In an attempt to identify proteins that might
underlie membrane trafficking processes in ciliates,
calcium-dependent, phospholipid-binding proteins were
isolated from extracts of Paramecium tetraurelia. The major
protein obtained, named copine, had a mass of 55 kDa, bound
phosphatidylserine but not phosphatidylcholine at micromolar levels of
calcium but not magnesium, and promoted lipid vesicle aggregation. The
sequence of a 920-base pair partial cDNA revealed that copine is a
novel protein that contains a C2 domain likely to be responsible for
its membrane active properties. Paramecium was found to
have two closely related copine genes, CPN1 and
CPN2. Current sequence data bases indicate the presence of
multiple copine homologs in green plants, nematodes, and humans. The
full-length sequences reveal that copines consist of two C2 domains at
the N terminus followed by a domain similar to the A domain that
mediates interactions between integrins and extracellular ligands. A
human homolog, copine I, was expressed in bacteria as a fusion protein with glutathione S-transferase. This recombinant protein
exhibited calcium-dependent phospholipid binding
properties similar to those of Paramecium copine. An
antiserum raised against a fragment of human copine I was used to
identify chromobindin 17, a secretory vesicle-binding protein, as a
copine. This association with secretory vesicles, as well the general
ability of copines to bind phospholipid bilayers in a
calcium-dependent manner, suggests that these proteins may
function in membrane trafficking.
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INTRODUCTION |
Molecular life at the interface of the cell membrane and the
cytoplasmic milieu may be regulated by proteins that attach to and
detach from the membrane surface in response to signals.
Calcium-dependent, membrane-binding proteins may play such
a role. Two major protein motifs that regulate
calcium-dependent interactions with membrane lipids have
been extensively characterized: The annexin fold (1, 2), and the C2
domain (3, 4). The annexin fold appears in quartets in the annexin
family of proteins. The structure of these proteins, which has been
solved (1), consists only of the calcium and lipid binding domains,
with the exception of generally short amino-terminal domains that
provide additional regulation of the annexin or binding sites for other
proteins. In contrast, the C2 domain, for which the structure is also
known (3), is a motif that is attached to a diverse array of enzymatic
or protein interaction domains to provide calcium and/or lipid
regulation of functions inherent in other portions of the
protein. Examples of C2 domain-containing proteins include protein
kinase C (5), phospholipase C (6), synaptotagmin (7), rabphilin (8), Doc2 (9), and Munc13 (10).
The absence of clear enzymatic activities has made the functions of the
annexins difficult to determine. Their interaction with membranes can
lead to modulation of other membrane-binding proteins such as
phospholipases (11). They also exhibit a "bivalent" activity in the
sense that they can bind to two membranes and therefore draw them
together into a complex that is subject to fusion with additional
perturbation of membrane structure (2, 12, 13). This activity, as well
as relevant localizations of some annexins (14, 15), has led to the
proposal that the annexins may mediate membrane-trafficking events.
However, some proteins containing C2 domains, such as the cytoplasmic
portion of synaptotagmin, are endowed with similar attributes (16), so
it is difficult to define activities unique to annexins.
We recently attempted to characterize calcium-dependent,
membrane-binding proteins from Paramecium tetraurelia
because of some of the unique cytological and genetic characteristics
of this organism. Our approach depended on the isolation of
membrane-binding proteins from EGTA extracts of homogenized cells, an
approach that has been very effective in isolating annexins from a wide variety of organisms (17-19). However, the major protein we obtained from Paramecium by this approach was not an annexin but a
novel protein with two copies of the C2 domain and one copy of a domain related to the A domain that mediates protein-protein interactions between integrins and their extracellular ligands.
Because the Paramecium protein associates with lipid
membranes, like a "companion," we have given the protein a name
reflecting this property: copine (pronounced "ko-peen
"), from the
French feminine noun copine, which means "friend."
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EXPERIMENTAL PROCEDURES |
Purification of Paramecium Copine--
P.
tetraurelia, wild-type stock d4-2, was grown at 27 °C in an
infusion of wheat grass powder (Pines International, Lawrence, KS)
inoculated with Klebsiella pneumoniae and supplemented with
-sitosterol (4 µg/ml) according to Sonneborn (20). Twelve to 18 liters of cell culture at a density of 2,000-4,000 cells/ml were
harvested by centrifugation yielding a wet cell pellet of 5-10 ml. All
subsequent steps were carried out on ice or at 4 °C. The cells were
washed once with spring water (Volvic) and resuspended in 3 volumes of
homogenization buffer (150 mM NaCl, 50 mM
HEPES, pH 7.4, 5 mM EGTA, 50 µM
phenylmethylsulfonyl fluoride, and 5 µM leupeptin). The
cells were homogenized with 40 strokes of a Potter-Elvehjem homogenizer
with a tightly fitting pestle. The volume was increased to 50 ml by the
addition of homogenizing buffer, and the cells were further homogenized
with five strokes of a tightly fitting Dounce homogenizer. By phase
microscopy it was found that this procedure resulted in the lysis of
all cells, but large organelles appeared intact. The homogenate was
centrifuged at 27,000 × g for 15 min, the pellet was
discarded, and the supernatant was centrifuged at 200,000 × g for 1 h to prepare a postmicrosomal supernatant.
Multilamellar liposomes were prepared from 200 mg of a bovine brain
lipid fraction enriched to 80% in phosphatidylserine (Sigma product
B-1502) by sonication in 150 mM NaCl, 50 mM
HEPES-NaOH, pH 7.4, as described previously (21). The vesicles were
pelleted at 200,000 × g for 1 h, and the
postmicrosomal supernatant was used to resuspend the lipid pellet with
10 strokes of a tightly fitting Dounce homogenizer. The free calcium
level of the suspension was adjusted to approximately 3 mM
by the addition of 8 mM CaCl2. The pH, reduced
by release of protons from EGTA, was returned to 7.4 by the addition of
NaOH. The vesicles were sedimented by centrifugation at 200,000 × g for 1 h, and the supernatant was discarded. The
pellet was resuspended in wash buffer (150 mM NaCl, 50 mM HEPES-NaOH, pH 7.4, 2 mM CaCl2,
50 µM phenylmethylsulfonyl fluoride, 5 µM
leupeptin) by 10 strokes with a Dounce homogenizer (first wash). The
vesicles were sedimented as above and then resuspended in wash buffer
without NaCl (second wash). After sedimentation, the vesicles were
resuspended in 10 ml of extracting buffer (25 mM
HEPES-NaOH, pH 7.4, 10 mM EGTA, 50 µM
phenylmethylsulfonyl fluoride) and sedimented as above. The
supernatant, containing calcium-dependent lipid-binding
proteins, was saved. The lipid pellet was resuspended in extracting
buffer again and sedimented as before, providing a second extract. In
some cases a third extraction was performed, yielding about 20%
additional protein. The extracts from the lipid vesicles were pooled
and applied to a Poros Q anion exchange column (PerSeptive Biosystems,
Cambridge, MA) equilibrated in 25 mM HEPES, pH 7.4. The
flow-through fractions from this column contained the purified copine.
Assay of the Lipid Binding and Aggregating Activities of
Copine--
Phospholipid binding activity of purified copine was
determined by incubation of 5 µg of copine with 0.5-1 mg of
phospholipid vesicles prepared as for the purification procedure above
in the presence of 2 ml of 25 mM HEPES, pH 7.4, 8 mM EGTA, with or without 10 mM
CaCl2 or MgCl2 at room temperature. After
sedimentation at 100,000 × g for 30 min, or
12,000 × g for 10 min, the supernatants were desalted
on Sephadex G-25 and lyophilized before applying to SDS-polyacrylamide
gels. The pellets were resuspended directly in gel starting buffer and
applied to SDS-polyacrylamide gels. The aggregation of lipid vesicles
was determined by measurement of the turbidity (absorbance at 540 nm)
of the vesicle suspensions in the binding assay after incubation with 5 µg of copine for 30 min to 1 h.
Determination of the Partial Sequence of Paramecium
Copine--
50 µg of purified copine was applied to an
SDS-polyacrylamide gel. The 55-kDa band was stained with
Coomassie Blue and excised, and the protein was isolated by
electroelution in an Amicon electroelution cell by application of 100 V
for 2 h. SDS was removed from the eluted protein by precipitation
of the protein in chloroform and methanol (23). The protein was then
subjected to digestion with lysyl endoprotease purified from
Achromobacter lyticus (Waco Products, Richmond, VA), and the
resulting peptides were isolated by HPLC, as described (24). Peptides
were sequenced by Edman degradation on an Applied Biosystems model 470A
gas phase sequencer coupled to a 120A phenylthiohydantoin analyzer.
Degenerate oligonucleotides were synthesized corresponding to the
peptide sequences, favoring codon usage in Paramecium (25). Successful PCR1 reactions
(see "Results") were obtained using the following primers, which
incorporated EcoRI restriction sites (underlined).
Note that the design of these primers takes advantage of the
almost exclusive use by Paramecium of AGA as the codon for arginine (25). Standard PCRs were conducted with Taq
polymerase and 30 cycles of 94 °C for 1 min, 52 °C for 2 min, and
72 °C for 3 min. cDNA from P. tetraurelia, prepared
as described by Madeddu et al. (26), was used as template.
The major product had an apparent size of 900 base pairs on an agarose
gel. It was eluted and subcloned into the plasmid Bluescript SK after
digestion with EcoRI. Because of the presence of internal
EcoRI restriction sites in the PCR product, interpretation
of the sequence was not straightforward. Therefore, a second PCR
product obtained using the same primers and cDNA sample was
subcloned into the TA cloning vector pCRII (Invitrogen, San Diego, CA)
for sequencing. The clones obtained by these two procedures arose from
different genes (see "Results").
Construction of Expression Vectors for Human Copine I--
Two
overlapping partial cDNAs corresponding to expressed sequence tags
(ESTs) for human copine I were obtained from the American Type Culture
Collection (IMAGE Consortium clone identification numbers 51016 and
487481, corresponding to GenBankTM accession numbers H19014
and AA043485, respectively). Sequencing of these cDNAs was
completed, permitting the development of the following strategies for
expression of full-length copine I and fragments of copine I. Numbering
the coding sequence from 1 to 1613, oligonucleotides were synthesized
to amplify copine I nucleotides 4-743 from clone AA043485,
incorporating an XbaI site at the 5
-end and an
XhoI site at the 3
-end: upstream primer,
5
-GCGCTCTAGAGGCCCACTGCGTGACCTTGG; downstream primer,
5
-CCAGCTCGAGCTACTTCTCAGGGTGGATGCATTG.
Using this primer pair and ligating the PCR product into pGEX-KG (27)
cut with XbaI and XhoI created an expression
vector that would produce a fusion protein of glutathione
S-transferase (GST) and most of the two C2 domains of copine
I.
To construct a vector to express the full-length copine,
oligonucleotides were constructed to amplify copine I nucleotides 528-1613 from clone H19014. These primers incorporate the naturally occurring BglII site at nucleotide 528 and an
XhoI site immediately following the termination codon:
upstream, 5
-GTGTACAGATCTGAGGTCATCAAG; downstream,
5
-CCAGCTCGAGCTAGGCCTGGGGGGCCTGTGC.
Using this primer pair and ligating the PCR product into the construct
described above for the C2 domains cut with BglII and XhoI created an expression vector (pGEX-copine I) for
full-length copine I fused to GST.
To express the C-terminal half of copine I, comprising the A domain, a
third upstream primer was designed corresponding to residues
759-783 and incorporating an XbaI site: upstream,
5
-GCGCTCTAGAAAGCTACAAGAACTCTGGAACTATCC.
Using this primer and the primer corresponding to the C terminus of the
protein, cDNA encoding the A domain was amplified from clone H19014
and ligated into pGEX-KG cut with XbaI and XhoI.
Expression of GST-Copine I A Domain and Production of an
Antiserum--
E. coli strain XL1 Blue (Stratagene)
harboring pGEX-KG with the copine I A domain was induced to produce the
GST-copine I A domain fusion protein as described (27, 28). Total
bacterial cell homogenates were run on an SDS-polyacrylamide gel. After staining briefly with Coomassie Blue, approximately 500 µg of the
GST-copine I A domain band of mass 55 kDa was excised, adjusted to pH
7.4 by incubation with phosphate-buffered saline, and then homogenized
with Freund's complete adjuvant for immunization of rabbits. Booster
injections were performed at 2 and 4 weeks using antigen similarly
prepared and mixed with Freund's incomplete adjuvant. Antiserum
reacting with copine I was initially detected by Western blot after 4 weeks.
Affinity-purified antibodies directed against the fusion protein or
specifically against the copine I A domain were prepared by incubation
of serum with the corresponding antigens immobilized on nitrocellulose
(29). The antigens were purified after solubilization in sarcosyl as
described by Frangioni and Neel (30) and binding to glutathione-agarose
beads (Sigma). After removal of the sarcosyl, the antigens were
insoluble and could not be eluted from the beads with glutathione.
Cleavage with thrombin could be effected (27), but the copine A domain
fragment was insoluble. Therefore, the antigen and bead complexes or
SDS extracts of the beads were applied to SDS gels to further purify
the antigens by electrophoresis.
Expression and Isolation of Full-length GST-Copine I and
Lipid-binding Studies--
E. coli strain XL1 Blue
(Stratagene) harboring pGEX-KG with full-length copine I was induced to
produce the GST-copine I fusion protein. The fusion protein was
extracted in sarcosyl (30) and bound to glutathione-agarose beads. A
portion of the fusion protein was eluted from the beads with 150 mM NaCl, 50 mM TRIS-HCl, pH 8.0, 1 mM EGTA, and 10 mM glutathione. The eluate was
clarified by centrifugation at 6,000 × g for 10 min.
Less than 5% of the protein bound to the beads initially was obtained
in the supernatant, suggesting poor solubility of the fusion protein or
poor extraction with glutathione. The supernatant was adjusted to pH
7.3 by the addition of HCl and then incubated in 135-µl aliquots
containing 100 ng of protein for 10 min at room temperature in the
presence or absence of 3.5 mM CaCl2 and
approximately 250 µg of phosphatidylserine-enriched brain lipid
vesicles (Sigma B-1502). After centrifugation at 6,000 × g for 10 min, the pellets and supernatants were examined by Western blotting with the affinity-purified anti-copine I
antibodies.
General Methods--
Standard methods of molecular biology and
recombinant DNA technology were as described in Ausubel et
al. (31). For Southern blotting (see Fig. 5), hybridization was
performed at 55 °C in Church buffer, and washes were at 55 °C in
2 × SSC, 0.1% SDS, followed by 0.2 × SSC, 0.2% SDS (31).
Macronuclear DNA for Southern blot analysis was prepared as described
(32). DNA sequencing was performed using the Sanger method on an ABI
Prism 377 automated DNA sequencer. SDS-polyacrylamide gels were run
according to Laemmli (22), and Western blotting was performed according
to Burnette (33) using horseradish peroxide-coupled secondary
antibodies and colorimetric detection with 4-chloronapthol (see Fig. 9)
or chemiluminescence (see Fig. 8; Pierce). Protein was assayed by the
Bradford (34) method, using bovine serum albumin as a standard. The
chromobindin fraction of adrenal medullary cytosol was prepared by
affinity chromatography as described (35).
 |
RESULTS |
Isolation of a 55-kDa Phospholipid-binding Protein from Extracts of
Paramecium--
Calcium-dependent, phospholipid-binding
proteins were isolated from the soluble fraction of homogenates of mass
cultures of P. tetraurelia by binding to multilamellar
vesicles prepared from brain lipid extracts enriched in
phosphatidylserine. Fig. 1 shows an SDS
gel of fractions obtained from a typical preparation. Lane S
is the postmicrosomal supernatant that was prepared in EGTA, representing all of the soluble proteins of the homogenate. Lane PC represents the supernatant remaining after adding lipid
vesicles and 3 mM excess free calcium to the postmicrosomal
supernatant and then sedimenting the vesicles by centrifugation. Note
that this procedure does not cause a visible reduction in any of the bands in the postmicrosomal supernatant. Thus, none of the major proteins of the cytosol appear to bind lipids. The vesicles were then
washed twice in high and low ionic strength buffers to remove nonspecifically bound proteins. Lanes E1 and E2
represent the proteins obtained by extracting the brain lipid vesicles
with a buffer containing 10 mM EGTA to remove proteins that
have bound to the lipids in a calcium-dependent manner.
Only small amounts of protein are obtained. In a typical preparation,
70 mg of protein was present in the initial postmicrosomal supernatant,
while the first extract from the lipid vesicles contained 360 µg of
protein, and the second extract contained 200 µg.

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Fig. 1.
Isolation of copine from P. tetraurelia. Fractions obtained during the isolation of
copine were examined by SDS-polyacrylamide gel electrophoresis.
Lane S, postmicrosomal supernatant. Lane PC,
postcalcium supernatant obtained from the postmicrosomal supernatant by
adding phosphatidylserine vesicles in the presence of 2 mM free calcium and sedimenting the vesicles. Lane E1, first
EGTA extract from the washed lipid vesicles, containing
calcium-dependent lipid-binding proteins. Lane
E2, second EGTA extract from the washed lipid vesicles, containing
additional lipid-binding proteins; C marks the position of
copine at 55 kDa. Lane QFT, flow-through of POROS-Q anion
exchange column obtained after applying the E1 and
E2 extracts. The single band represents purified copine. The numbers on the left are the molecular masses in
kDa of the marked migration positions of prestained molecular weight
standards.
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The major protein in the EGTA extracts from the lipid vesicles had an
apparent mass of 55 kDa. Interestingly, the protein was obtained in
greatest amount in the second EGTA extract. This is indicative of a
very high sensitivity to calcium, since two washes in 10 mM
EGTA were necessary to reduce the concentration of calcium sufficiently
(10
7 M or less) to remove the protein.
The 55-kDa protein was purified to homogeneity (Fig. 1, lane
QFT) by passage over the fast protein liquid chromatography anion exchange medium Poros-Q, since it did not adhere to this resin, while
other proteins in the extracts were retained. The typical yield of the
purified protein was 50-70 µg, thus representing about 0.1% of the
protein in the initial postmicrosomal supernatant. This purified 55-kDa
protein is henceforth referred to as "copine," as discussed in the
Introduction.
Characterization of the Interaction of Purified Copine with
Phospholipids--
The small amounts of copine that could be obtained
limited the degree of characterization possible. Emphasis was put on
comparing the calcium and lipid specificities of copine in relation to
those of the annexins and C2 domain-containing proteins. These
specificities were tested in a centrifugation assay using multilamellar
brain lipid vesicles. The purified copine was incubated with the
vesicles under various conditions. The vesicles were then sedimented,
and the supernatants and pellets were analyzed for copine by SDS gel electrophoresis.
Fig. 2 illustrates an SDS gel of the
supernatants from a typical binding experiment, and Table
I summarizes the data from several
binding experiments. It was found that calcium alone (i.e., in the absence of phospholipids) caused the copine to pellet at high
g force (100,000 × g), implying that a
calcium-dependent self-association of the protein was
occurring. This self-association was calcium-specific, in that
magnesium did not promote the pelleting of copine. At a lower
centrifugal force, 12,000 × g, sufficient to sediment
the lipid vesicles, copine was not pelleted unless phospholipids were
present, suggesting the copine bound to the vesicles. The copine was
evidently not destroyed, since it could be recovered from the pellets,
as it was during the initial copine isolation procedure. The
association with the lipid vesicles did not occur when magnesium was
substituted for calcium (Fig. 2 and Table I) or when
phosphatidylcholine vesicles were used instead of phosphatidylserine
(Table I). All of these characteristics are typical of annexins or
proteins that contain C2 domains.

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Fig. 2.
Calcium-dependent sedimentation
of purified copine. Copine (2.5 µg/ml) was incubated with
divalent cations and phosphatidylserine vesicles (250 µg/ml; ~300
µM) in a volume of 2 ml and assayed for sedimentation at
100,000 × g. SDS-polyacrylamide gels of the resulting
supernatants are shown. Lane ENS, copine in 8 mM
EGTA without centrifugation (control for total amount of copine in the
assay). Lane E, copine in 8 mM EGTA, supernatant
after centrifugation. Lane Ca, copine in 2 mM
free calcium after centrifugation. The protein has pelleted. Lane
E + L, copine plus phosphatidylserine vesicles in EGTA after
centrifugation. Lane Ca + L, copine plus phosphatidylserine
vesicles in 2 mM free calcium. Lane Mg + L, copine plus phosphatidylserine vesicles in 2 mM free
magnesium. C marks the position of the copine band. The
migration positions of molecular weight standards are marked on the
left with the corresponding masses in kDa.
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Table I
Binding of Paramecium copine to lipids
Table entries indicate whether copine was sedimented (+) or remained in
the supernatant ( ) when centrifuged at the indicated g
force in the presence of EGTA, calcium (Ca), magnesium (Mg), phosphatidylserine vesicles (PS), or phosphatidylcholine vesicles (PC).
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It was also observed that under conditions where copine bound to the
lipid vesicles, it exhibited a "bivalent" activity. The protein
promoted the aggregation of the vesicles, which could be detected as an
increase in the turbidity of the vesicle suspension (Fig.
3).

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Fig. 3.
Copine promotes calcium-dependent
aggregation of phosphatidylserine vesicles. Phosphatidylserine
vesicles (250 µg/ml; ~300 µM) were incubated with 8 mM EGTA or 2 mM free calcium in the presence or
absence of 2.5 µg/ml of copine. The A540 of
the suspension was measured after 30 min. The initial
A540 of all suspensions was approximately
0.15.
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Cloning of a Partial cDNA for Copine--
Purified copine was
excised from a Coomassie-stained SDS gel, eluted, and subjected to
hydrolysis with lysyl-endoprotease to generate peptides for direct
sequencing. Six peptides isolated by HPLC were sequenced (Table
II). These short sequences did not show
significant similarity to known protein sequences.
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Table II
Peptides derived from Paramecium copine
The initial lysine in parentheses (K) was assumed to be present due to
the use of lysyl endoprotease to generate the peptides.
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The peptide sequences were used to design degenerate oligonucleotides
to amplify corresponding sections of DNA by the polymerase chain
reaction from P. tetraurelia cDNA. Since the order of
the peptides in the copine sequence was unknown, oligonucleotides in
both orientations were prepared corresponding to three peptides (Table
I, peptides p55.1, p55.2, and p55.3) and were used in PCR reactions in
all possible combinations. Possibly due to the high degeneracy of the
oligonucleotides (256-512-fold), many amplified products were seen on
agarose gels of the reaction products. To narrow down the range of
candidates for further study, particular attention was paid to
amplification products that were of a size corresponding to the sum of
the sizes of products using other primer pairs. However, the most
promising products obeying such rational rules were determined to be
false positives by subcloning and sequencing. Redesign of the
oligonucleotides and additional peptide sequence information (p55.4)
finally led to the very strong amplification of a 920-base pair product
with one set of primers representing portions of peptides P55.2 and
P55.4 (Table II; also see "Experimental Procedures"). Subcloning
and sequencing of this product verified that it contained a single open
reading frame incorporating the sequence of peptide P55.4 used for
primer design beyond the region used for the primer per se
(Fig. 4). In addition, the PCR product
contained the sequence of a third peptide (P55.6, Table II). The primer
corresponding to peptide P55.2 was designed using sequence at the N
terminus of the peptide and was incorporated at the 3
-end of the
amplified cDNA; thus, no verification of the sequence at this end
was possible. However, the amino acid residues encoded by the primer
sequence were in the same reading frame as the other two peptides.
These data, as well as additional features of the Paramecium
and homologous sequences described below, verified that the correct
product had been obtained. In retrospect, the most obvious
characteristic of the correct PCR product compared with the false
positives was that it was obtained in much greater amounts, as
indicated by ethidium bromide staining on agarose gels.

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Fig. 4.
Sequence of Paramecium
copine. The nucleotide sequence of the partial cDNA
corresponding to gene CPN1 is numbered 1-920 (GenBankTM accession number U64872). Nucleotide
substitutions present in the sequence of the cDNA corresponding to
gene CPN2 are indicated above the CPN1
cDNA sequence. The translation of the CPN1 cDNA, using the ciliate genetic code, is given below the
nucleotide sequence. Substitutions in the translated sequence of the
CPN2 cDNA are given below the amino sequence
for CPN1. Portions of the amino acid sequence present in
peptides (Table II) are underlined. The peptide at the N
terminus has been added to maximize the alignment with other C2 domains
(see text). Underlined nucleotide sequences at the 5 - and
3 -ends are in the positions of the degenerate primers used for
amplification and are therefore not necessarily identical to the native
sequences.
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Comparison of the Paramecium copine sequence with current
data bases indicated strong similarity with C2 domain-containing proteins in the region of the C2 domain. This similarity could be
extended if an additional peptide (P55.1 in Table II) were added to the
N terminus of the sequence encoded by the partial cDNA (Fig. 4).
The presence of the C2 domain provided a rational explanation for the
biochemical properties of copine and thus was further evidence that the
correct cDNA had been amplified.
Two Copine Genes Are Present in Paramecium--
Peptides P55.4 and
P55.5, in their region of overlap, are identical except for the
substitution of one arginine for a lysine (Table II). This suggested
that there may be at least two closely related copine gene products.
Additional evidence for this came from the cDNA cloning.
Two different strategies were used to subclone the copine cDNA
product produced by PCR. The two strategies yielded two different copine sequences encoded by different genes (CPN1 and
CPN2). In one case the PCR product was cut with
EcoRI, taking advantage of the EcoRI sites that
had been designed into the oligonucleotide primers to facilitate
subcloning. However, due to the presence of internal EcoRI
sites in the PCR product and the formation of concatamers during the
ligation process a subclone was obtained that contained four pieces of
the cDNA in random orientations. To verify that the correct
sequence had been visualized from this complex clone and that no small
fragments had been omitted, the subcloning was repeated by performing a
new PCR and subcloning the uncut insert using the TA cloning method
(Invitrogen). The nucleotide sequence of the clone obtained this way
(corresponding to the gene we have named CPN1) was 91%
identical to the original reconstructed clone (corresponding to gene
CPN2). Of the 79 base differences between these clones, only
13 result in changes in the encoded amino acids (Fig. 4), indicating a
strong evolutionary pressure to retain the amino acid sequence, which
is 96% identical between the two clones.
To investigate the total number of closely related copine genes in the
Paramecium genome, a Southern blot analysis was performed on
Paramecium macronuclear DNA cleaved with various restriction enzymes using the copine cDNA as a probe and conditions of high stringency for hybridization. As seen in Fig.
5, in most digests two genomic
restriction fragments bound to the probe with equal intensity,
indicating the presence of two closely related genes for copine.

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Fig. 5.
Southern blot of Paramecium
macronuclear DNA probed with the copine cDNA. DNA from
P. tetraurelia was cleaved with the restriction enzymes
marked, resolved by electrophoresis in an agarose gel, transferred to
nitrocellulose, and probed with the copine cDNA. Two bands of
similar intensity are seen in most lanes, suggesting the presence of
two closely related copine genes. Numbers on the
right indicate the migration distances of mass standards in
kilobases (kb).
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The Copine Family of Proteins--
Probing current data bases with
the copine cDNA sequence revealed the existence of a number of
uncharacterized sequences from genomic sequencing projects that are
similar to that of copine. These included multiple human ESTs, open
reading frames found in nematode genomic sequences, and ESTs and
genomic sequences from rice and Arabidopsis.
Representative cDNAs corresponding to the human ESTs were obtained
from the American Type Culture Collection and sequenced. The sequences
could be organized into groups representing five different human genes
(Fig. 6), which we refer to as human
copines I-V. The degrees of identity between the amino acid sequence
of copine I and the other human copines, in the known regions of overlap, are as follows: copine II, 60%; copine III, 78%; copine IV,
53%; copine V, 56%.

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Fig. 6.
Amino acid sequences of five human
copines. The full-length sequence of human copine I is aligned
with the partial sequences of human copines II, III, IV, and V. The two
C2 domains are shaded in dark gray. A consensus
sequence consisting of residues present in at least 50% of 65 previously characterized C2 domains (4) is given in the top
line. The A domain is shaded in light gray.
Residues that are identical in all sequences (where known) are
boxed. X, residues thought to chelate calcium (in
the C2 domains) or magnesium (in the A domain). O, conserved
histidine present in the copine A domain. The GenBankTM
accession number for the copine I sequence is U83246. The accession
numbers of representative ESTs corresponding to these copines are as
follows: copine I, H19014; copine II, R87434; copine III, N72351;
copine IV, H29499; copine V, H09181.
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Five different nematode copine genes have also been analyzed
(GenBankTM accession numbers Z80223, Z73911, U21317,
Z68213, and U28941). An alignment of the inferred amino acid sequences of representative copines from Paramecium, human, nematode,
and Arabidopsis is given in Fig.
7. The degree of identity between the
human sequence and the other sequences, in the region of overlap, is as
follows: nematode, 40%; Arabidopsis, 40%;
Paramecium, 33%. The greatest degree of conservation is
seen in residues characteristic of the C2 domain and the integrin A
domain (see "Discussion"). Curiously, the conceptualized
Arabidopsis sequence begins in the middle of the first C2
domain. No additional exons that could complete the sequence of this
domain are apparent in the cosmid clone (GenBankTM
accession number AC000106) upstream of the copine gene and downstream
of the preceding gene. However, it is not known if this conceptualized
protein is indeed expressed.

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Fig. 7.
Amino acid sequences of copines from
divergent organisms. The partial sequence of copine from P. tetraurelia (GenBankTM accession number U64872) is
aligned with the full-length human copine I sequence (U83246) and
copine sequences from the nematode Caenorhabditis elegans
(Z80223) and the green plant Arabidopsis thaliana
(AC000106). The two C2 domains are shaded in dark gray. A consensus sequence consisting of residues present in at least 50% of 65 previously characterized C2 domains (4) is given in
the top line. The A domain is shaded in light
gray. Residues that are identical in the majority of sequences
where known (3 of 4, 2 of 3, or 2 of 2) are boxed.
X, residues thought to chelate calcium (in the C2 domains)
or magnesium (in the A domain). O, conserved histidine
present in the copine A domain. To achieve optimal alignment of the
Arabidopsis sequence, minor adjustments were made to the
intron/exon boundaries previously interpreted in the
GenBankTM entry.
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Expression of Recombinant Human Copine I--
Portions of two
cDNAs corresponding to ESTs for human copine I were ligated into
the expression vector pGEX-KG (27) to create vectors for the expression
of fusion proteins consisting of GST fused to copine I or domains of
copine I (see "Experimental Procedures"). The fusion proteins were
expressed to a high level upon induction of the tac promoter
in E. coli; however, the proteins were only poorly soluble
(less than 5%). A fusion protein of GST and the C-terminal half of
copine I was partially purified by electrophoresis in SDS and used for
the immunization of rabbits. The antiserum produced, as well as
antibodies purified by adsorption on copine I immobilized on
nitrocellulose, reacted strongly with recombinant human copine I and,
in a preliminary survey, with a 55-kDa protein in a variety of rat
tissues, including heart, lung, kidney, liver, and skeletal muscle.
Calcium-dependent Binding of Recombinant Human Copine I
to Phosphatidylserine Vesicles--
The soluble portion of the
full-length copine I-GST fusion protein produced in bacteria was tested
for the ability to bind lipid vesicles in a
calcium-dependent manner. Similar to the behavior of
Paramecium copine, the recombinant human protein bound to
and sedimented with vesicles enriched in phosphatidylserine in a
calcium-dependent fashion when tested in a centrifugation
assay (Fig. 8). Also similar to the
Paramecium protein, calcium caused the apparent
self-association of the human copine, since a portion of the protein
sedimented in a calcium-dependent manner in the absence of
lipid (Fig. 8). GST by itself did not sediment with or without lipid in
a calcium-dependent manner (not shown), suggesting that the
calcium-dependent behavior of the fusion protein was due to
the copine moiety.

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Fig. 8.
Calcium-dependent binding of
recombinant human copine I to phosphatidylserine vesicles.
Approximately 100 ng of copine I-GST fusion protein was incubated with
250 µg of (~2 mM) phosphatidylserine vesicles
(PS) and 1 mM EGTA (EGTA), or 2.5 mM free Ca2+ (Ca) as detailed under
"Experimental Procedures." After sedimentation by centrifugation,
the supernatants and the lipid vesicle pellets were examined for the
presence of copine by Western blotting with an affinity-purified
anti-copine I antibody. Migration positions of molecular weight
standards are marked on the left with the corresponding
masses in kDa. Note that the copine is partially pelleted in the
presence of calcium alone and is completely pelleted in the presence of
calcium and lipid together.
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Identification of Chromobindin 17 as Copine--
The properties of
copine suggested that it might be a member of the chromobindins, a
class of soluble proteins that bind to chromaffin granule membranes in
the presence of calcium (35). Accordingly, the antiserum to human
copine I was used to probe a Western blot of the chromobindin fraction
obtained from bovine adrenal medullary cytosol. The band corresponding
to the protein catalogued in 1983 as chromobindin 17 (35) reacted
strongly with the copine antiserum as well as with antibodies purified from the serum by adsorption on the recombinant GST-copine I A domain
fusion protein (Fig. 9). The same result
was obtained with antibodies purified by adsorption on the copine I A
domain purified after cleavage from the GST (not shown). Since
chromobindin 17 has been characterized as a protein that binds to
phospholipids as well as intact chromaffin granule membranes (35), is
of the appropriate mass (55 kDa), has a particularly high affinity for calcium (35), and, like copine I, has a broad tissue
distribution,2 we conclude
that chromobindin 17 is a copine. Because of sequence similarities
among the various mammalian copines that might result in immunological
cross-reaction, we are not certain whether chromobindin 17 corresponds
to copine I or another member of the copine family. Chromobindin 17 is
frequently present in chromobindin fractions from various tissues as
the upper band of a doublet (as seen in Fig. 9). It is possible that
the lower band represents another member of the copine family.

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Fig. 9.
Identification of chromobindin 17 as a
copine. Chromobindins, calcium-dependent chromaffin
granule-binding proteins from bovine adrenal medulla, were eluted from
a chromaffin granule membrane affinity column with EGTA,
electrophoresed in SDS, and transferred to a nitrocellulose strip that
was stained in Ponceau S (lane P). 67, the
position of annexin VI; CB17, the position of chromobindin
17 (the upper band of a closely spaced doublet); 32-36, the
positions of annexins I, II, IV, and V. Chromobindins from a parallel
gel were transferred to nitrocellulose and probed with the copine I
preimmune (lane PI) or immune (lane I) serum. The
single protein reacting with the antiserum (C) corresponds to chromobindin 17 (CB17). A weakly staining band is seen in
the preimmune serum but is of higher mobility than the band reacting with the immune serum. Lanes A1 and A2 represent
chromobindins stained with antibodies from two different rabbits
affinity-purified by adsorption to recombinant copine I.
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DISCUSSION |
Calcium-dependent, membrane-binding proteins have been
isolated by affinity techniques from a number of different organisms (e.g. Refs. 17-19 and 35). Typically, these techniques
yield a complex mixture of proteins. The proteins obtained in the
greatest yield when using lipids or biological membranes as the
affinity reagent and the soluble fractions from either plants or
animals have always been annexins, although protein kinase C (35),
phospholipase C (36), and other unrelated proteins (37) have been
obtained in smaller amounts. Paramecium is thus unusual in
that the major protein, copine, falls into a different class. Although
no annexin has been definitively characterized from
Paramecium, there are reports of Paramecium
proteins that cross-react with anti-annexin antisera
(38).3 It is possible that
some of the additional proteins obtained in the lipid-binding fraction
in this study are annexins; however, we detected no immunological
cross-reaction between these proteins and antisera raised against
mammalian annexin I, II, IV, VI, or VII or nematode Nex-1 annexin (data
not shown). Copine is the first Paramecium protein to be
characterized that possesses C2 domains, thus extending the breadth of
this family of lipid-binding proteins to include ciliates.
Organization of Domains in the Structure of
Copine--
Examination of the full-length sequences of a human
copine, a nematode copine and a plant copine (Fig. 7) reveals a very
interesting domain structure for copine. There are two C2 domains in
the N-terminal half of the molecule. Both C2 domains appear to be
functional (with the exception of the plant sequence; see
"Results") in the sense that they contain acidic residues
implicated in the binding of calcium by the first C2 domain of
synaptotagmin (3, 4).
In general, there are two distinct topologies for C2 domains (4).
Residues corresponding to the first
-strand in the C2 domain of
topology type I are found in the same structural position as the eighth
-strand of the C2 domain of topology type II. The topologies can be
recognized on the basis of primary sequence depending upon whether the
amino acid residues corresponding to this strand are found before (as
in synaptotagmin type I) or after (as in phospholipase C type II) the
sequences representing the rest of the domain. In copine, both C2
domains are type II. For example, in human copine I these wandering
-strands occur at residues 125-133 and 265-273, at the trailing
ends of the C2 domains (Fig. 6). Thus, the organization of the C2
domains of copine obeys two previously recognized generalizations
concerning C2 domains (4): 1) if present at the N terminus of a
protein, the C2 domain adopts the type II topology; and 2) if two C2
domains are adjacent to one another, they have the same topology.
The sequence of the C-terminal half of the copine molecule shows a
distant relationship to the A domain found in a number of extracellular
proteins or the extracellular portions of membrane proteins such as
integrins; von Willebrand factor; complement factor C2; L-type calcium
channels; collagens VI, VII, XI, and XIV; and the plasmodial surface
protein thrombospondin-related anonymous protein (39, 40).
Interestingly, there are no other examples of A domains in
intracellular proteins, so copine represents a unique fusion of a
domain typically found in intracellular proteins, the C2 domain, and a
domain typically found in extracellular proteins, the A domain. There
are no signal sequences or hydrophobic transmembrane sequences
evidently coded for by the cDNAs or genomic sequences of the
various copine homologs. Furthermore, the Paramecium protein could not be extracted unless the cells were lysed, suggesting that
copine is an intracellular protein.
The three-dimensional structure of the A domain (also called I domain)
from the
-subunit of integrin CR3 (CD11b/CD18) has recently been
determined (40). This integrin is a member of the
-2 integrin family
and is the major integrin of phagocytic cells. The A domain appears to
mediate the binding of the integrin to extracellular ligands in a
magnesium-dependent fashion. The similarity between copine
and the integrin A domain is strongest in the C-terminal
of
the domain (Fig. 10). The hydrophobic
motifs at the end of the D and E
-strands of the integrin domain are
particularly characteristic in the copine family. In addition, residues
that participate in the coordination of magnesium, either directly or
through intervening water molecules, in the A domain crystal structure
are also present in the copine A domain (Fig. 10). The presence of the
corresponding metal-chelating residues suggests that this domain of
copine may bind target molecules in a magnesium dependent fashion just
as magnesium is required for the binding of the integrin A domain to
target proteins in the extracellular matrix.

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Fig. 10.
Sequence and structural comparison of the
copine C-terminal domain and the integrin A domain. Portions of
the human copine I sequence (residues 292-512) are aligned with
portions of the sequence of the A domain of the -subunit of human
integrin CR3 (residues 8-191). The alignment was generated using the
FASTA program; colons mark amino acid identities;
periods marks amino acid similarities. The positions of
-helices and -strands as determined by crystallography are marked
below the A domain sequence. -Helices and -strands
predicted to be present in copine by the algorithm of Chou and Fasman
(42) are marked above the copine sequence. Residues in the A
domain sequence that participate in the chelation of magnesium are in
boldface type, underlined, and labeled according
to residue number. Aligned residues in the copine sequence that may
play an analogous role are similarly marked.
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In addition to the sequence similarities, it is notable that the
secondary structure predictions obtained for this portion of the copine
molecule using the algorithm of Chou and Fasman (41) are in excellent
agreement with the actual secondary structure of the corresponding
portions of the A domain as determined by crystallography, particularly
near the metal-chelating residues (Fig. 10). This agreement suggests
that this portion of copine may indeed adopt secondary and tertiary
structures similar to those of the A domain fold. However, as shown in
Fig. 10, the upstream metal-chelating residues (DGSGS in the integrin;
hypothetically DFTGS in human copine I) are displaced relative to the
downstream chelating residues by an additional 29 amino acids, and the
sequence and structural predictions for this intervening region of
copine cannot readily be aligned with the A domain. Thus, it is likely
that in copine there are significant additions to the loops that may
extend from the core of the A domain-like structure.
The putative copine A domain has a histidine near the beginning of the
domain within a sequence block (SLH) that is conserved in all species,
from plants to humans (Fig. 7). No corresponding residue or sequence
motif is found in other A domains. It is possible that this histidine
is involved in a catalytic function when copine binds a target through
its A domain.
The
-
-
organization of the structure of the integrin A domain
has been recognized as a classical "Rossman fold" (40). This
structural motif is also the basis of the formation of nucleotide- or
dinucleotide-binding pockets in a large number of intracellular enzymes
(42). The copine sequence does not show significant similarity to
nucleotide-binding folds that can be recognized by conventional
sequence comparison algorithms or by the presence of sequence
"fingerprints" characteristic of nucleotide-binding proteins (42).
However, it is of great interest that rabbit muscle copine I was
recently found to bind to an ATP affinity column in a
calcium-dependent
manner.4 Thus, if copine does
possess an enzymatic function in its A domain, the function may require
ATP or a related nucleotide as a cofactor or cosubstrate.
Following the putative A domain, the copines have a variable length
C-terminal domain that is relatively enriched in prolines. This domain
ranges from 34 to 45 residues in the human copines in which it has been
sequenced (Fig. 6) and up to 86 residues in one of the nematode copines
(Fig. 7). This domain may confer unique characteristics on the
different family members and may provide an additional site for
protein-protein interactions.
Functions of the Copines--
The high degree of conservation of
the copines, present in plants, animals, and ciliates, suggests they
play a fundamental role in eukaryotic cell biology. However, there are
no obvious copine homologs encoded by the yeast genome, so they are not
necessary for essential processes in yeast. The diversity of copines in
any one organism, such as the nematode or the human, suggests that the
family members have radiated early in evolution to perform distinct
functions.
Many proteins that contain C2 domains play roles in membrane
trafficking. Well documented examples include synaptotagmin (7),
rabphilin (8), and Munc13 (10), all of which have multiple C2 domains
(two or three). Additional C2 domain-containing proteins carry out
enzymatic functions in diverse calcium-dependent cell
signaling processes, such as phosphorylation by protein kinase C,
release of diacylglycerol and inositol trisphosphate by phospholipase
C, release of arachidonic acid by cytosolic phospholipase A2 (43), and
ubiquitination of proteins targeted for destruction (44).
Our identification of copine I as a secretory vesicle-binding protein,
as well as the general property of calcium-dependent
association with membranes, suggests that the copine family of proteins
may also be involved in membrane trafficking. Such a role would be
consistent with a need for distinct variants of copine to mediate
membrane trafficking in distinct pathways. To fully understand the cell
biological roles of the copines it will be important to determine if
the C-terminal portion of copine acts as an enzyme, perhaps
nucleotide-dependent, or, like the integrin A domain,
mediates interactions with other proteins.
We are indebted to Lon Aggerbeck for
providing access to fast protein liquid chromatography facilities in
his laboratory, to John Shannon for peptide sequence analysis, to
Laurence Vayssie for preparation of Paramecium cDNA, and
to Jacques Retief for assistance with sequence alignment and
presentation.