not change
calhepatin affinity for Ca2+, but it is not unlikely that in
some cases calhepatin biological activity could be regulated
by Cu2+ instead of Ca2+ [5] Preliminary cross-linking
experiments show that both Ca2+and Cu2+increased the
dimer/monomer ratio, suggesting that, like Ca2+, Cu2+
also enhance calhepatin dimerization
Surprisingly, according to the BLASTPprogram [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
Ca2+-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
A C K N O W L E D G E M E N T S
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.
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