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INTRODUCTION |
Grancalcin is a recently described protein present in some cells
of hematopoietic origin and especially abundant in human neutrophils
(1-3). Grancalcin belongs to the calpain subfamily of EF-hand
Ca2+-binding proteins, comprising calpain light and heavy
chain, sorcin, grancalcin, ALG-2, peflin, and YG25-yeast (4, 5). So
far, the members of this subfamily seem to have diverse functions: calpain functions as a protease and can regulate adhesion, sorcin binds
to and regulates the cardiac ryanodine calcium channel, ALG-2 plays a
role in apoptosis, and the function of grancalcin is unknown. From
amino acid sequence comparison, it was initially deduced that these
proteins possess four EF-hand motifs (6). However, recent studies on
the three-dimensional structure of dVI, the Ca2+-binding
domain of calpain light chain (7, 8), surprisingly revealed the
presence of a fifth EF-hand N-terminal to the previously identified
EF-hands. This EF-hand is of a novel type and has therefore escaped
detection by sequence similarity analysis. This group of proteins is
thus characterized by five EF-hands and has accordingly been named the
penta-EF-hand (PEF)1
subfamily (4). In addition, the members of the PEF subfamily contain N
termini of varying length and sequence but all rich in glycine and
hydrophobic residues. In calpain, EF1 (although of an unusual sequence
composition) and EF2 are paired and bind two Ca2+ with high
affinity. EF3 and EF4 are also paired and possess one site of high
affinity and one of low affinity for Ca2+. Finally, EF5,
which does not bind Ca2+, is paired with a similar EF5 of
another monomer and is thus a dimerization module. Native calpain is a
heterodimer of the calpain light and heavy chain (9). The recent
crystallization of apograncalcin (10, 11) has shown great overall
similarity to the crystal structure of calpain. Grancalcin was also
found to form dimers by paring of cognate EF5s. Unfortunately,
grancalcin precipitated in the presence of Ca2+ and the
topology of active Ca2+-binding sites could not be
determined, except for binding of Ca2+ to EF3 from one
monomer. Moreover, the N terminus of grancalcin is disordered in the
crystals, and the first well defined residue is Ser53. In
molecular sieve chromatography, purified grancalcin migrates as a
homodimer (1) and ALG-2 as a monomer (12, 13). However, when using a
chemical cross-linker, ALG-2 could be shown to exist also as a dimer
(13).
The cDNA for grancalcin is 1.65 kilobase pairs long and contains an
open reading frame for 217 amino acids; the first 14 amino acids have
been reported to be removed posttranslationally to give rise to a
203-residue-long functional protein (2). This presumably functional
grancalcin has a calculated molecular mass of 22.4 kDa, while
grancalcin migrates as a 28-kDa protein in SDS-PAGE (1, 2). The reason
for this discrepancy is unknown, but it is not due to glycosylation
(1). Grancalcin translocates to membranes upon binding of
Ca2+ (2, 3, 14). This is a common feature of several
EF-hand proteins. Translocation is often mediated by exposure of
hydrophobic patches of amino acids subsequent to Ca2+
binding, as is the case for calpain (15) and calretinin (16). In other
instances, such as in the recoverin family, a covalently bound fatty
acid becomes exposed after Ca2+-induced conformational
changes, and this leads to translocation of the protein to membranes, a
mechanism also called the myristoyl switch (17). Which of these
molecular schemes prevails for grancalcin is at present unknown.
EF-hand proteins have affinities for Ca2+ varying from
108 to 103
M
1. Moreover, some show great
selectivity toward Ca2+, such as calmodulin, S100 proteins,
and calretinin, whereas others also bind Mg2+, such as
parvalbumin and recoverin (for a review, see Ref. 18). Neither the
affinity nor the selectivity can be deduced from the sequence of the
EF-hands. Except for a positive 45Ca2+ overlay,
no information on the ion-binding and conformational changes of
grancalcin is available.
Because the biochemical characterization of grancalcin has been very
sparse and nothing is known about the function of grancalcin, we
decided to address these issues. In this report, we have identified a
N-terminal posttranslational modification of wild-type grancalcin by
mass spectroscopy and sequencing. Because grancalcin precipitates in
the presence of Ca2+ in most experiments, we have generated
three mutants of grancalcin with varying N-terminal deletions. We show
that these mutants do not precipitate to the same degree as recombinant
grancalcin. We have also compared critical properties of wild type and
recombinant grancalcin, determined the binding characteristics of
grancalcin for Ca2+ by flow dialysis, and probed cation
induced conformational changes by fluorometry of the intrinsic Trp
residues. Finally, using affinity chromatography, we identify the
actin-bundling protein L-plastin as binding partner of grancalcin and
document the Ca2+ dependence of this interaction.
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EXPERIMENTAL PROCEDURES |
Isolation of Human Neutrophils--
Human neutrophils were
isolated from volunteer donors as described (19). In short,
erythrocytes were sedimented by 2% dextran (Amersham Pharmacia
Biotech) in saline, and the leukocyte-rich supernatant was submitted to
400 × g density centrifugation for 30 min on
Lymphoprep (Nycomed, Oslo, Norway). The pellet was submitted to
hypotonic lysis of contaminating erythrocytes for 30 s in pure water, after which tonicity was restored by the addition of NaCl. The
neutrophils were washed once, counted, and resuspended in saline.
SDS-PAGE and Immunoblotting--
SDS-PAGE (20) and
immunoblotting (21) were performed on Bio-Rad systems according to the
instructions given by the manufacturer (Bio-Rad) and as described (3),
except for visualization of immunoblots, which was by metal-enhanced
diaminobenzidine tetrahydrochloride (Pierce).
Protein Measurement--
Crude protein concentration was
determined by the method of Bradford according to the instructions
given by the manufacturer (Bio-Rad), and catalase ranging from 0.05 to
0.5 mg/ml was used as a standard. Grancalcin was quantified by
enzyme-linked immunosorbent assay as previously described (3). Purified
grancalcin was quantified by the ultraviolet absorption spectrum using
a molar extinction coefficient
278 nm of 28,000 M
1
cm
1.
Subcellular Fractionation of Human Neutrophils--
Isolated
neutrophils at 3 × 107 cells/ml were incubated in
saline with 5 mM diisopropyl fluorophosphate (Sigma) for 5 min on ice and centrifuged at 200 × g for 6 min. Cell
pellets were resuspended at 2 × 108 cells/ml in
binding buffer (100 mM KCl, 3 mM NaCl, 10 mM PIPES (pH 7.2)) containing 0.5 mM
phenylmethylsulfonyl fluoride, 200 units/ml aprotinin, and 100 µg/ml
leupeptin (all three Sigma-Aldrich) and disrupted by nitrogen
cavitation at 400 p.s.i. for 5 min at 4 °C as described (22).
The cavitate was ultracentrifuged at 100,000 × g for
45 min, and the supernatant was kept as cytosol.
Purification of Wild-type and Recombinant Grancalcin--
Human
neutrophil cytosol (prepared as above) in binding buffer plus 0.5 mM CaCl2 was purified on a self-packaged
phenyl-Sepharose (Amersham Pharmacia Biotech) column at room
temperature. After binding of cytosolic proteins, the column was washed
thoroughly in binding buffer plus 0.5 mM CaCl2.
Bound proteins were eluted in fractions of 2 ml with binding buffer
plus 5 mM EGTA. Fractions containing grancalcin (as
determined by SDS-PAGE) were submitted to a buffer change to 20 mM Trizma (pH 7.4) using a Centriprep column (Amicon,
Beverly, MA) and passed through a Mono-Q (Amersham Pharmacia Biotech)
column coupled to an FPLC instrument (Amersham Pharmacia Biotech).
Bound proteins were eluted with a 0-1 M NaCl nonlinear
gradient, and 0.5-ml fractions were evaluated by SDS-PAGE and
immunoblotting. Recombinant grancalcin was produced and purified as
previously described (3).
Cloning and Purification of Grancalcin Mutants--
N-terminal
deletion mutants were cloned from the grancalcin clone using the
following primers for the N terminus:
42-grancalcin, 5'-CGC
GGA TCC GCA TAT TCA GAC ACT TAT TCC-3';
50-grancalcin, 5'-CGC GGA
TCC GCT GGT GAC TCC GTG TAT AC-3'; and
53-grancalcin, 5'-CGC GGA TCC
TCC GTG TAT ACT TTC AGT G-3'. For all three mutants, the following
primer was used for the C terminus: 5'-CCG GAA TTC TCA AAT TGC CAT AGT
GCC CTG C-3'. The obtained clones and the vector (pGEX2T (Pharmacia
Amersham Biotech)) were cut with BamHI and EcoRI
and, following ligation, transformed into Escherichia coli. The expressed clones were checked by sequencing (model 377 DNASequencer (PE Biosystems, Foster City, CA)), and only clones with
correct sequences were used for protein expression. Procedures for
expression and purification of N-terminal deletion mutants were as
described for recombinant grancalcin (3).
Proteolytic Cleavages--
For the identification of the
N-terminal modification in wild type grancalcin, 20 µg of both wild
type and recombinant grancalcin was incubated with 0.5 µg of
endoproteinase Glu-C (Roche Molecular Biochemicals) in 500 µl 50 mM NH4HCO3 (pH 7.8) including 10%
acetonitrile for 20 h at room temperature. The reaction was
stopped by the addition of 1 ml 1% trifluoroacetic acid. The resulting
fragments were purified by HPLC on a 2.1 × 150-mm C-4 column
(Vydac, Hesperia, CA) eluted with a linear gradient (1% per min) from
solvent A (0.1% trifluoroacetic acid) to solvent B (0.1%
trifluoroacetic acid in acetonitrile). Peak fractions were collected
manually. The putative N-terminal fragment was localized by mass
spectrometry, dried, and submitted to cleavage with 0.1 µg of
thermolysin (Roche Molecular Biochemicals) in 100 µl of buffer (100 mM NH4HCO3, 5 mM
CaCl2 (pH 7.8) including 10% actonitrile) for 3 h at
37 °C. The reaction was stopped by the addition of 400 µl of 1%
trifluoroacetic acid. The resulting fragments were purified by HPLC on
a 2.1 × 150-mm C-8 column (Vydac, Hesperia, CA) as described above.
Mass Spectrometry and Sequence Analysis--
The purified
peptides were analyzed in a matrix-assisted laser desorption/ionization
time-of-flight mass spectrometer (Biflex, Bruker-Franzen, Bremen,
Germany). For analysis, 0.5 µl of the sample was mixed with 0.5 µl
of matrix solution (
-cyano-4-hydroxycinnamic acid in
acetonitrile/methanol; Hewlett Packard), and 0.5 µl of the mixture
was applied to the probe and allowed to dry. The purified proteins
(wild-type grancalcin and recombinant grancalcin) were diluted 4-10
times with a 20 mM octyl-
-glucopyranoside (OG) (Roche Molecular Biochemicals) solution in formic
acid/H2O/acetonitrile (1:3:2, by volume) before mixing with
the matrix. Most samples were analyzed in the positive mode. Spectra
were recorded for 20-100 laser shots. The method has an accuracy of
0.1%.
The amino acid sequences of the purified peptides were determined
employing an automatic protein sequencer (model 494A; PerkinElmer Life Sciences) equipped with an online HPLC system for detection of the amino acid-phenylthiohydantoin derivatives. All chemicals and
solvents were sequence or HPLC grade supplied by PerkinElmer Life Sciences.
Size Exclusion Chromatography--
200 µl of recombinant
protein (10 µM) or cytosol in binding buffer with or
without 0.5 mM Ca2+ was applied to a Superdex
75 column (FPLC system; Amersham Pharmacia Biotech) equilibrated in
binding buffer with or without 0.5 mM Ca2+, and
eluted fractions were analyzed by absorbance and/or grancalcin enzyme-linked immunosorbent assay. The following markers were used to
calibrate the column: blue dextran 2000 (>2,000,000), ribonuclease A
(13,700), chymotrypsinogen A (25,000), and ovalbumin (43,000).
Chemical Cross-linking--
Proteins were cross-linked with the
chemical cross-linker DSG (Pierce) essentially as described (13). In
short, proteins were incubated for 30 min at room temperature in 100 µl of cross-linking buffer (20 mM HEPES (pH 8.0), 50 mM NaCl, and 1 ml of dithiothreitol) to which 5 µl of DSG
was added (from 10 mM stock in N,
N-dimethylformamide). The reaction was quenched by the addition of
125 µl of 1 M Tris-HCl, pH 7.5, and cross-linking was
evaluated by immunoblotting.
Ca2+-dependent Precipitation of
Grancalcin and N-terminal Deletion Mutants--
Recombinant grancalcin
at 2 or 10 µM and
42-grancalcin and
53-grancalcin
at 10 µM in binding buffer (with or without 25 mM OG) were supplemented with varying concentrations of
Ca2+ and rotated end over end for 30 min at room
temperature. The tubes were centrifuged at 14,800 × g
for 5 min. at 4 °C, and the supernatants were assayed by absorption
to determine protein concentrations.
Metal Ion Removal and Cation Binding--
Since the method of
trichloroacetic acid precipitation (14) irreversibly modified the Trp
fluorescence spectra of grancalcin, the concentrated protein sample was
supplemented with 1 mM EGTA, dialyzed overnight against
buffer A containing 0.1 mM EGTA, and passed through
a Sephadex G25 column (0.8 × 40 cm) equilibrated in buffer A (50 mM Tris-HCl buffer (pH 7.5), 150 mM KCl). The protein contained less than 0.1 mol of Ca2+/mol.
Ca2+ binding was measured on the recombinant protein at
25 °C by the flow dialysis method (23) in buffer A. Protein
concentrations were 25 µM. Processing of data and
evaluation of the binding constants were as described (18).
Optical Methods to Probe the Trp Environment--
Emission
fluorescence spectra were obtained in a PerkinElmer Life Sciences LS-5B
spectrofluorometer. The measurements were carried out at 25 °C on 4 µM wild-type or recombinant grancalcin in buffer A in the
presence of 37.5 mM OG with an excitation wavelength at 278 nm and both slits at 5 nm. Measurements were performed in the presence
of either 50 µM EGTA, 0.5 mM
CaCl2 or 4 M guanidine-HCl, respectively.
Isolation and Identification of Binding Partner for
Grancalcin--
Recombinant grancalcin at 1 mg/ml in the presence of
0.5 mM CaCl2 or 0.5 mM EGTA was
coupled to CNBr-activated SepharoseTM according to the manufacturer's
instructions (Amersham Pharmacia Biotech). Three columns were packaged:
one containing recombinant grancalcin in the presence of
Ca2+, one containing recombinant grancalcin without
calcium, and one without any protein present. Purified human
neutrophils at 3 × 107 cells/ml were solubilized in
solubilization buffer (10 mM HEPES (pH 7.4), 100 mM KCl, 25 mM OG, 0.2% cetyltrimethylammonium
bromide, 1 mM phenylmethylsulfonyl fluoride, 200 units/ml
aprotinin, 100 µg/ml leupeptin, and 100 µM EGTA) and
incubated overnight at 4 °C. Unsolubilized material were spun down
(5000 × g for 10 min), and 10 ml of supernatant,
supplemented with 0.5 mM EGTA, were applied to each to each
of the three columns. The columns were washed extensively in binding
buffer plus 0.5 mM EGTA, and bound proteins were eluted
with binding buffer plus 5 mM CaCl2. Fractions of 1 ml were collected and evaluated by SDS-PAGE. Fractions with a
visible protein band were pooled and concentrated by centrifugation and
subjected to SDS-PAGE. The identified band was cut out of the SDS gel
and digested with trypsin essentially as described by Wilm et
al. (24). In short, the gel piece was cut into small cubes and
washed for 1 h in 100 mM
NH4HCO3 (AB), and excess liquid was removed.
Then the proteins were reduced for 30 min at 60 °C in 100 µl of 4 mM dithiothreitol in 100 mM AB, cooled, and
alkylated by the addition of 10 µl of 100 mM
iodoacetamide followed by a 30-min incubation at room temperature in
the dark. Excess liquid was removed, and the gel pieces were washed for
1 h in 50% acetonitrile in 100 mM AB followed by
shrinkage in acetonitrile and vacuum centrifugation. The tube with the
dry gel pieces was placed in an ice bath and allowed to swell in 25 mM AB including 2 µg/100 µl modified trypsin (Promega).
After 45 min, the excess liquid was removed, and buffer was added to
cover the gel pieces during overnight incubation at 37 °C. The next
day, the liquid was removed and combined with two consecutive
extractions with 50 µl of 0.1% trifluoroacetic acid in 60%
acetonitrile. The extract was dried in a vacuum centrifuge to near
dryness followed by the addition of 5 µl of 0.1% trifluoroacetic
acid in 30% acetonitrile. For analysis, 0.4 µl of the sample was
mixed with 0.1 µl of internal standard mixture (angiotensin II and
dynorphin, 0.06 and 1 pmol/µl, respectively) and 0.5 µl matrix
solution (
-cyano-4 hydroxycinnamic acid) and measured by mass
spectrometry as described above, except the instrument was upgraded
with the time lag focusing option (delayed extraction). The peptide
molecular mass fingerprint was used for a search of the SWISS-PROT and
TrEMBL data base using the "PeptIdent" tool on the ExPASy Molecular
Biology Server of the Swiss Institute of Bioinformatics (25).
Immunoprecipitation--
Cytosol from human neutrophils
(prepared as described above) was diluted with binding buffer to a
final concentration of 1 mg/ml (as determined by Bio-Rad). 5 µl of
anti-grancalcin or anti-L-plastin antibodies (two different antibodies;
LPL4A1, which binds to L-plastin irrespective of Ca2+ load,
and LPL7,2, which only binds to the Ca2+-loaded form of
L-plastin (described in Ref. 26), a kind gift from Dr. Eric J. Brown
(Division of Infectious Disease, Howard Hughes Medical Institute, St.
Louis, MO)) were added to 1 ml of the diluted cytosol in the presence
of 0.5 mM CaCl2 or 5 mM EGTA, respectively. The solution were rotated end over end overnight at
4 °C. Next, 100 µl of protein A-Sepharose®
beads (Amersham Pharmacia Biotech) (prepared in binding buffer as
described by the manufacturer) were added, and the slurry was rotated
end over end for 2 h at 4 °C. The tubes were centrifuged to
pellet beads, and the supernatant was gently removed. The pellets were
washed three times in binding buffer with either 0.5 mM CaCl2 or 5 mM EGTA present
before reconstitution into the initial volume. Samples were run on
SDS-PAGE and immunoblotted as described above. For immunoblotting with
L-plastin and MRP14 antibodies, the following dilutions were used:
LPL4A1 primary antibody, 1:1000; secondary antibody (goat anti-mouse
(D0486) (Dako A/S, Glostrup, Denmark), 1:1000; LPL7,2 primary antibody,
1:1000; secondary antibody (rabbit anti-rat (P0450) (Dako A/S), 1:1000;
MRP14 primary antibody (Bachem, Bubendorf, Switzerland), 1:2000
(biotinylated); secondary layer (avidine horseradish peroxidase (P0347)
(Dako A/S)), 1:1000.
 |
RESULTS |
Calcium-induced Hydrophobicity and Purification of Native
Grancalcin--
One aim of this study was to determine if native
grancalcin has a lipid anchor or other posttranslational modifications.
Furthermore, we wanted to verify the reported N terminus of grancalcin.
Since many EF-hand proteins expose hydrophobic areas upon binding of Ca2+, we tested if grancalcin also becomes hydrophobic in
the presence of high concentrations of Ca2+. Human
neutrophil cytosol was passed through a phenyl-Sepharose column
in the presence of 0.5 mM Ca2+, and the column
was washed with Ca2+ buffer. Replacement of
Ca2+ by an excess of EGTA led to elution of a limited
number of proteins (Fig. 1A).
By Coomassie staining, several bands can be seen, which all are
expected to be Ca2+-binding proteins or proteins that
interact with Ca2+-binding proteins. Indeed, several well
known Ca2+-binding proteins could be identified (data not
shown). The band at 28 kDa, showing retarded EGTA elution, was
identified as grancalcin by immunoblotting. Fractions containing
grancalcin were further purified by anion-exchange chromatography as
described under "Experimental Procedures." This resulted in pure
grancalcin as evaluated by SDS-PAGE and immunoblotting (Fig.
1B). Thus, we here present a novel and fast protocol for
purification of grancalcin from the cytosol of human neutrophils, based
on its Ca2+-dependent binding to a hydrophobic
matrix.

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Fig. 1.
Elution profile of neutrophil cytosolic
proteins separated on a phenyl-Sepharose column (A)
and purity of wild-type grancalcin (B). Human
neutrophilic cytosol was loaded onto a phenyl-Sepharose column in the
presence of 0.5 mM Ca2+ and washed in a
Ca2+ buffer. Bound proteins were eluted with EGTA and
collected in fractions of 2 ml. A, 14% SDS-PAGE profile of
eluted proteins (Coomassie stain). Lane 1,
molecular weight markers; lanes 2-9, fractions
2-9 of the EGTA eluate. Fractions containing grancalcin (28 kDa) were
pooled and separated by ion exchange chromatography. B, ion
exchange chromatography fractions containing grancalcin were
concentrated and submitted to SDS-PAGE on 10% gels and identified by
Coomassie staining (lane 1) and immunoblotting
with anti-grancalcin antibodies (lane 3).
Lane 2 shows molecular weight markers.
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Purification of Recombinant Grancalcin and Three Deletion
Mutants--
Recombinant grancalcin was produced and purified as
described (3). The grancalcin clone was checked by conventional
sequencing techniques and found to be identical with the published (2) sequence (data not shown). Recombinant grancalcin is synthesized as a
glutathione S-transferase fusion protein linked by a
cleavage site for thrombin and differs from prograncalcin (we use the
term prograncalcin for the hypothetical protein corresponding to the coding sequence, including the N-terminal Met) by an additional Gly-Ser
dipeptide at the N terminus, as was confirmed by N-terminal sequence
analysis (data not shown). The molecular mass of purified recombinant
grancalcin was determined to be 24,124 Da, in good agreement with the
theoretical mass of 24,154 Da.
In order to solve solubility problems (see below), we also generated
three mutants with varying deletions in the N terminus, named after the
starting amino acid according to the nomenclature for prograncalcin:
42-grancalcin,
50-grancalcin, and
53-grancalcin, respectively.
All three mutants were easily expressed and purified to electrophoretic
homogeneity (Fig. 2) following the same
procedure as for recombinant grancalcin. Our polyclonal (rabbit)
anti-grancalcin antibodies reacted with all three mutants (data not
shown).

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Fig. 2.
Recombinant grancalcin and N-terminal
mutants. Three N-terminal deletion mutants were generated and
expressed in E. coli. The recombinant mutant proteins were
isolated and purified as for recombinant grancalcin. Lane
1, molecular weight markers; lane 2,
recombinant grancalcin; lane 3, 42-grancalcin;
lane 4, 50-grancalcin; lane
5, 53-grancalcin. The three mutants have, as expected, an
increasingly higher mobility in SDS-PAGE as compared with recombinant
grancalcin, due to their smaller molecular weight.
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Peptide and Oligonucleotide Sequencing and Mass Spectroscopy of
Wild-type and Recombinant Grancalcin--
The purified wild-type
grancalcin gave no signal in protein sequence analysis, indicating that
the N terminus is blocked. Moreover, by mass spectrometry wild-type
grancalcin was found to have a molecular mass of 23,856 Da as compared
with 24,010 Da calculated for prograncalcin (hypothetical protein,
corresponding to the coding sequence), suggesting that the N terminus
of wild-type grancalcin is modified. Cleavage of wild-type grancalcin
with endoproteinase Glu-C resulted in a number of fragments, one of which had a measured molecular mass (2829.4 Da, obtained in the negative mode) matching that of fragment 2-28 of prograncalcin if it
is N-terminally acetylated (calculated 2828.2 Da). To confirm that
this peptide was indeed the N-terminal fragment, it was further digested with thermolysin, resulting in two fragments with molecular masses of 1101.2 and 1746.8 Da, respectively. Sequence analysis of the
latter showed the first 6 residues to be Phe-Ser-Ile-Gln-Val-Pro. This
sequence and the molecular mass indicate that this fragment represents prograncalcin 13-28 (calculated 1745.1 Da). The
molecular mass of the former fragment (obtained in the negative mode,
using internal calibration) fits precisely to the cDNA-deduced
sequence of prograncalcin 2-12 bearing an N-terminal acetyl group
(1101.1 Da). Including this modification, the theoretical molecular
mass of wild-type grancalcin becomes 23916 Da.
Dimerization--
It was originally found that purified
grancalcin exists as a homodimer (1), as also confirmed by the crystal
structure (11). We wanted to examine if also cytosolic grancalcin and N-terminal mutants existed as dimers. Molecular sieve chromatography studies gave an apparent molecular mass of 40 kDa for recombinant grancalcin (Fig. 3A), and when
cytosol was subjected to the same procedure and grancalcin was
identified by immunoblotting, grancalcin also eluted as a 40-kDa
protein. When the chromatography was performed in the presence of
calcium, grancalcin eluted slightly later, with an apparent mass of 36 kDa. Thus, grancalcin is a dimer under all conditions, but the
Ca2+-loaded form is slightly smaller, indicating
conformational changes that make the complex more compact. N-terminal
mutants all ran as dimers on molecular sieve chromatography (data only
shown for
42-grancalcin), verifying that the N terminus does not
play a role in dimer formation. By chemical cross-linking with DSG, we could also verify that grancalcin is a functional dimer under all
conditions tested (Fig. 3B) (data only shown for recombinant grancalcin and
53-grancalcin).

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Fig. 3.
Molecular mass determination of grancalcin by
molecular sieve chromatography and chemical cross-linking of grancalcin
by DSG. A, 200 µl of recombinant grancalcin (10 µM) and cytosol in the absence and presence of
Ca2+ and 200 µl of 42-grancalcin were subjected to
molecular sieve chromatography by FPLC. Grancalcin was detected by
absorbance and enzyme-linked immunosorbent assay, and its peak elution
volume (Ve) was used to calculate
Kav according to the equation,
Kav = (Ve V0)/(Vt V0). Ovalbumin (43 kDa), chymotrypsinogen
A (25 kDa), and ribonuclease A (13.7 kDa) served as standards, and blue
dextran 2000 was used to determine void volume
(V0 = 8.04 ml) of the column, which had a total
volume (Vt) of 24 ml. B, chemical
cross-linking by DSG followed by immunoblotting of grancalcin.
Lane 1, recombinant grancalcin and DSG;
lane 2, recombinant grancalcin without DSG;
lane 3, 53-grancalcin and DSG.
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Ca2+-dependent Precipitation of Grancalcin
and N-terminal Mutants--
Precipitation of grancalcin was dependent
on both the concentration of protein and Ca2+, and the
presence of detergent increased the threshold for
Ca2+-induced precipitation (Fig.
4A). Therefore, we have
performed subsequent experiments at low protein concentrations and
repeated the experiments in the presence of detergent. Furthermore,
because the first 52 amino acids are disordered in the crystals of
grancalcin (11) and contain many hydrophobic residues that might be
responsible for the pronounced precipitation, we tested N-terminal
deletion mutants. The precipitation experiment of Fig. 4B
illustrates that, as predicted, the N-terminal mutants are more soluble
in the presence of Ca2+ than the full-length protein (only
data for
42- and
53-grancalcin are shown).

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Fig. 4.
Ca2+ precipitation of grancalcin
and N-terminal deletion mutants. A, two concentrations
of recombinant grancalcin, 10 and 2 µM (with or without
25 mM OG), were incubated with the indicated
Ca2+ concentrations for 30 min. The tubes were then
centrifuged, and the grancalcin concentration in the supernatant was
determined by absorbance. The concentration values are normalized to
incubations without Ca2+. B, 42-grancalcin
and 53-grancalcin at 10 µM (with or without 25 mM OG) were incubated with the indicated Ca2+
concentrations for 30 min. The tubes were then centrifuged, and the
protein concentration was determined as in A.
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Direct Cation-binding Studies--
Evaluation of Ca2+
binding to recombinant grancalcin and N-terminal mutants by flow
dialysis (Fig. 5) revealed two
Ca2+-binding sites per monomer. In the absence of detergent
(Fig. 5A), both recombinant grancalcin and the N-terminal
mutants precipitated, but the N-terminal mutants precipitated only at
the very end of the titration. In the presence of the detergent OG
(Fig. 5B), the N-terminal mutants did not precipitate at
all, whereas recombinant grancalcin precipitated at the very end. In
the absence of detergent, recombinant grancalcin displayed a
[Ca2+]0.5 value of 83 µM and
moderate positive cooperativity (nH = 1.18). The
truncated forms displayed [Ca2+]0.5 values of
140 µM (for
42- and
50-grancalcin) and 174 µM (for
53-grancalcin) with no cooperativity
(nH = 1.0). In the presence of 25 mM OG,
recombinant grancalcin has a [Ca2+]0.5 of 25 µM with an nH of 1.1, whereas the
truncated forms displayed a [Ca2+]0.5 of 35 µM (for
42-grancalcin) to 50 µM (for
50- and
53-grancalcin) without any cooperativity or even slight
negative cooperativity for
53-grancalcin (nH = 0.94). Protein precipitation is a confounding factor in these flow
dialyses, and the presence of detergent increases the affinity by a
factor of 3, but it seems safe to conclude that grancalcin shows
optimal Ca2+ sensitivity in the 25-83 µM
range. The presence of 2 mM Mg2+ did not
influence the binding curve (data not shown), indicating that the two
sites are of the so-called Ca2+-specific type.
Ca2+-specific sites seem to be common in the other proteins
of the PEF family, as was also reported for ALG-2 (12). The selectivity of the calpain light chain EF-hands is not known, but the affinity of
the recombinant protein for Ca2+ corresponds to
[Ca2+]0.5 values of 60-150 µM
(27), thus similar to that of grancalcin. ALG-2 has two
Ca2+ binding sites of high affinity around 1-3
µM and one site of low affinity of 300 µM
(28).

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Fig. 5.
Ca2+ binding to recombinant
grancalcin and N-terminal deletion mutants. Ca2+
binding was measured by the flow dialysis method at 25 °C in 50 mM Tris-HCl, 150 mM KCl in the absence
(A) and presence (B) of 25 mM OG,
respectively. Protein concentration was 25 µM. and
, recombinant grancalcin (two experiments); , 42-grancalcin;
, 50-grancalcin; , 53-grancalcin.
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Intrinsic Trp Fluorescence--
Grancalcin contains two Trp
residues (Trp118 and Trp124) as useful probes
to monitor differences between wild-type and recombinant grancalcin as
well as structural changes upon binding of Ca2+. After
excitation at 278 nm of a solution of 1 µM wild-type or recombinant grancalcin in buffer A plus 37.5 mM OG, the
emission fluorescence spectra of metal-free and
Ca2+-saturated proteins show maxima between 325 and 335 nm
(Fig. 6). This is characteristic for
hiding of several Trp residues in a hydrophobic environment and was
confirmed by the ~3-fold intensity decrease and 20-nm red shift of
the fluorescence maximum when guanidine HCl was added. Saturating
Ca2+ concentrations moderately increased the fluorescence,
but Mg2+ had no influence (data not shown). It should be
noted that all of the spectra of wild-type and recombinant grancalcin
are very similar, suggesting that their secondary structure and
hydrophobic cores must be very similar. Moreover, the signal change of
the metal-free proteins as a function of the concentration of guanidine HCl yielded [Gua-HCl]0.5 values of 1.4 and 1.5 M for wild-type and recombinant protein (data not shown),
respectively, indicating that both are equally stable.

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Fig. 6.
Trp fluorescence spectra of wild-type and
recombinant grancalcin. Thin and thick
lines, comparison of wild-type and recombinant grancalcin,
respectively. Spectra were recorded at 25 °C on 1 µM
protein in buffer A containing 37.5 mM OG and either 50 µM EGTA (gray line) or 1 mM Ca2+ (solid line). The
spectra of the denatured forms (dashed line) were
recorded after the addition of up to 4 M guanidine
HCl.
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Binding Partner of Grancalcin--
In order to identify proteins
that might interact with grancalcin, we made three affinity columns:
one containing recombinant grancalcin in the presence of
Ca2+, one containing recombinant grancalcin without
calcium, and one column with just the matrix as a negative control. At
first, we applied a whole cell homogenate of human neutrophils to the
columns in the presence of Ca2+ and eluted with EGTA, but
this did not result in any visible protein. However, when we applied
the homogenate to the columns in the presence of EGTA, washed
extensively, and then eluted with Ca2+, a band of ~67 kDa
was found in both the eluate from the Ca2+ (Fig.
7) and EGTA column but not from the mock
column (not shown). The 67-kDa protein was concentrated by
SDS-PAGE, cut out of the gel, and digested with trypsin, and the
mixture of fragments was analyzed by mass spectroscopy. A data base
search with this set of molecular masses identified L-plastin with a
high score, covering 37% of the sequence distributed among 19 peptides
with molecular masses matching the theoretical values within 50 ppm.
L-plastin belongs to the plastin group of proteins, and L stands
for leukocyte, indicating that it is the isotype present in leukocytes.
Plastins are EF-hand proteins with actin-binding motifs, and they are
known to have actin bundling activities. L-plastin has a molecular
mass of 67 kDa and is well described in human neutrophils (26,
29, 30)

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Fig. 7.
Ca2+ eluate from grancalcin
column, to which a whole neutrophil homogenate was applied. Whole
cell homogenate of human neutrophils was loaded onto a grancalcin
column in the presence of 0.5 mM EGTA and washed in an EGTA
buffer. Bound proteins were eluted with 5 mM
Ca2+ and collected in fractions of 1 ml. Fractional
distribution of eluted proteins shown by SDS-PAGE on 10% gels followed
by Coomassie staining (fractions 1-7 shown as lanes
2-8). A band of ~67 kDa (arrow) can bee seen
in fractions 2-5 (lanes 3-6).
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Immunoprecipitation--
We substantiated the interaction between
grancalcin and L-plastin by co-immunoprecipitation experiments followed
by visualization by immunoblotting with anti-grancalcin and
anti-L-plastin antibodies, respectively. When evaluating the
immunoblots, it is important to remember that grancalcin will
precipitate in the presence of Ca2+ (as shown above), and
this might introduce an artifact in the immunoprecipitation
experiments. Immunoprecipitation of isolated cytosol from human
neutrophils with L-plastin antibodies was, in the absence of
Ca2+, able to also pull down grancalcin and vice versa as
visualized by immunoblotting (Fig. 8,
1VI and 3VI). On the contrary, L-plastin could
not be detected in the pellet, when cytosol was immunoprecipitated with
anti-grancalcin antibodies in the presence of Ca2+ (Fig. 8,
3III). We therefore suggest that the fact that grancalcin could be found in the anti-L-plastin pellet is due to precipitation rather than immunoprecipitation. The Ca2+ specificity of
the interaction between grancalcin and L-plastin is further supported
by the fact that the L-plastin antibody LPL7,2, which only recognizes
the Ca2+-loaded form of L-plastin, was not able to pull
down grancalcin in the absence of Ca2+ (Fig. 8,
2VI). As a negative control, we also probed the
immoprecipitates by immunoblotting with anti-MRP14 antibodies. MRP14 is
a 14-kDa EF-hand protein that is highly enriched in the cytosol of
human neutrophils (31). We did not detect any immunoprecipitation of
MRP14 with either anti-grancalcin (Fig. 8, 4III and
4VI) or anti-L-plastin antibodies (data not shown). The
specific co-immunoprecipitation of grancalcin and L-plastin in the
absence of Ca2+ further corroborates the interaction
between the two proteins.

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Fig. 8.
Immunoblots of fractions of human
neutrophilic cytosol immunoprecipitated by anti-grancalcin and
anti-L-plastin antibodies. Isolated human neutrophilic cytosol in
the presence of either 0.5 mM CaCl2
(I, II, and III (+Ca2+))
or 5 mM EGTA (IV, V, and
VI ( Ca2+)) were immunoprecipitated by
anti-L-plastin (LPL4A1 (1) and LPL7,2 (2),
respectively) and anti-grancalcin (3 and 4)
antibodies, respectively. The starting material (I and
IV), the supernatant (II and V), and
the pellet (III and VI) were probed by
immunoblotting with anti-grancalcin (1 and 2),
anti-L-plastin (LPL4A1) (3), and anti-MRP14 antibodies
(4). The LPL7,2 antibody only recognizes the
Ca2+-loaded form of L-plastin.
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DISCUSSION |
Purification and N Terminus--
The purpose of this study was to
provide a detailed molecular description of the interaction of
grancalcin with Ca2+ and the ensuing conformational changes
and to identify a possible binding partner to grancalcin. The
Ca2+-dependent binding of grancalcin to a
phenyl-Sepharose column was used as a very efficient first step in the
purification of wild-type grancalcin from the cytosol of human
neutrophils. In this single chromatographic step, a limited number of
proteins, all expected to be Ca2+-binding proteins, were
highly enriched. Immunoblotting showed grancalcin to be present, and
wild-type grancalcin could be separated from the other proteins in the
eluate by subsequent ion exchange chromatography.
Sequence analysis of pure wild-type grancalcin suggested that the
N-terminal amino group is blocked. The molecular mass of wild-type
grancalcin is in agreement with posttranslational removal of the
N-terminal methionine and acetylation of the adjacent alanine without
any further modification of the protein, such as lipid attachment or
glycosylation. N-terminal acetylation is a common posttranslational
modification of cytosolic proteins (32), especially if the amino acid
succeeding the initiating methionine is glycine, threonine, serine, or
alanine (33). The N terminus of grancalcin has been reported to be
Ile15 (1); however, our data strongly suggest that this is
an artifact due to proteolysis during the purification procedure.
Initially, we isolated grancalcin that started at Ser14,
but inclusion of the protease inhibitors aprotinin and leupeptin prevented this proteolysis. Recombinant grancalcin was generated to
pursue a systematic study of grancalcin. Recombinant differs from
wild-type grancalcin by an additional Gly-Ser-Met tripeptide insert at
the N terminus, which is not acetylated. Nevertheless, our data suggest
that both proteins have very similar properties; moreover, they have
the same stability toward the denaturing agent guanidine HCl and
interact in a Ca2+-dependent manner with
biological membranes (3).
Precipitation--
One of the most salient features of grancalcin
is its high solubility in the metal-free form, whereas the
Ca2+-saturated form is partly insoluble, depending on the
protein concentration. Exposure of hydrophobic residues is probably the reason, since the protein is much more soluble in nonionic detergents and binds in a Ca2+-dependent way to
phenyl-Sepharose. Grancalcin is much more soluble in the presence of
Ca2+ than ALG-2 (12), and with working concentrations of
grancalcin below 2 µM, Ca2+ precipitation
does not seem to occur. Several EF-hand proteins bind to
phenyl-Sepharose upon binding of Ca2+, but it is unusual
for them to aggregate. It may be typical for the PEF subfamily, since
ALG-2 (12, 13) and calpain (34) also form insoluble aggregates upon
binding of Ca2+. The PEF proteins all have hydrophobic N
termini, which could be of importance for precipitation. We therefore
decided to generate N-terminal deletion mutants in order to test if
this would improve the solubility in the presence of Ca2+.
Indeed, we found that all three mutants were more soluble than the
intact protein. We conclude that the N terminus is of importance for
Ca2+-mediated precipitation. This suggests that
conformational changes upon binding of Ca2+ are transduced
into the N terminus to give important altered tertiary structure.
Unfortunately, the crystal structure of grancalcin cannot help us in
this regard, because the N terminus was disordered (11).
Ca2+ Binding--
Comparison of the sequence of
EF-hands in grancalcin with those in well known
Ca2+-binding proteins (6) led to the following predictions
about their capacity to bind Ca2+: EF1 is very
similar (64% identity) to that of calpain dVI and should bind
Ca2+; EF2 is abortive, since the -Z
position is occupied by Ala instead of Glu; EF3 is a canonical EF-hand
with the correct residues for the coordination of Ca2+; EF4
is completely abortive due to lack of oxygen-carrying residues in three
critical Ca2+-coordinating positions; and EF5 is very
similar to that of calpain dVI (i.e. an abortive
Ca2+-binding site) but well suited for dimerization. Flow
dialysis confirmed the prediction that in grancalcin only two EF-hands, likely to be EF1 and EF3, bind Ca2+ with moderate affinity
([Ca2+]0.5 = 25-83 µM) and
positive cooperativity (nH = 1.18). The
crystallization of apograncalcin revealed that EF1 is not in a
conformation that allows for Ca2+ binding, since the
X and Y liganding oxygens are directed
away from the center of the loop (11). EF3 displays a
Ca2+-binding conformation, but even in the "calcium
grancalcin" crystal only one Ca2+ ion was bound to one
EF3 in the dimer. The corresponding site in the second molecule of the
dimer is unoccupied and shows a much higher mobility. The conformations
of apograncalcin and grancalcin with one Ca2+ are very
similar. Moreover, the EF3-bound Ca2+ still seems to have a
high mobility. Thus, the following binding model can be postulated. The
first Ca2+ binds to one EF3 hand with rather low affinity
and without provoking substantial conformational changes. Then the
second EF3 binds Ca2+, leading to a reorientation of the
hydroxyl group of Ser136, which supposes a major
conformational change. The latter may activate EF1 in each subunit to
bind Ca2+. Once activated, the affinity of EF1 may be
higher than that of EF3, thus explaining the positive cooperativity.
The forms with the truncated N-terminal domain still bind two
Ca2+ ions/monomer but with an approximate 2-fold lower
affinity and absence of positive cooperativity, suggesting that this
domain is needed for cross-talk between EF1 and EF3 in fully
Ca2+-saturated grancalcin. Since the structure of
grancalcin with more than one bound Ca2+ could not be
resolved due to precipitation of the protein (11), we expect that
structural studies on the more soluble N-terminal mutants will allow
the proposed binding model to be tested. The moderate affinity of
grancalcin for Ca2+ is within the physiological range,
especially at locations near the plasma membrane or near internal
stores where the local Ca2+ changes can exceed 500 µM in activated human neutrophils (35). Furthermore, it
is likely that the Ca2+ affinity for grancalcin increases
in a hydrophobic environment as indicated by the prolonged elution of
grancalcin from the hydrophobic column as compared with other
Ca2+-binding proteins and by the higher
Ca2+-affinity in the presence of detergent. Enhanced
Ca2+ affinity when bound to membranes has been shown for
other Ca2+-binding proteins (e.g. protein kinase
C (36)).
Binding Partner--
We found that L-plastin interacts with
grancalcin and that Ca2+ regulates the interaction in a
negative fashion. L-plastin, like other plastin isoforms and fimbrin,
is a mosaic protein containing 2 EF-hands, a calmodulin-binding domain,
and two actin-binding domains (37). In resting cells, the
Ca2+-binding sites are unoccupied, and most of L-plastin is
involved in the cross-linking of F-actin fibers. Following stimulation of leukocytes with inflammatory stimuli such as
formyl-methionine-leucine-phenylalanine or immune complexes that bind
to Fc
receptors, L-plastin is phosphorylated at Ser5,
and this in turn leads to integrin activation and subsequent increased
adhesion (26, 29). At present, it is not known if the
L-plastin-grancalcin complex in the absence of Ca2+ still
has actin-bundling activity and if, in this complex, Ser5
can still be phosphorylated. Cell stimulation leads to a cytoplasmic Ca2+ rise with two consequences for L-plastin: 1) its
actin-bundling activity is inhibited, at least in in vitro
experiments (26); and 2) the L-plastin-grancalcin complex dissociates
(this study). Thus, Ca2+ regulates the activity of
L-plastin in a complex way, via endogenous EF-hand motifs and via
grancalcin. At present it is not clear if binding of Ca2+
to L-plastin, to grancalcin, or to both, is necessary to inhibit the
interaction between the two proteins. It is intriguing that calpain,
the close relative of grancalcin, is known to regulate adhesion and
thus migration through interaction with
1- and
3-integrins in Chinese hamster ovary cells (38), whereas
calpain does not affect
2-integrins, the subtype present
in human neutrophils (39). Grancalcin might thus have a role in human
neutrophils similar to that of calpain in other cell types, namely
regulation of adherence and migration through interaction with
L-plastin. Further experiments will hopefully clarify the functional
role of grancalcin and the importance of the interaction with L-plastin as well as the structural aspects of this interaction.