©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium-dependent Binding of S100C to the N-terminal Domain of Annexin I (*)

(Received for publication, August 8, 1995; and in revised form, October 6, 1995)

William S. Mailliard Harry T. Haigler (§) David D. Schlaepfer (¶)

From the Department of Physiology and Biophysics, University of California, Irvine, California 92717

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The annexin family of proteins is characterized by a conserved core domain that binds to phospholipids in a Ca-dependent manner. Each annexin also has a structurally distinct N-terminal domain that may impart functional specificity. To search for cellular proteins that interact with the N-terminal domain of annexin I, we constructed a fusion protein consisting of glutathione S-transferase fused to amino acids 2-47 of human annexin I (GST-AINT; AINT = annexin I N-terminal). Extracts from metabolically labeled A431 cells contained a single protein (M(r) 10,000) that bound to GST-AINT in a Ca-dependent manner. A synthetic peptide corresponding to amino acids 2-18 of annexin I inhibited the binding of the 10-kDa protein to GST-AINT with half-maximal inhibition occurring at 15 µM peptide. In cellular extracts, endogenous annexin I and the 10-kDa protein associated in a reversible Ca-dependent manner. Experiments with other annexins and with N-terminal truncated forms of annexin I indicated that the 10-kDa protein bound specifically to a site within the first 12 amino acids of annexin I. The 10-kDa protein was purified from human placenta by hydrophobic and affinity chromatography. Amino acid sequence analysis indicated that the 10-kDa protein is the human homologue of S100C, a recently identified member of the S100 subfamily of EF-hand Ca-binding proteins.


INTRODUCTION

Annexins are a family of intracellular proteins that bind in a reversible Ca-dependent manner to phospholipids preferentially located on the cytosolic face of plasma membranes (see (1) and (2) for a review). Annexins are structurally distinct from the EF-hand family of Ca-binding proteins. The Ca and phospholipid binding sites reside in a core domain that shares approximately 50% amino acid sequence identity between different annexin gene products(1) . Each annexin family member also contains an N-terminal domain that is not structurally related in the different annexins(1) .

Various members of the annexin family have been implicated in a number of different intracellular processes including vesicular trafficking (3) , membrane fusion during exocytosis(4) , signal transduction(1) , and ion channel formation(5, 6, 7) . However, the physiological function has not been established for any of the annexins. The different annexin gene products show cell-specific expression(1) , thereby implying that each annexin performs a different biological function. Since the N-terminal domains are not related, this domain may impart functional specificity.

Extensive research has focused on the interaction of annexins with phospholipid bilayers and cell membranes(2) . The hypothesis that annexins exert their biological effects by interacting with other proteins has received less attention. If this hypothesis is valid, we speculate that the annexin-protein interaction would most likely be mediated by their unique N-terminal domains. In fact, the N-terminal domain of annexins II and XI are involved in interactions with p11 (8) and calcyclin(9) , respectively. Calcyclin and p11 belong to the S100 subgroup of the EF-hand family of Ca-binding proteins (see (10) for a review). Most S100 proteins are small (M(r) = 9,000-12,000), although recent research detected functional S100 domains in profilaggrin (11) and trichohyalin(12) , which are much larger proteins. S100 proteins have been proposed to play roles in cellular proliferation, cellular differentiation, and inflammation, but neither the exact physiological function nor the molecular mechanisms by which these putative roles are accomplished are known(10) . Since these proteins appear to lack enzymatic activity, it is widely assumed that they bind to other cellular proteins and change their activity or cellular location. Like the annexins, S100 proteins are expressed only in certain cell types.

Annexins I and II have attracted considerable interest because of their possible involvement in cell growth and differentiation. Annexin I is phosphorylated on Tyr-21 in a Ca-dependent manner by the epidermal growth factor receptor protein kinase (13, 14) and at an adjacent site by protein kinase C(15) . Annexin II is phosphorylated at analogous sites by pp60(16) and protein kinase C (17) in response to extracellular effectors. The N-terminal domain of annexin I is also the site of selective proteolytic clips (13, 18) and of transglutaminase-catalyzed dimerization(19) .

To test the hypothesis that annexin I interacts with other cellular proteins via its N-terminal domain, we searched for proteins in cellular extracts that would bind to a fusion protein containing amino acids 2-47 of human annexin I. We detected Ca-dependent binding of a 10-kDa protein to the fusion protein and to intact annexin I. Amino acid sequence analysis identified the protein as S100C(20, 21, 22) , a recently identified member of the S100 family of proteins.


EXPERIMENTAL PROCEDURES

Materials

The human annexin I cDNA was a generous gift from Dr. Tony Hunter. The following reagents were obtained from commercial sources: glutathione-agarose (Sigma), phenyl-Sepharose and the pGEX-2T expression vector (Pharmacia Biotech Inc.), oligonucleotides (Operon Technologies), and the [S]Met/Cys TranS-label (DuPont NEN). Annexin I, des-1-12-annexin I, des-1-26-annexin I and annexin V were purified from human placenta as described(15, 23) . Recombinant hydra annexin XII was purified as described(24) . Polyclonal antiserum to human annexin I was generated as described(15) . Large unilammelar vesicles were formed from a 2:1 mixture of phosphatidylserine (Avanti) and L-alpha-phosphatidylcholine (Sigma) as described (14) . Peptides AI 2-18 and CPEP were synthesized at the University of California Irvine protein synthesis facility.

Methods

[S]Met/Cys Metabolic Labeling and Protein Extraction

Growing A431 epidermoid carcinoma cells were labeled with [S]Met/Cys for 16 h as described previously(25) . The labeled cells (in a 10-cm culture dish) were washed in phosphate-buffered saline (4 °C), and lysates were prepared at 4 °C with 1 ml of HNG buffer (20 mM Hepes, pH 7.4, 100 mM NaCl, 10% glycerol) containing 2 mM MgCl(2), 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 100 µg/ml RNase, 100 µg/ml DNase I, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The cells were collected by scraping and lysed with 15 strokes of a motorized Dounce homogenizer, and the insoluble material was removed by centrifugation (200,000 times g, 30 min). The cell supernatant was preincubated with purified GST (^1)attached to glutathione-agarose to reduce nonspecific binding. The GST beads were removed by centrifugation, and the cell lysate was filtered (0.2 µM), aliquoted, and stored frozen at -70 °C.

Production of Glutathione S-Transferase-Annexin I N-terminal Fusion Protein (GST-AINT)

Polymerase chain reaction with the human annexin I cDNA as a template using the following sense (5`-GCGTGGATCCGCAATGGTATCAGAGTTCCTCAAGC-3`) and antisense (5`-CGATGAATTCCGAGGATGGATTGAAGGTAGG-3`) primers was used to generate the annexin I N-terminal fragment. The amplified 155-base pair polymerase chain reaction fragment was digested with BamHI and EcoRI and ligated into pGEX-2T. The resulting construct (pGEX2T-AINT) creates a fusion protein between GST and amino acids 2-47 of human annexin I. The identity of the construct was verified by dideoxy sequencing.

Isolation of GST-AINT Fusion Protein

BL21(DE3) bacteria were transformed with pGEX2T-AINT and an ampicillin-resistant colony was used to inoculate 500 ml of Terrific Broth. The cells were grown in the presence of ampicillin (100 µg/ml) at 37 °C. When the cells reached an OD 1.0, protein expression was induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside (0.5 mM). The cells were incubated in a shaker overnight at room temperature and then harvested by centrifugation (5,000 times g, 10 min). The cells were lysed with a French press in the presence of 1% Triton, insoluble matter was removed by centrifugation (12,000 times g, 10 min), and the fusion protein in the supernatant was isolated by glutathione-agarose affinity chromatography. GST-AINT was eluted with reduced glutathione (10 mM), dialyzed against Tris-buffered saline containing 10% glycerol, concentrated, and stored frozen at -70 °C. SDS-PAGE followed by Coomassie Blue staining of the purified GST-AINT preparation revealed one major protein (M(r) = 31,000), which was recognized by polyclonal antiserum to annexin I.

Cellular Protein Binding to GST-AINT

S-Labeled total cell lysate (1 ml, 1 mg of protein) was incubated (2 h, 4 °C) in the presence of 1 mM free Ca with either 20 µg of GST or GST-AINT prebound to glutathione-agarose. The beads were collected by centrifugation and washed four times with 1 ml of HNGT buffer (HNG buffer containing 0.1% Triton X-100) containing 1 mM CaCl(2). Certain samples were resuspended directly into SDS sample buffer to analyze the total bound protein. Other samples were extracted with HNGT containing EGTA (5 mM), followed by centrifugation to separate the EGTA eluate from the protein remaining bound to the beads. The samples were analyzed by 16% SDS-PAGE gel. The gel was stained with Coomassie Blue and dried, and the associated radioactive proteins were visualized by autoradiography.

Synthesis of Annexin I N-terminal Peptide

A peptide corresponding to amino acids 2-18 of human annexin I plus a C-terminal cysteine, AI 2-18 (Ac-AMVSEFLKQAWFIENEEC), was synthesized. The N terminus of the AI 2-18 peptide was acetylated to mimic the native state of the annexin I N terminus. A control peptide, CPEP (FQTDSPNNKGDLKEF), of similar charge and hydrophobicity was used as a control in the indicated incubations. The peptides were purified by HPLC chromatography and stored lyophilized at -70 °C. The peptides (amount determined by dry weight) were solubilized in 20 mM Hepes, pH 7.4, at 1 µg/µl, prior to experimentation.

Peptide Competition for GST-AINT Binding

Total S-labeled cell lysate (approximately 0.25 mg of protein) was incubated with a synthetic annexin I N-terminal peptide (AI 2-18) or control peptide (CPEP) in 350 µl total volume at 4 °C. After 30 min, GST-AINT (20 µg) bound to glutathione-agarose was added to each binding reaction. After 1.5 h at 4 °C, the reactions were washed four times with 500 µl of HNGT buffer containing 1 mM CaCl(2) by repeated centrifugation and resuspension. The reversibly bound protein was extracted (5 min on ice) with 50 µl of HNGT buffer containing 5 mM EGTA and analyzed by 16% SDS-PAGE, followed by autoradiography.

Cellular Protein Association with Phospholipid-bound Annexins

Phosphatidylserine-phosphatidylcholine vesicles (1 mg) were used to selectively bind and remove cellular annexins from S-labeled cell lysates. The vesicle-bound annexins and any associated proteins were extracted (5 min on ice) with 75 µl buffer B (20 mM Hepes, pH 7.4, 100 mM NaCl, and 2 mM MgCl(2)) containing 10 mM EGTA. The reactions were centrifuged, and the EGTA-extractable proteins were analyzed by SDS-PAGE followed by autoradiography. Individual binding assays (1 ml) also were performed with the addition (25 µg/ml) of purified annexin I, des-1-12-annexin I, des-1-26-annexin I, annexin V, and annexin XII.

Immunoprecipitation of the Annexin Ibullet10-kDa Protein Complex

S-Labeled cell lysates (500 µl, 0.5 mg of protein) were preincubated (30 min, 4 °C) with 50 µl (15 mg/ml) protein A-Sepharose. The beads were removed by centrifugation and the supernatants saved. The supernatants were incubated (overnight, 4 °C) with annexin I antiserum with or without 1 mM free Ca or with preimmune serum with 1 mM free Ca. Protein A-Sepharose was added and the reactions were centrifuged. The pellet was washed twice with 1 ml of Tris-buffered saline containing 0.1% Triton X-100 and either 1 mM Ca or 1 mM EGTA. The washed pellets were resuspended directly into SDS sample buffer and analyzed by 16% SDS-PAGE and autoradiography.

Phospholipid Vesicle Association and Peptide Inhibition Assay

Cellular annexins were partially purified from extracts of S-labeled A431 cells using phospholipid vesicles as described above. Cell lysates were preincubated (30 min, 4 °C) with vesicles (2 mg of vesicles/1 ml of extract) in the presence of 1 mM EGTA. The vesicles and nonspecific binding proteins were removed by centrifugation (20,000 times g), and the supernatant was saved. Aliquots (500 µl) of the supernatant were adjusted to 1 mM free Ca and incubated (1 h at 4 °C) with fresh phospholipid vesicles. The vesicles were pelleted by centrifugation and washed twice with 500 µl of buffer B containing 1 mM CaCl(2). The washed vesicle pellet was incubated (1 h, 4 °C) with 100 µM AI 2-18 peptide, 100 µM CPEP peptide, or no peptide in 75 µl of buffer B containing 1 mM CaCl(2). The vesicles were pelleted by centrifugation, and the supernatants were saved. The pellets were washed once with 500 µl of buffer B containing 1 mM CaCl(2) and then extracted (5 min on ice) with 75 µl buffer B containing 10 mM EGTA. The reactions were centrifuged, and the supernatants were saved. Aliquots were analyzed by 16% acrylamide SDS-PAGE, followed by autoradiography.

Purification of the 10-kDa Protein

Soluble proteins from a fresh human placenta were isolated as described previously(6) . Briefly, after homogenization and centrifugation (100,000 times g, 30 min) in the presence of EGTA, CaCl(2) (2 mM free Ca) and NaCl (0.5 M) were added to the supernatant and it was centrifuged again (100,000 times g, 15 min). The supernatant was loaded directly onto a phenyl-Sepharose column (22 times 1.5 cm) equilibrated in Tris buffer (20 mM Tris, pH 7.5, 0.5 M NaCl) containing 1 mM CaCl(2). After washing the column in the same buffer, bound proteins were eluted with Tris buffer containing 5 mM EGTA and dialyzed against 20 mM Tris, pH 7.5, and 0.1 M NaCl. CaCl(2) (2 mM) and 5 mg of GST-AINT, bound to glutathione-agarose, were added to the protein fraction (2 h, 4 °C). The GST-AINT-associated agarose beads were pelleted by centrifugation, washed twice with 40 ml of HNGT containing 1 mM CaCl(2), and four times with 40 ml of 20 mM Tris, pH 7.5, containing 1 mM CaCl(2). The GST-AINT-associated proteins were extracted twice (5 min, on ice) with 3 ml of 20 mM Tris-HCl, pH 7.5, containing 5 mM EGTA and stored at -70 °C.

HPLC Purification and Amino Acid Sequence Analysis

The GST-AINT-associated proteins from human placenta were concentrated in a Centricon (YM-3), and 200 µl of the sample was loaded onto a Vydac C4 column (15 cm times 4.6 mm). The bound proteins were eluted by an acetonitrile gradient while monitoring the elution profile by absorbance at 280 nm. Peak fractions were analyzed by 16% SDS-PAGE and Coomassie staining. Peaks at 47% and 50% acetonitrile contained proteins with apparent M(r) of 6,000 and 10,000, respectively. The 6- and 10-kDa proteins were each digested with cyanogen bromide and the resultant peptides purified by reverse phase HPLC (Vydac C18 column). The peptide fragment that eluted at 23% acetonitrile from each digest was subjected to automated Edman degradation sequence analysis.


RESULTS

Ca-dependent Binding of a 10-kDa Protein to GST-AINT

To search for cellular proteins that interact with annexin I via its N-terminal domain, we constructed a fusion protein consisting of glutathione S-transferase fused to amino acids 2-47 of annexin I (GST-AINT). GST-AINT or GST bound to glutathione-agarose beads was incubated with extracts from the human epidermoid carcinoma cell line, A431, that had been metabolically labeled with [S]Met/Cys. If Ca (1 mM) was present in the extract, a 10-kDa protein bound to GST-AINT, but not to GST (Fig. 1, lanes a and b). The 10-kDa protein could be eluted from GST-AINT with EGTA and was the only protein detected by SDS-PAGE and autoradiography (Fig. 1, lane d). Several proteins bound to both GST-AINT and GST in the presence of Ca, but were not extracted by EGTA (Fig. 1, lanes c, e, and f). No binding was observed when the binding reaction was performed in the presence of the divalent cation Mg instead of Ca (data not shown). These data suggest that the 10-kDa protein interacts with the N-terminal portion of annexin I in a Ca-dependent manner.


Figure 1: Ca-dependent binding of a 10-kDa protein to GST-AINT. Lysates from S-labeled A431 cells were incubated with GST or GST-AINT in the presence of Ca. Glutathione-agarose was added and the beads, along with any associated proteins, were pelleted and washed by repeated centrifugation. The proteins bound to GST (lanes a, c, and e) or GST-AINT (lanes b, d, and f) were either analyzed directly (lanes a and b) or eluted with EGTA and then analyzed (lanes c and d). The proteins remaining associated with GST (lane e) or GST-AINT (lane f) after extraction with EGTA also were analyzed. Samples were analyzed by SDS-PAGE and were visualized by autoradiography. Methods are described under ``Experimental Procedures.'' The arrow indicates the position of the 10-kDa protein that bound to GST-AINT.



Synthetic Peptide Inhibition of the GST-AINTbullet10-kDa Protein Interaction

A peptide corresponding to the first 18 amino acids of annexin I (AI 2-18) was synthesized and tested to determine whether it would inhibit the binding of the 10-kDa protein to GST-AINT. S-labeled cell extracts were incubated with increasing concentrations of AI 2-18, and the GST-AINT binding reaction was performed as described above. AI 2-18 (100 µM) nearly completely inhibited the interaction between GST-AINT and the 10-kDa protein (Fig. 2A, compare lanes a and b). A peptide (CPEP) of similar length, charge, and hydrophobicity as AI 2-18 had no detectable effect on the interaction between the 10-kDa protein and GST-AINT (Fig. 2A, lane c). Quantitative densitometric analysis showed that the inhibition by the AI 2-18 peptide was dose-dependent (Fig. 2B) with half-maximal inhibition observed at 15 µM AI 2-18.


Figure 2: Inhibition of 10-kDa protein binding to GST-AINT by an annexin I N-terminal peptide (AI 2-18). Panel A, lysates from S-labeled A431 cells were incubated with GST-AINT in the presence of Ca and either no peptide (lane a), 100 µM AI 2-18 (lane b), or 100 µM of a control peptide (CPEP, lane c). The GST-AINT bound proteins were eluted with EGTA, analyzed by SDS-PAGE, and visualized by autoradiography. Methods are described under ``Experimental Procedures.'' The arrow indicates the position of the 10-kDa protein. Panel B, the reactions were performed, as described in panel A, with increasing concentrations of AI 2-18 peptide in the binding assay. The samples were analyzed by SDS-PAGE, and the 10-kDa protein was visualized by autoradiography. The intensity of the band at 10-kDa was analyzed by densitometry, and the results are expressed as a percent of the intensity observed in the absence of peptide. Experimental points shown are the average of duplicate reactions.



The 10-kDa Protein Binds to Endogenous Annexin I

We exploited the reversible Ca-dependent association of annexins with phospholipid vesicles to determine whether endogenous annexin I interacted with the 10-kDa protein in cell extracts. Phospholipid vesicles were incubated with S-labeled cell extract in the presence of Ca, and the vesicle-annexin complexes, along with any other associated proteins, were pelleted by centrifugation. The washed pellet contained a 10-kDa band (Fig. 3, lane b) as well as the expected bands between 33 and 40 kDa, representing the endogenous A431 annexins. When the cell extract and phospholipid vesicle mixture was incubated in the presence of AI 2-18 (100 µM), the association of the 10-kDa protein with the vesicle pellet was disrupted (Fig. 3, lanes c and d), but the annexins remained associated with the vesicle pellet (Fig. 3, lane d). A control peptide, CPEP (100 µM), was unable to disrupt the vesicle-annexin Ibullet10-kDa complex (Fig. 3, lanes e and f). These results show that the 10-kDa protein that bound to GST-AINT in the experiments described above (Fig. 1, lane d) has properties similar to the 10-kDa protein in cellular extracts that associated with the vesicles (Fig. 3, lane b).


Figure 3: An endogenous annexin Ibullet10-kDa protein complex binds to phospholipid vesicles. Lysates from S-labeled A431 cells were incubated with phospholipid vesicles in the presence of Ca. The vesicles and any associated proteins were pelleted by centrifugation, washed in the presence of Ca, and then incubated with either no peptide (lanes a and b), 100 µM AI 2-18 (lanes c and d), or 100 µM CPEP (lanes e and f). The vesicles were removed by centrifugation, and the supernatants containing protein eluted by the peptide were saved for analysis (lanes a, c, and e). The proteins that remained associated with the vesicle pellet after the peptide incubation also were analyzed (lanes b, d, and f). Methods are described under ``Experimental Procedures.'' The samples were analyzed by SDS-PAGE, and the proteins were visualized by autoradiography.



To confirm that the 10-kDa protein associated with endogenous annexin I, a polyclonal antiserum was used to immunoprecipitate annexin I from S-labeled lysates of A431 cells. In the presence of Ca, a 10-kDa protein co-immunoprecipitated with antibodies to annexin I (Fig. 4, lane c), but not with a rabbit preimmune serum control (Fig. 4, lane a). In the presence of EGTA, no 10-kDa protein was detected associated with immunoprecipitated annexin I (Fig. 4, lane b).


Figure 4: Co-immunoprecipitation of a 10-kDa protein with annexin I. Rabbit preimmune control antiserum (lane a) or antiserum against annexin I (lanes b and c) were used to immunoprecipitate S-labeled proteins from extracts of A431 cells (see ``Experimental Procedures''). The incubations were in buffers containing either Ca (lanes a and c) or EGTA (lane b). The immunoprecipitated proteins were resolved by SDS-PAGE and visualized by autoradiography. Immunoprecipitated annexin I (apparent M(r) = 35,000) and the associated 10-kDa protein are indicated by the arrows on the right.



Structural Requirements for the 10-kDa Protein Binding to Annexins

The structural elements of annexins that were required for interaction with the 10-kDa protein were investigated. In these experiments, exogenous annexins and phospholipid vesicles were added to incubations containing S-labeled cell extracts and the association of the 10-kDa protein with the annexin-vesicle complex was measured. Some 10-kDa protein associated with vesicles due to endogenous annexin I; however, the amount increased 10-fold (as measured by densitometry) when exogenous annexin I was added to the incubation (Fig. 5, compare lanes a and b). The addition of the AI 2-18 peptide (100 µM) reduced the amount of the 10-kDa protein bound to exogenous annexin I by over 90% (data not shown). No binding of the 10-kDa protein was detected to two truncated forms of annexin I, which were missing either the first 12 or 26 amino acids of the N-terminal domain (Fig. 5, lanes c and d). Likewise, this assay did not detect any binding of the 10-kDa protein to exogenous annexins V or XII (Fig. 5, lanes e and f), two annexins whose structurally conserved core domains share similar Ca and phospholipid binding properties with annexin I. Taken together, these results indicate that the 10-kDa protein interacts specifically with the first 12 amino acids of annexin I and not any other region of this protein nor with other annexins.


Figure 5: A cellular 10-kDa protein binds to annexin I. Lysates from S-labeled A431 cells were incubated in the presence of phospholipid vesicles, Ca, and the indicated purified annexin protein (25 µg/ml). The vesicles and any associated proteins were pelleted by centrifugation, washed in the presence of Ca, and the bound proteins were eluted with EGTA (see ``Experimental Procedures''). Aliquots of the eluted proteins were analyzed by SDS-PAGE, the gel was stained with Coomassie Blue, and S-labeled proteins were visualized by autoradiography. The Coomassie Blue-stained bands confirmed that the indicated annexins bound to the vesicles (data not shown). Additions: lane a, none; lane b, annexin I; lane c, des-1-12-annexin I; lane d, des-1-26-annexin I; lane e, annexin V; lane f, annexin XII.



Purification and Amino Acid Sequence Analysis of the 10-kDa Protein

Tissue extracts from human placenta were used to obtain large amounts of the 10-kDa protein for sequence analysis. Using a series of chromatography steps involving Ca-dependent binding to phenyl-Sepharose (Fig. 6, lane a) followed by Ca-dependent binding to glutathione-agarose-bound GST-AINT, two major Coomassie Blue-stained proteins (apparent M(r) values of 10,000 and 6000) were obtained (Fig. 6, lane b). The 10- and 6-kDa proteins from the GST-AINT affinity column were separated by reverse phase HPLC chromatography (Fig. 6, lanes c and d). Approximately 200 µg of the 10-kDa protein and 600 µg of the 6-kDa protein were obtained from 500 g (wet weight) of placental tissue.


Figure 6: Purification of GST-AINT-binding proteins from human placenta. The proteins that bound to GST-AINT were purified from extracts of human placenta, and aliquots from the purification steps were analyzed by SDS-PAGE and Coomassie Blue staining (see ``Experimental Procedures''). Lane a, proteins eluted from the phenyl-Sepharose column with EGTA. Lane b, proteins eluted from the GST-AINT affinity column with EGTA. Lanes c and d, HPLC reverse phase chromatography was used to separate the 10-kDa (lane c) and 6-kDa (lane d) proteins.



Edman degradation of the 10- and 6-kDa proteins resulted in no sequential release of phenylthiohydantoin amino acids, implying the 10- and 6-kDa proteins have blocked N termini. To obtain sequence information from the 10- and 6-kDa proteins, samples were digested with CNBr and the resulting peptide fragments were fractionated by HPLC chromatography. The two major peak fractions from the HPLC column were chosen for Edman degradation sequencing. The sequence XXELAAFT was obtained for the 10-kDa protein, and the sequence NTELAAFTKN was obtained for the 6-kDa protein (Fig. 7). Due to the exact match between these sequences, the 6-kDa protein is proposed to be a proteolytic fragment of the 10-kDa protein. Since the 6-kDa protein appeared to have a blocked N terminus, the proteolytic clip probably occurred toward the carboxyl side of the sequenced peptide. When the partial sequence of the 10- and 6-kDa proteins were compared to the protein sequence data base, an exact match was found to a member of the S100 protein family called calgizzarin from chicken (21) and rabbit(22) . The same protein from pig, termed S100C(20) , had only a single amino acid difference from the 10- and 6-kDa partial sequence. The amino acid sequence of human S100C (calgizzarin) has not been reported. However, the partial sequence and functional properties of the 10-kDa protein that binds to annexin I leave little doubt that it is human S100C (calgizzarin). Since the 6-kDa protein retains the ability to interact with GST-AINT, it appears that the annexin I binding site on S100C is located in the N-terminal half of the protein (see Fig. 7).


Figure 7: Partial amino acid sequence of the 10-kDa and 6-kDa GST-AINT-binding proteins. Peptide fragments of the HPLC-purified 10-kDa and 6-kDa proteins from placenta were generated by CNBr digestion, purified by HPLC, and subjected to Edman degradation sequencing. The same sequence was obtained for both peptides (except the first two and last cycles from the 10-kDa-derived peptide were not interpretable). The peptide sequence obtained is compared to the sequence of S100C/calgizzarin from rabbit (22) chicken (21) and pig (20) . Features of porcine, rabbit, and chicken proteins are diagrammed with the numbers corresponding to the amino acids in porcine S100C. The site of the proposed C-terminal proteolytic clip that generates the 6-kDa protein is indicated by the arrow.




DISCUSSION

We detected a single protein in extracts of cultured A431 cells that underwent reversible Ca-dependent binding to a fusion protein containing the N-terminal domain of annexin I (Fig. 1). We purified this protein from placenta and obtained a partial amino acid sequence, which identified it as the human form of an S100 protein known as porcine S100C (20) and also as chicken (21) or rabbit (22) calgizzarin (Fig. 7). Partial amino acid sequence analysis of the 10-kDa protein purified from A431 cells (data not shown) confirmed that the placental and A431 proteins were the same protein. A previous study using labeled S100C and a gel overlay technique detected a number of bands in extracts from porcine heart including a 36-kDa band that may have been annexin I(26) . We speculate that this interaction occurs in intact cells, since the association of endogenous annexin I and S100C in extracts of A431 cells could be detected by immunoprecipitation (Fig. 4) and by an assay that exploited Ca-dependent interaction of annexin I with phospholipid vesicles (Fig. 3).

Based on the limited information that is available for S100C, there is a good correlation between the tissue-specific expression of S100C and annexin I. Both proteins are highly expressed in lung and kidney but show minimal expression in brain and liver(20, 27, 28) . We also detected a 10-kDa protein in extracts from diploid human fibroblasts (data not shown) that interacted with GST-AINT in a manner indistinguishable from the S100C protein detected in A431 cells.

Previous studies detected high affinity specific interactions between two other members of the annexin and S100 families; annexin II-p11 (8) and annexin XI-calcyclin(29) . Annexin I is structurally more similar to annexin II than other annexins, and p11 is structurally more similar to S100C than other S100 proteins. Another similarity between annexins I and II is that they both are phosphorylated by tyrosine kinases (the epidermal growth factor receptor (14) and pp60(16) , respectively) and by protein kinase C(15, 17) . Because of these structural and functional similarities, it is particularly interesting to compare and contrast the annexin I-S100C interaction with the annexin II-p11 interaction. Annexin II and p11 form a heterotetramer (2:2 molar ratio) and the site of interaction is an amphipathic helix formed by the first 14 amino acids of annexin II(8) . In parallel with the annexin II-p11 interaction, our studies localized the S100C binding site to the first 12 amino acids of annexin I (Fig. 5). Chou-Fasman analysis of the first 19 residues of annexin I suggest a high probability for alpha-helical formation with residues Met-3, Val-4, Phe-7, Leu-8, Ala-11, and Ile-14 aligning onto one side of a theoretical amphipathic helix (Fig. 8). In comparison, residues Val-4, Ile-7, Leu-8, and Leu-11 of the N-terminal domain of annexin II are similarly aligned on one side of an amphipathic helix (Fig. 8), and these hydrophobic residues are critical for p11 binding (8, 30) . Based on similarities with annexin II, we speculate that the hypothetical annexin I hydrophobic face is the binding site for S100C. The first 14 amino acids of annexin I are highly conserved from humans to sponges, thereby indicating that this putative S100C binding site on annexin I plays a conserved and critical role in its biological function(1) . Since previous studies established that cellular annexin I is sensitive to partial proteolysis at both Trp-12 (13, 18) and Lys-26(13) , it will be of interest to determine whether the cellular interaction between annexin I and S100C is regulated by proteolysis.


Figure 8: Helical wheel presentations N-terminal 19 residues of annexins I and II. The single-letter amino acid code for the N-terminal regions of annexins I and II are shown. Hydrophobic amino acids aligned on one side of the helix have been circled.



Although the site of interaction with S100 proteins is in the N-terminal domains of both annexins I and II, the sites of interaction with annexins on S100C and p11 appear to be in different regions of the proteins. Site-directed mutagenesis localized the annexin II binding site to hydrophobic residues in the C-terminal portion of p11(31, 32) . In contrast, the annexin I binding site of S100C appears to be in the N-terminal half of the protein as evidenced by the ability of the 6-kDa protein, which is missing the carboxyl portion of the protein, to interact with GST-AINT ( Fig. 6and Fig. 7).

Most S100 proteins that have been studied undergo a conformational change when they bind Ca that results in the exposure of a hydrophobic domain, which often interacts with other proteins(10) . Consistent with these studies, we found that S100C interacts in a Ca-dependent manner with endogenous annexin I ( Fig. 3and Fig. 4) and GST-AINT (Fig. 1). Since the GST-AINT construct lacks the annexin I Ca binding sites, we speculate that the Ca binding site required for this interaction resides on S100C. Previous studies with purified S100C have shown that it binds Cain vitro(21) . In contrast, the annexin II and p11 interaction is Ca-independent(33) . Amino acid substitutions and deletions in the two p11 EF-hand motifs prevent p11 from binding Ca(33, 34) , and these changes have been proposed to lock p11 into an exposed conformation even in the absence of Ca.

Like the interaction of annexin I with S100C, the interaction of annexin XI with calcyclin is dependent on Ca(29) and occurs in the N-terminal half of calcyclin (35) and within the N-terminal domain of annexin XI(9) . Unlike annexin II and possibly annexin I, Chou-Fasman analysis of the binding site on annexin XI indicates that it is unlikely to adopt a helical conformation(9) .

Based on length, the N-terminal domains of the annexin family in mammals can be divided into three categories. Annexins VII and XI are long (170 residues); annexins I, II, and XIIIb are intermediate (45 residues); and the others are short (15 residues). Since the three annexins that have been shown to interact with S100 proteins are in the long and intermediate category, it is tempting to speculate that the annexins with short N-terminal domains do not undergo this interaction. Indeed, we did not detect cellular proteins that associated with the short N-terminal domain of annexin V (Fig. 5). Furthermore, it will be interesting to determine whether annexin VII, which has a long N-terminal domain, will interact with an S100 protein.

The N-terminal domains of the annexins are structurally unrelated, and there is no evidence that they evolved from a common precursor. Thus, it would appear that interaction with S100 proteins evolved independently in the three different annexins. This implies that these annexin-S100 complexes serve an important cellular function.

In summary, this study shows the interaction with S100C may be involved in the cellular function of annexin I. Because little is known concerning the physiological function of either the annexins or the S100 family of proteins, it is difficult to speculate on the physiological function of their interaction. Previous studies of annexin II showed that interaction with p11 affected the cellular localization of annexin II. The heterotetramer of annexin II and p11 is primarily associated with cytoskeletal (36, 37) and Triton-insoluble complexes(38) , whereas annexin II monomers are primarily cytoplasmic (36) . Annexin XI localized to nuclear structures even though it lacks an obvious nuclear localization sequence, and this localization is speculated to be mediated by calcyclin(39) . Based on these parallels, it is possible that the interaction between annexin I and S100C may target this complex to particular regions of the cell in a Ca-dependent manner. Alternatively, the binding of S100C to the N-terminal domain of annexin I may modulate the activity of the core domain. Parallel studies of annexin II showed that the half-maximal Ca concentration required for membrane binding and aggregation is significantly higher for the annexin II monomer than for the annexin II-p11 heterotetramer(40, 41) . Although the properties of the annexin I-S100C complex have not been characterized, previous studies showed that post-translational modifications to the N-terminal domain of annexin I modulated the Ca and phospholipid binding properties of the core domain(14, 42) . The N-terminal domain also is known to modulate annexin I-induced phospholipid vesicle aggregation, even though this activity does not directly reside in the N-terminal domain(43, 44, 45) . The elucidation of a definitive role for S100C in the targeting or regulation of annexin I requires more information concerning the biological function of annexin I.


FOOTNOTES

*
This work was supported by Grant BE-196 from the American Cancer Society and by Predoctoral Training Grant 5T32CA09054 (to W. S. M.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 714-824-6304; Fax: 714-824-8540.

Present address: The Salk Institute for Biological Studies, Dept. of Molecular Biology and Virology, 10010 N. Torrey Pines Rd., La Jolla, CA 92027.

(^1)
The abbreviations used are: GST, glutathione S-transferase; AINT, annexin I N-terminal; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.


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