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Originally published online as doi:10.1189/jlb.0203083 on June 16, 2003

Published online before print June 16, 2003
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(Journal of Leukocyte Biology. 2003;74:379-388.)
© 2003 by Society for Leukocyte Biology

Tissue expression of copines and isolation of copines I and III from the cytosol of human neutrophils

Jack B. Cowland*, Daniel Carter*, Malene D. Bjerregaard*, Anders H. Johnsen{dagger}, Niels Borregaard* and Karsten Lollike*,1

The Granulocyte Research Laboratory, Departments of
* Hematology and
{dagger} Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark

1Correspondence at current address: Novo Nordisk Health Care AG, Andreasstrasse 15, 8050 Zurich, Switzerland. E-mail: kalo{at}novonordisk.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Copines are a recently identified group of proteins characterized by two Ca2+-binding C2-domains at the N terminus and an A-domain at the C terminus. Although pEST sequences indicate the existence of at least seven copines in man, only copines I, III, and VI have been identified at protein level. Here, we describe the isolation of copines I and III in the cytosol of human neutrophils by use of Ca2+-induced hydrophobic chromatography. This is the first demonstration that copines are coexpressed in the same cell. We found that copine III exists in the cytosol of human neutrophils as a monomer with a blocked N terminus. Copines I and III undergo conformational changes upon Ca2+ binding that lead to exposure of hydrophobic patches. Examination of RNA from 68 human tissues demonstrated that copines I–III are ubiquitously expressed whereas copines IV–VII each has a more restricted and individual expression profile. Expression of copines I–III was also demonstrated in neutrophil precursors from bone marrow. Copine I was uniformly expressed at all stages of neutrophil differentiation, whereas copine II and even more so, copine III were expressed in the more immature neutrophil precursors, which indicates an individual function of these copines.

Key Words: granulopoiesis • calcium-binding proteins • mRNA profiles • differentiation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+ is the most important intracellular secondary messenger with effect on virtually every cellular process. The specificity of the Ca2+ signal is mediated by a plethora of Ca2+-binding proteins, most belonging to one of three major Ca2+-binding groups: EF-hand proteins, annexins, and/or C2-domain proteins. The three protein groups coordinate the Ca2+ in a pentagonal bipyramid. However, the arrangement of the amino acids participating in Ca2+ binding is quite different among the three groups. In EF-hand proteins, a consecutive loop of most frequently 12 amino acids surrounded by two {alpha} helixes binds the Ca2+ ions. In C2-domains, the participating amino acids are far apart in the primary sequence but are positioned close together in the tertiary structure at the top of an eight-stranded, antiparallel ß-sandwich [1 , 2 ]. In annexins, a stretch of six amino acids together with a residue 38 amino acids downstream is responsible for Ca2+ binding [3 ].

The copine protein family is a recently identified group of C2-domain proteins [4 ]. This family is unique in having two C2-domains at the N terminus, whereas in most other C2-proteins, the domains are at the C terminus or in the middle [5 ]. Furthermore, copines possess an A-domain (or von Willenbrand domain) at the C terminus, thus being the first intracellular protein for which an A-domain has been described. A-domains usually mediate protein–protein interactions of extracellular proteins. It has recently been shown that copines can bind to a number of target proteins via the A-domain [6 ]. Copine genes have been found in Paramecium, Aradopsis, as well as in man [4 ]. pEST sequences indicate that at least seven human copine genes exist [4 , 7 ], but so far, only copines I, III, and VI (or N-copine) have been identified at the protein level [4 , 8 , 9 ]. Copines VI and VII seem to be expressed exclusively in the brain [7 , 9 ]. It is not known if two or more copines can be coexpressed and if so, whether the copines have redundant or variant functions. The exact function of copines is unknown, but copines I and VI bind to membranes in the presence of Ca2+ [4 , 10 ], as is common for other C2-domain proteins [5 ], and this has been suggested to have a functional significance. Two-hybrid screening has identified OS9 as a binding partner for copine VI [11 ], but the functional significance of this is obscure, as the function of OS9 is unknown. Initial indications that copine VII plays a role in breast cancer were not confirmed [7 ]. Recently, copine III was found to be a phosphoprotein with associated kinase activity, and a novel nucleotide-binding motif was identified in the C terminus of copines [8 ], suggesting that copines might be a novel, unconventional kinase family.

Human neutrophils are the most numerous of the circulating leukocytes. They play an important role in the immune defense. Neutrophils primarily kill fungi and bacteria but may also be active against vira and can recruit other leukocytes to sites of inflammation. Human neutrophils possess a multitude of highly toxic proteins and peptides that are securely stored in different granule populations to ensure a specific release only when needed. At least three different granule populations can be isolated: azurophilic, specific, and gelatinase granules, respectively. In addition, human neutrophils contain an easily mobilizable compartment, the secretory vesicles. Mobilization of the four different membrane-bound organelles is highly hierarchic and strongly dependent on Ca2+ [12 ], but the molecular mechanisms are unknown.

In a systematic search for new Ca2+-binding proteins from the cytosol of human neutrophils, we identified two members of the recently recognized copine group, namely copines I and III. This is the first demonstration of coexpression of two copines in one cell type. The isolation and initial characterization of copines I and III from human neutrophils are described. Furthermore, we show the mRNA expression profiles of all known copines (copines I–VII) in 68 human tissues and at four different stages of neutrophil differentiation in bone marrow (BM).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of human neutrophils
Human neutrophils were isolated from blood from volunteer donors after consent as described [13 ]. In short, erythrocytes were sedimented by 2% Dextran (Pharmacia, Uppsala, Sweden) in saline, and the leukocyte-rich supernatant was centrifuged at 400 g 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 addition of NaCl. The neutrophils were washed once, counted, and resuspended in saline.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting
SDS-PAGE [14 ] and immunoblotting [15 ] were performed on Bio-Rad systems according to the instructions given by the manufacturer (Bio-Rad, Hercules, CA) and as described previously [16 ]. Visualization of immunoblots was by metal-enhanced diaminobenzidinetetrahydrochloride (Pierce, Rockford, IL). Copine I: Anticopine I antibodies (a generous gift from Carl E. Creutz and Jose L. Tomsig, Department of Pharmacology, University of Virginia, Charlottesville) were diluted 1:5000 in washing buffer; copine VI: anticopine VI antibodies (a generous gift from Takashi Nakayama, CNS Research Laboratories, Shionogi and Co., Osaka, Japan) were diluted 1:1000 in washing buffer.

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 standard.

Subcellular fractionation of human neutrophils
Isolated neutrophils at 3 x 107 cells/ml were incubated in saline with 5 mM diisopropyl fluorophosphate (Aldrich, Milwaukee, WI) for 5 min on ice and were centrifuged at 200 g for 6 min. Production of cytosol cell pellets was resuspended at 2 x 108 cells/ml in binding buffer [100 mM KCl, 3 mM NaCl, 10 mM piperazine-N,N'-bis(2-ethanesulfonate; PIPES; pH 7.2)] containing 0.5 mM phenylmethylsulfonyl fluride, 200 U/ml aprotinin, and 100 µg/ml leupeptin (all three from Sigma Chemical Co., St. Louis, MO) and was disrupted by nitrogen cavitation at 400 psi for 5 min at 4°C as described [17 ]. The cavitate was ultracentrifuged at 100,000 g for 45 min, and the supernatant was kept as cytosol.

Isolation and purification of copines I and III
Human neutrophil cytosol (10 ml prepared as above) in binding buffer plus 0.5 mM CaCl2 was loaded onto a self-packaged phenyl-Sepharose (Amersham Pharmacia Biotech AB, Little Chalfont, UK) column (2 ml) at room temperature. After binding cytosolic proteins, the column was washed thoroughly in binding buffer plus 0.5 mM CaCl2. Bound proteins were subsequently eluted in fractions of 2 ml with binding buffer supplemented with 5 mM EGTA. Fractions containing relevant proteins (as determined by SDS-PAGE) were submitted to a buffer change to 20 mM Tris(hydroxymethyl)aminomethane, hydrochloride (Sigma, St. Louis, MO, USA) (pH 7.4) using a centriprep column (Amicon, Beverly, MA) and were passed through a Mono-Q (Amersham Pharmacia Biotech AB) column coupled to a fast protein liquid chromatography (FPLC) instrument (Amersham Pharmacia Biotech AB). Bound proteins were eluted with a 0–1 M NaCl nonlinear gradient in 20 mM TRIZMA (pH 7.4), and 0.5 ml fractions were evaluated by SDS-PAGE and immunoblotting. Fractions containing copine III were subjected to molecular sieve chromatography, also using FPLC in 20 mM TRIZMA (pH 7.4; Superdex 75, Amersham Pharmacia Biotech AB), and were evaluated by SDS-PAGE.

Protein identification
Proteins separated by SDS-PAGE were identified by analysis of peptides obtained by trypsin digestion of the bands cut out from the gel. The method was modified from Wilm et al. [18 ] as described previously [16 ].

For the initial identification of copine III, the tryptic digest was separated by reversed-phase high-pressure liquid chromatography (HPLC) using a Vydac C8 column (1x150 mm). The column was eluted with a gradient from 10 to 40% acetonitrile with 0.1% trifluoroacetic acid added to both solvents. The absorbance at 214 mm was monitored, and peak fractions were collected manually. The molecular masses of the peptides were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on a Biflex instrument (Bruker, Billerica, MA) operated in the linear mode using {alpha}-cyano-4-hydroxy-cinnamic acid as matrix (Hewlett Packard, Palo Alto, CA) and external calibration. Three peptides were selected for identification by amino acid sequence analysis using an automatic protein sequencer (model 494A, ABD, Perkin Elmer, Foster City, CA) equipped with on-line HPLC systems for detection of the amino acid phenylthiohydanthoin derivatives. All chemicals and solvents were sequence- or HPLC-grade delivered by Perkin Elmer.

For the subsequent verification of the two "60 kDa" components as copines I and III, respectively, the mixtures of tryptic peptides were analyzed directly by mass spectrometry as described above. However, for this purpose, the instrument was operated with delayed extraction in the reflected mode using internal calibration by mixing with a solution of angiotensin II and dynorphin.

Cloning of copine probes
The relevant pEST clones containing copine cDNAs were obtained from MHPG (Cambridge, UK; copines I–VI). The plasmid p10-BS containing the 3'-end of copine VII was kindly donated by Dr. Anna Savoia (Servizio di Genetica Medica, San Giovanni Rotondo, Italy) [7 ]. The following primers were used to amplify part of the 3'-untranslated region of the copine transcripts for use as probes: copine I, 5'-GGAGGCTGTGGCAAGTCC-3' and 5'-TATTGAATGAGGGTTGTCAGG-3'; copine II, 5'-CACAAGGAAGCAGAGTGAGC-3' and 5'-TTGCTGGGGGAAGCTGCC-3'; copine III, 5'-TTGTGTTATGTGGAGCAATGCC-3' and 5'-CCACAGTGTCTAAACTGTAGC-3'; copine IV, 5'-CAGATACCATTTTATTTCAAGGC-3' and 5'-AAAAACCCCAACAACAAATACC-3'; copine V, 5'-GCAACAGGGATTGGCATGC-3' and 5'-CTGGGGCTCTGGCTTCTG-3'; copine VI, 5'-CCTCCGGACCGACACTCC-3' and 5'-GATAGGAGCAGGACCCACC-3'; copine VII, 5'-GGGGGCAGTGAGGAATGG-3' and 5'-CACTTTATTACTCCTGAAGCC-3'. The amplified fragments were cloned in pCRII (Invitrogen, San Diego, CA), and the correctness of the inserts was confirmed by dideoxy dye-terminator sequencing with AmpliTaq® DNA polymerase, FS (Perkin Elmer, Applied Biosystems Division). The specificity of the probes was confirmed by a BLAST search, which demonstrated that cross-reaction with other genes should not be possible.

Isolation of cells from BM and peripheral blood neutrophils
BM cells (15 ml) were obtained by aspiration from the posterior–superior iliac crest from healthy volunteers. The sample was immediately anticoagulated by acid citrate dexdrose (ACD; 25 mmol/l sodium citrate, 126 mmol/l glucose), and an equal volume of 2% Dextran T-500 (Pharmacia) in 0.9% NaCl was added to induce sedimentation of the erythrocytes at room temperature. Following this, the cells were kept at 4°C. The leukocyte-rich supernatant was centrifuged at 200 g for 10 min at 4°C. The pelleted cells were resuspended in phosphate-buffered saline, layered on top of a two-layer PercollTM (Pharmacia) gradient containing 9 ml Percoll of 1.080 density layered below 9 ml Percoll of 1.065 density, and centrifuged at 1000 g for 20 min at 4°C. This resulted in separation of BM cells into three bands containing neutrophil precursors of different maturity: myeloblasts and promyelocytes (MB and PM), myelocytes and metamyelocytes (MC and MM), and band cells and segmented neutrophil cells (BC and SC), respectively, as described previously (refs. [19 20 21 ]). Contaminating erythrocytes in the BC and SC cell population were lysed by subjecting the cells to 30 s of hypotonic lysis in distilled water. Mature neutrophils were isolated from peripheral blood anticoagulated in ACD. The erythrocytes were sedimented by 2% Dextran T-500 at room temperature. Following this, the cells were kept at 4°C. The leukocyte-rich supernatant was pelleted and resuspended in saline for subsequent centrifugation on Lymphoprep (Nygaard, Oslo, Norway) at 400 g for 30 min to separate polymorphonuclear cells from platelets and mononuclear cells [13 ]. The remaining erythrocytes were lysed by hypotonic lysis as above.

Non-neutrophil cells were removed by incubation with immunolabeled magnetic beads as described previously [22 ]. Briefly, this involves a depletion of non-neutrophil BM cells from the three populations of neutrophil precursors by incubating with mouse antibodies against human CD2, CD3, CD14, CD19, CD56, CD61, and glycophorin A. For the BC + SC population, mouse antibodies against human CD49d were also included. Lymphoprep-purified neutrophil granulocytes [polymorphonuclear neutrophils (PMNs)] from peripheral blood were depleted for contaminating eosinophils by incubation with CD49d antibodies. All antibodies were purchased from PharMingen (BD Biosciences, Franklin Lakes, NJ) and were incubated with the cells according to the manufacturer’s recommendations. Following incubation with the primary antibody, the cells were incubated with immunomagnetic goat anti-mouse immunoglobulin G microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations and were applied to a magnetic cell sorter column (Miltenyi Biotech). The run-through was collected, and the cells were used for RNA purification. The purity of the neutrophil cell populations was >95%.

Dot-blot of multiple tissue expression (MTE) array
A MTE array (Clontech, Palo Alta, CA) was hybridized to copines I–VII at 65°C in ExpressHyb (Clontech) supplemented with sheared salmon sperm DNA (7.5 µg/ml) and human Cot-1 DNA (1.5 µg/ml) according to the manufacturer’s recommendations. Following hybridization, the filter was washed 5 x 20 min at 65°C in 2 x saline sodium citrate (SSC; 1xSSC=150 mM NaCl, 15 mM sodium citrate, pH 7.0), 1% SDS, and 2 x 20 min at 65°C in 0.1 x SSC, 0.5% SDS. The blot was developed and quantified by a phosphoimager (Fuji Imager Analyzer BAS-2500, Image Reader ver. 1.4E, Image Gauge ver. 3.01 software, Fuji, Stockholm, Sweden). The membranes were stripped by boiling in 0.5% SDS for 10 min before rehybridization. The probes used for hybridization were radiolabeled with [{alpha}-32P] dCTP using the Random Primers DNA labeling system (Gibco-BRL, Grand Island, NY). The filter was first hybridized with the copine III probe. Following hybridization to the remaining six copine probes, the filter was again hybridized to copine III to ensure that the relative hybridization signals were not altered after the eight hybridizations.

RNA isolation and Northern blotting
Between 0.5 x 107 and 2 x 108 cells, depending on the cell population, were used for preparation of total RNA with Trizol (Gibco-BRL). The RNA was precipitated with ethanol and resuspended in 0.1 mM EDTA. The concentration was determined by spectrophotometric measurement, and the integrity of the RNA was assessed by running 2 µg RNA on an agarose gel. RNA from whole brain was purchased from Clontech. For Northern blotting, 5 µg RNA was run on a 1% agarose gel with 6% formaldehyde dissolved in 1 x 20 mM 3-(N-morpholino)-propane-sulfonic acid, 5 mM sodium acetate, 1 mM EDTA, pH 7.0, for size separation. Ethidium bromide staining ensured the presence of equal amounts of RNA in each lane. The RNA was transferred to a Hybond-N membrane (Amersham, Little Chalfont, UK) by capillary blotting and was fixed by UV-irradiation. The filters were prehybridized for 1–2 h at 42°C in 6 ml ULTRAhyb (Ambion, Austin, TX), preheated to 68°C, and hybridized overnight at 42°C after addition of further 4 ml containing the 32P-labeled probe and sheared salmon sperm DNA (10 µg/ml). The membranes were washed for 2 x 15 min at 42°C in 2 x SSC, 0.1% SDS, followed by 1 x 15 min in 0.2 x SSC, 0.1% SDS, and 2 x 15 min in 0.1 x SSC, 0.1% SDS at 42°C. The blot was developed and quantified by a phosphoimager. The sizes of the mRNAs were determined by reference to 18S and 28S rRNA, which were visualized by ethidium bromide staining. The membranes were stripped by boiling in 0.1% SDS before rehybridization. The probes used for hybridization were radiolabeled with [{alpha}-32P] dCTP using the Random Primers DNA labeling system (Gibco-BRL). For quantitative assessments, the intensities of the copine signals were normalized to the hybridization intensity from a probe against ß-actin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hydrophobic Ca2+-binding proteins in human neutrophilic cytosol
To isolate EF-hand Ca2+-binding proteins from the cytosol of human neutrophils, we set up a purification procedure exploiting the hydrophobic nature of many EF-hand proteins following Ca2+-induced conformational changes [16 ]. We passed isolated human neutrophilic cytosol, supplemented with 0.5 mM Ca2+, over a phenyl sepharose column, washed the column extensively, and analyzed EGTA-eluted fractions by SDS-PAGE. Several well-known EF-hand proteins could readily be identified among the lower molecular weight (MW) proteins by immunoblotting or N-terminal peptide amino sequence analysis, e.g., calmodulin, grancalcin, and calcyclin (Fig. 1 ). The protein constituting a prominent band of ~60 kDa (Fig. 1) was, however, not immediately identifiable, as the intact protein did not give any signal in amino acid sequencing, indicating that the N terminus is blocked. We therefore subjected the protein to trypsin digestion and obtained and sequenced three fragments (see Fig. 2 ), which surprisingly, identified the 60-kDa protein as a C2-domain Ca2+-binding protein belonging to the copines, a recently identified protein family with two C2-domains and an A-domain [4 ]. As originally described by Creutz et al. [4 ], pEST sequences suggested the existence of at least six human copine genes, and these were termed copines I–VI. Later, this list has been expanded to also encompass copine VII [7 ]. From sequence comparison, we concluded that the unidentified protein was copine III (see Fig. 2 ). Copine III was originally described as one (termed KIAA0636) of 100 deduced protein sequences based on cDNA clones from a human brain library [23 ]. Recently, copine III was purified and its cDNA cloned from human Jurcat cells [8 ]. Both of these reports showed that mRNA for copine III was highly expressed in all tissues tested (heart, brain, lung, liver, skeletal muscle, kidney, pancreas, spleen, testis, and ovary). Copine III could be isolated by ion-exchange chromatography followed by gel-filtration using FPLC. On gel-filtration, copine III ran as a 61-kDa protein (data not shown), indicating that the protein under our FPLC condition exists as a monomer.



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  		Figure 1. EGTA eluate from a hydrophobic column to which human neutrophilic cytosol was applied in the presence of Ca2+. Human neutrophilic cytosol was supplemented with 0.5 mM Ca2+ and passed over a phenyl sepharose column. The column was washed extensively, and Ca2+-bound proteins were eluted by addition of a surplus of EGTA and collected in 2-ml fractions that were evaluated by SDS-PAGE. Depicted are the first nine eluted fractions (lanes 1–9). The procedure is clearly specific for isolating a limited number of proteins, which are all expected to be Ca2+-binding proteins or proteins that interact with Ca2+-binding proteins. Several well-known Ca2+-binding proteins could be identified in the eluate by N-terminal amino acid sequence analysis or immunoblotting (a, calcyclin; b, calmodulin, where it is well described that Ca2+-binding induces a conformational change in calmodulin, resulting in an increased mobility in SDS-PAGE; c, grancalcin). The prominent band at ~60 K (arrow) was cut off the gel and identified by amino acid sequence analysis of several tryptic fragments. This band was found to be copine III.

 


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  		Figure 2. Sequence alignment of copines I and III. Sequence alignment of copines I and III as performed by the program BLAST. The three sequences of copine III obtained by amino acid sequence analysis and mass spectrometry of the trypsin-generated fragments are underlined. Identities: 356/540 (65%); positives: 432/540 (79%); gaps: 12/540 (2%).

 
Identification of copines I and III
Besides copine III, only two other copines have been described at the protein level, namely copine I [4 ] and copine VI or N-copine [9 ]. Antibodies toward copine VI did not react with human neutrophilic cytosol (we tested antibodies directed toward the A-domain and the first C2-domain; data not shown). Copine I antibodies, conversely, did react with human neutrophilic cytosol to give a single band of ~60 kDa (data not shown). Therefore, we initially suspected that anticopine I antibodies exhibited cross-reactivity toward copine III. However, when we performed immunoblotting of ion-exchange fractions, the anticopine I antibodies did not react with the band we had originally identified but rather with a protein band of slightly lower electrophoretic mobility (Fig. 3 ). This indicated that copines I and III might be coexpressed in human neutrophilic cytosol. We therefore analyzed both bands from the gel by digestion with trypsin followed by mass spectroscopy of the resulting peptide mixture (Table 1 ). All the observed molecular masses from each of the bands were unique for copine I or copine III, respectively. The presence of copines I and III in human neutrophils was further substantiated by the expression of mRNA for both proteins in human neutrophils (see Fig. 6 ) as evaluated by Northern blotting.



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  		Figure 3. SDS-PAGE and anticopine I immunoblot of fractions obtained by an ion-exchange chromatography of pooled EGTA eluate. Fractions 2–6 from the EGTA eluate (Fig. 1) were pooled, had a change of buffer, and were subjected to ion-exchange chromatography on a Mono-Q column and eluted into 0.5-ml fractions with a 1-M NaCl gradient. (A) SDS-PAGE of fractions 12–18, showing a band of 60 K in fractions 12–16 and a band of 63 K in fractions 15–18. (B) Fraction 15 (from another FPLC run) subjected to SDS-PAGE (left) or immunoblotting with anticopine I antibodies (right).

 

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  		Table 1. Distinction of Copines I and III

 


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  		Figure 6. Northern blot of copines I–III in neutrophil precursors from BM and from peripheral blood. (Upper) Northern blot of total RNA from three populations of human BM cells containing MB and PM, MC and MM, and BC and SC and mature granulocytes from PMNs. The blots were hybridized with probes against copines I–III and ß-actin. (Lower) Schematic representation of hybridization intensities. For each probe, the cell population showing maximal expression is given the value 1; the expression levels in the other cell populations are shown relative to this. All expression levels are shown as the mean values of ß-actin-normalized transcript levels from three different persons. SD is shown as error bars.

 
Calcium-binding mechanism of copines
Calcium-binding proteins belonging to all three Ca2+-binding families (EF-hand, annexin, and C2-domain) are found in the cytosol of human neutrophils. As an initial characterization of copines I and III, we compared the Ca2+-binding mechanism of the copines to those of other neutrophilic Ca2+-binding proteins. Notably, the EF-hand migration inhibitory factor-related proteins (MRP)-8 and MRP-14 (S1008 and S1009), which are very prominent in the cytosol of human neutrophils [24 ], did not bind to the phenyl sepharose column (Fig. 4 ). Furthermore, none of the five annexins, present in neutrophil cytosol [25 ], bound to the hydrophobic matrix (Fig. 4) , as evaluated by immunoblotting. In contrast to MRPs and annexins, the EF-hand proteins calcyclin, calmodulin, and grancalcin and also copines I and III bound to the hydrophobic phenyl sepharose column in a Ca2+-dependent manner (Fig. 1) .



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  		Figure 4. Immunoblotting of cytosol, wash, run-through, and eluate from phenyl sepharose column. Four fractions (cytosol, run-through, wash, and eluate, equivalent to pooled lanes 1–9 from Fig. 1 ) from a phenyl sepharose Ca2+-binding experiment were immunoblotted with antibodies toward Ca2+-binding proteins known to be present in human neutrophils. (Top two boxes) Pan antiannexin antibodies. (Middle) Anti-MRP-8 and -14 antibodies. (Bottom) Antigrancalcin antibodies.

 
Expression of copines in human tissues
As very limited information is available on the tissue distribution of copines, we examined the expression of copines I–VII by hybridization to MTE arrays containing mRNA from 68 normal human tissues. As the amino acid sequences (and thus, the coding regions) of copines are highly conserved, we used part of the 3'-untranslated region of the copine transcripts as probes. This approach has previously been used for Northern blotting of copine VII [7 ]. The data demonstrated individual expression profiles for each of the seven copines (Fig. 5 ). All copines were expressed in most or all regions of adult brain (lanes 1–21) and in fetal brain (lane 62) but varied considerably in their expression levels in non-neuronal tissues. Copines I–III were expressed in all the normal tissues represented, although the expression levels did vary between tissues. In most tissues, the expression level was comparable with or higher than that of whole brain (lane 1, indicated by a dotted line). Copines IV–VII showed a more limited expression. Compared with whole brain, a strong expression of copine IV was also found in aorta and heart tissues (lanes 22–29), BM (lane 47), and prostate (lane 53). High expression of copine V was seen in heart tissue (including fetal heart, lane 63) but not aorta (lane 23). Expression of copine V was furthermore detected in stomach (lane 31), spleen (lane 43), lymph node (lane 46), and testis (lane 54). Copine VI was almost exclusively expressed in the brain, and only weak expression could be measured in kidney (lane 41), liver (lane 56), and fetal heart (lane 63). Expression of copine VII comparable with that of whole brain was found in duodenum and jejunum (lanes 32 and 33), thymus (lane 44), testis (lane 54), fetal brain (lane 62), and fetal thymus (lane 67).




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  		Figure 5. Expression of copines I–VII in normal human tissues. Panels I–VII: A MTE array (Clontech) was hybridized with probes specific for copines I–VII (copines I–IV, A; and copines V–VII, B). For each hybridization experiment, the level of expression is shown relative to the tissue with highest expression. For each probe, the level of expression for whole brain (lane 1) is also indicated by a dotted line. RNA from the following tissues are represented: 1–10, Whole brain, cerebral cortex, frontal lobe, parietal lobe, occipital lobe, temporal lobe, paracentral gyrus of cerebral cortex, pons, cerebellum left, cerebellum right; 11–21, corpus callosum, amygdala, caudate nucleus, hippocampus, medulla oblongata, putamen, substantia nigra, accumbens nucleus, thalamus, pituitary gland, spinal cord; 22–29, heart, aorta, atrium left, atrium right, ventricle left, ventricle right, interventricular septum, apex of heart; 30–40, esophagus, stomach, duodenum, jejunum, ileum, ilocecum, appendix, colon-ascending, colon-transverse, colon-descending, rectum; 41–47, kidney, skeletal muscle, spleen, thymus, peripheral blood leukocytes, lymph node, BM; 48–55, trachea, lung, placenta, bladder, uterus, prostate, testis, ovary; 56–61, liver, pancreas, adrenal gland, thyroid gland, salivary gland, mammary gland; 62–68, fetal brain, fetal heart, fetal kidney, fetal liver, fetal spleen, fetal thymus, fetal lung.

 
Strong expression of copines I–IV and a weak expression of copine V were measured in BM (lane 47). High expression of copines I and III and weak expression of copines II and V were also found in peripheral blood leukocytes (lane 45). The expression of copines IV, VI, and VII in peripheral blood leukocytes and of copines VI and VII in BM was at background level.

Expression of copines in neutrophil precursors and peripheral blood neutrophils
As the MTE data showed that copines I–V were expressed in BM and except for copine IV, also in peripheral blood leukocytes, we decided to examine the expression of copine mRNAs at different stages of neutrophil maturation in BM and in peripheral blood. Neutrophil precursors from human BM were separated into three populations containing cells of different maturity by density centrifugation on a Percoll gradient. The three cell populations were enriched in MB and PM, MC and MM, and BC and SC, respectively. Following removal of contaminating blood cells by use of magnetic beads labeled with antibodies against non-neutrophil cells, RNA was extracted from the neutrophil precursors and analyzed for copine expression by Northern blot. RNA from PMNs was also included on the blot. Expression of all seven copines was examined by hybridization with specific probes, and in all cases, RNA of cells prepared from three unrelated donors was tested. Only expression of copines I–III was detected in neutrophils (Fig. 6 ). Copine I was rather uniformly expressed during neutrophil maturation, whereas copine II and even more so, copine III, were more strongly expressed in the less mature neutrophil precursors with peak transcript levels in MC and MM. The nondetection of transcripts for copines IV and V in neutrophils was not a result of an unsuccessful hybridization, as RNA from brain, which was included on the blot as a positive control, was able to bind the probes (data not shown). The MTE data showing expression of copines IV and V in BM and of copine V in peripheral blood leukocytes may therefore represent an expression in non-neutrophil blood cells. This may also explain the strong expression of copine III in peripheral blood leukocytes on the MTE blot.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Identification of copines in human neutrophils
To isolate EF-hand proteins from the cytosol of human neutrophils, we have set up an efficient separation encompassing hydrophobic interaction [16 ]. As expected, a number of well-known EF-hand proteins appeared in the hydrophobic eluate. To our surprise, we could, however, also identify two members of the novel copine protein family. The expression of copines I and III in human neutrophils and their precursors was verified by Northern blotting. This is the first time that copines have been demonstrated in human blood cells and also the first time that it has been demonstrated that two copines can be coexpressed in one cell. Although copine I and copine III show great overall sequence similarity (Fig. 2) anticopine I antibodies did not react with copine III. Aticopine VI antibodies did not react with copine I or copine III either, which could indicate that the tertiary structure of each copine is unique.

Copine I and copine III ran as monomers in SDS-PAGE with an electrophoretic mobility in accordance with their calculated MW, indicating that no major post-transcriptional modification takes place. The monomeric nature of copine III was confirmed by its behavior in molecular sieve chromatography. Copine III gave no signal by N-terminal sequence analysis, indicating that the N-terminal is modified, like many cytosolic proteins [26 ].

Ca2+-binding mechanism of copines
Copine I and copine VI bind to membranes in a Ca2+-dependent manner in in vitro studies [4 , 10 ], and likewise, copine I was also shown to bind to liposomes [27 ]. The EF-hand proteins MRP-8 and MRP-14, which are present in the cytosol of human neutrophils in high amounts, did not bind to the hydrophobic column, in accordance with the theory that MRPs do not undergo Ca2+-induced conformational changes. Annexins do not bind membranes via hydrophobic interaction but rather by a mechanism known as Ca2+-bridging [5 ]. This fits well with our findings that annexins do not bind to a phenyl sepharose column (Fig. 4) , whereas they do bind to membrane fractions [28 ]. Conversely, it is described that calmodulin and grancalcin undergo marked conformational changes when binding Ca2+ and that this leads to exposure of hydrophobic patches [16 ]. Indeed, we could identify calmodulin and grancalcin in the EGTA eluate, indicating that they do bind to the hydrophobic column. As copine I and copine III were identified in the EGTA eluate, we hypothesize that these two copines undergo conformational changes upon Ca2+ binding and that these conformational changes expose hydrophobic patches in a manner similar to some EF-hand proteins. The hydrophobic patches can mediate binding to a hydrophobic enviroment, e.g., phenyl sepharose, biological membranes, or liposomes.

Copine expression
The RNA data show that copines I and III were expressed in neutrophil precursors from BM and in PMNs, from peripheral blood. This is in accordance with our isolation of both proteins from the cytosol of human neutrophils. Additionally, mRNA for copine II was found in BM and peripheral blood neutrophils. However, we did not identify copine II among the proteins eluted from the phenyl sepharose column. This might be because the expression level of copine II in human neutrophils is too low to allow detection by this technique or because the copine II protein is not synthesized in human neutrophils. It could also be hypothesized that copine II does not bind Ca2+ or that it does not undergo conformational changes that will allow it to interact with a hydrophobic column. The final determination of a potential presence of copine II in human neutrophils and precursors must therefore await the development of specific copine II antibodies.

We have detailed the expression profiles of all copines and find that they can be divided into two groups: copines I–III, which are ubiquitously expressed, and copines IV–VII, which all have a more restricted expression profile. We found that copine I has a high and uniform expression in BM neutrophilic precursors and in mature neutrophils. Copines II and III, conversely, have the highest mRNA levels in the more immature neutrophil precursor cells.

The function of copines is unknown. Recently, copine III has been demonstrated to have kinase activity. The different copines could be redundant and have similar functions, or they might mediate phosphorylation of different targets. The fact that copines seem to have variant tertiary structure and demonstrate a differentiated expression in maturing neutrophils indicates that they have seperate functions. As Ca2+ obviously induces important conformational changes in copines, this might have a functional consequence by inducing a translocation to membranes or a binding to potential targets, which only have been recognized following such structural changes.


   ACKNOWLEDGEMENTS
 
The Danish Medical Research Council, The Carlsberg Foundation, and the Danish Cancer Society supported this work. J. B. C. and D. C. contributed equally to the experimental part of this work. Hanne Kristensen, Inge Kobbernagel, and Allan Kastrup are thanked for expert technical assistance.

Received February 25, 2003; revised April 25, 2003; accepted April 28, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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