Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M605798200 on October 8, 2006

J. Biol. Chem., Vol. 281, Issue 50, 38905-38917, December 15, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/50/38905    most recent
M605798200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sturchler, E.
Right arrow Articles by Heizmann, C. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sturchler, E.
Right arrow Articles by Heizmann, C. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

S100A16, a Novel Calcium-binding Protein of the EF-hand Superfamily*

Emmanuel Sturchler{ddagger}, Jos A. Cox§, Isabelle Durussel§, Mirjam Weibel{ddagger}, and Claus W. Heizmann{ddagger}1

From the {ddagger}Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Steinwiesstrasse 75, 8032 Zurich, Switzerland and the §Department of Biochemistry, University of Geneva, 1211 Geneva, Switzerland

Received for publication, June 16, 2006 , and in revised form, September 21, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
S100A16 protein is a new and unique member of the EF-hand Ca2+-binding proteins. S100 proteins are cell- and tissue-specific and are involved in many intra- and extracellular processes through interacting with specific target proteins. In the central nervous system S100 proteins are implicated in cell proliferation, differentiation, migration, and apoptosis as well as in cognition. S100 proteins became of major interest because of their close association with brain pathologies, for example depression or Alzheimer's disease. Here we report for the first time the purification and biochemical characterization of human and mouse recombinant S100A16 proteins. Flow dialysis revealed that both homodimeric S100A16 proteins bind two Ca2+ ions with the C-terminal EF-hand of each subunit, the human protein exhibiting a 2-fold higher affinity. Trp fluorescence variations indicate conformational changes in the orthologous proteins upon Ca2+ binding, whereas formation of a hydrophobic patch, implicated in target protein recognition, only occurs in the human S100A16 protein. In situ hybridization analysis and immunohistochemistry revealed a widespread distribution in the mouse brain. Furthermore, S100A16 expression was found to be astrocyte-specific. Finally, we investigated S100A16 intracellular localization in human glioblastoma cells. The protein was found to accumulate within nucleoli and to translocate to the cytoplasm in response to Ca2+ stimulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
S100 proteins represent the largest subgroup of Ca2+-binding proteins of the EF-hand type with 21 identified members (1, 2). The number of S100 genes, their chromosomal localization, transcriptional direction, as well as their expression pattern are highly conserved between human, mouse, and rat (3). Among the different human S100 genes, 17 are clustered on the human chromosome 1q21 (3), a region frequently involved in chromosomal rearrangement in cancers (4-6). Expression patterns of some members, including S100A3, -A8, -A9, -A12, and -B, are tissue-and cell-specific (7-10), whereas S100A2, -A13, and -A16 are expressed in a wide variety of tissues (2, 11-13). S100 proteins play a role in Ca2+ homeostasis, cell proliferation, migration, differentiation, and apoptosis. A total of ten S100 family members are reported to be expressed in the central nervous system in a temporal, spatial, and cell type-specific manner. In response to Ca2+ oscillation, S100 proteins interact with distinct target proteins and/or relocate to different cellular compartments (for review see Ref. 14). Thereby, they are implicated in multiple intra- as well as extracellular activities. The brain-specific S100B regulates enzyme activity by rendering certain phosphorylation sites inaccessible to their interaction partners as was reported for neurogranin (15), tau protein (16, 17), GFAP2 (18), and p53 transcription factor (19-22). More recently, S100A10 has been shown to interact with serotonin 1B receptor and to modulate its membrane presence and availability (23). In addition, several S100 proteins have been shown to be secreted (24-27) via different mechanisms, including the classic endoplasmic reticulum-Golgi pathway, and alternative pathways involving cytoskeletal components such as actin and tubulin. S100B secretion from astroglial cells is induced by serotonin 1a receptor agonist binding (28). Moreover, the release of S100 protein from apoptotic cells following brain injury may be sufficient to induce cellular responses. In vitro, extracellular S100B and S100A4 promote neurite outgrowth and neuronal survival by acting through the receptor for advanced glycation end products and heparan sulfate proteoglycans, respectively, both of which are present at the cell surface (29). Moreover, extracellular S100B has been shown to modulate long term neuronal synaptic plasticity (30), glutamate uptake of hippocampal astrocytes (31), and inflammation (32) (for review see Ref. 33). Several S100 proteins have been linked to brain diseases like multiple sclerosis, Down's syndrome, Alzheimer's disease, or depression, usually due to altered levels of protein expression.

S100 proteins are small acidic proteins (Mr ~ 10-13 kDa) characterized by distinctive homo- or hetero-dimeric architecture and two highly conserved Ca2+-binding domains: a classic C-terminal EF-hand with a canonical Ca2+-binding loop and an S100-specific N-terminal EF-hand (for reviews see Refs. 34-38). In general the affinities of S100 proteins for Ca2+ are low with a [Ca2+]0.5 value of 100-500 µM (39). Binding of Ca2+ to S100B induces helix rearrangement within each subunit of the dimer, resulting in the exposure of two hydrophobic surfaces (one in each monomer) (38, 40, 41), which are involved in target protein recognition (36, 37). Beside Ca2+, a number of S100 proteins can also bind Zn2+ or Cu2+ (42, 43) that can influence the affinity of Ca2+ binding (38). Thus, S100 proteins display variable transition metal-binding properties in agreement with their highly diversified and specialized functions.

Among the S100 protein family, the S100A163 is a novel member. S100A16 gene was isolated from astrocytoma and is located in the S100A cluster on human chromosome 1q21. Interestingly, the mouse S100A16 protein differs from its human orthologue by the presence of five additional QQE(C/S) repeats at the C-terminal end. S100A16 has been described to be ubiquitously expressed and has been shown to be up-regulated in human tumors (2). To gain first insights on the role and the molecular mechanism by which it exerts its function in the central nervous system, we expressed and purified both human and mouse S100A16 recombinant proteins. We compared their Ca2+- and Zn2+-binding properties and the effect of these cations on protein conformation. Furthermore, we used 2-p-toluidinylnaphtalene 6-sulfonate (TNS) to analyze the formation of Ca2+- and Zn2+-induced hydrophobic patches. S100A16 localization in mouse brain was investigated by in situ hybridization and immunostaining. Area-restricted distribution as well as an astrocyte-specific expression was observed. Finally, using a specific purified antibody, we focused on the subcellular localization of S100A16 in glioblastoma cell lines under different conditions. Our results demonstrate the presence of S100A16 protein in nucleoli and cytoplasm of the cells. Furthermore, [Ca2+]i influences the nuclear export/import of S100A16.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
His Tag S100A16 Constructs—The human and the mouse S100A16 cDNA were obtained from the IMAGE consortium. The coding region of both human and mouse S100A16 was amplified by PCR using specific oligonucleotides containing, respectively, a 5' BamHI site (5'-GGTGGATCCATGTCAGACTGCTACACGGA-3'/5'-GGTGGATCCATGGCTGACTGCTATACAGA-3') and a HindIII site (5'-GGTAAGCTTCTAGCTGCTGCTCTGCTGC-3'/5'-GGTAAGCTTCTAGCTGCTGCCCTGCTGG-3'). PCR products were cut with the corresponding restriction enzymes and cloned into the pET20-nHisT vector downstream of the His tag.

Generation of S100A16-GFP Fusion Construct—The coding region of human S100A16 cDNA was amplified by PCR using oligonucleotides containing a 5' Xho site (5'-CTCGAGATGTCAGACTGCTAC-3') and a BamHI site (5'-GGATCCCGGCTGCTGCTCTGCTG-3'). PCR products were cut with the corresponding restriction enzymes and cloned in-frame into the pEGFP-N1 vector (Clontech). Plasmids were sequenced by using a sequencer (ABI Prism 310, Applied Biosystems, Foster City, CA) to verify the integrity of ORF.

Expression and Purification of the Human and Mouse Recombinant S100A16 Proteins—The S100A16-pET20-nHisT constructs were transformed into the BL21(DE3)pLysS strain. After induction of the expression with 1 mM isopropyl-1-thio-beta-D-galactopyranoside and cell lysis, the purification of the proteins was performed on a nickel-nitrilotriacetic acid column (Invitrogen) according to the manufacturer's protocol. The His-tagged S100A16 fusion proteins were then cleaved with thrombin (Roche Diagnostics, Mannheim, Germany) overnight at room temperature. Recombinant S100A16 was further purified by using a HiTrap benzamidine FF (high sub) column (Amersham Biosciences) to remove thrombin. Fractions were collected, and the presence of S100A16 proteins was identified by Western blotting and mass spectrometry. The flow-through of the HiTrap benzamidine column containing the human S100A16 protein was dialyzed against 50 mM Tris-HCl, pH 7.5, 500 mM KCl, 1 mM beta-mercaptoethanol, 50 µM EGTA. The mouse S100A16 protein was dialyzed against 50 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM beta-mercaptoethanol, 50 µM EGTA. The protein concentrations were determined by spectrophotometry using the extinction coefficients {epsilon}278 nm of 11590 M -1cm-1 for the human S100A16 and 11960 M-1cm-1 for the mouse S100A16, both based on their Trp and Tyr content.

Metal Removal and Cation Binding—Human S100A16 and mouse S100A16 were dialyzed against 50 µM EGTA or precipitated with 3% trichloroacetic acid to reduce the Ca2+ contamination and passed through a 40 x 1 cm Sephadex G-25 column equilibrated in 50 mM Tris-HCl, pH 7.5, 150 mM KCl (buffer A) for mouse S100A16 or 50 mM Tris-HCl, pH 7.5, 500 mM KCl (buffer B) for human S100A16. Typically the contamination represents <2% of the total binding capacity. Total Ca2+ was determined with a PerkinElmer Life Sciences 2380 atomic absorption spectrophotometer.

Flow dialysis on 100-200 µM S100A16 were carried out on mouse S100A16 in buffer A and on human S100A16 in buffer B, both in the absence or presence of 50 µM Zn2+ at 25 °C according to the modified method of Colowick and Womack (44). Treatment of the raw data and evaluation of the binding parameters was done as previously described (45).

Direct Zn2+ binding was determined with the method of equilibrium gel filtration on a Sephadex G-25 (column of 40 x 1 cm) equilibrated in buffer A containing a fixed concentration of Zn2+. The protein concentration was determined by spectrophotometry, and the free and protein-bound Zn2+ were quantified with atomic absorption.

Trp Fluorescence—Emission fluorescence spectra were taken with a PerkinElmer LS-5B spectrofluorometer on 12 µM of the metal-free form in buffer A or B at 25 °C with excitation at 278 nm and slits of 5 nm. 20 µM EGTA, or 1 mM Ca2+, or 20 µM Zn2+, or 4 M guanidine-HCl were added to monitor the effect of the respective cations or to obtain the spectrum of the denatured protein. Solution backgrounds were subtracted.

Interaction with Hydrophobic Probes—The Ca2+-dependent changes in the hydrophobic core of the S100A16 proteins were followed by monitoring the fluorescence properties of TNS as described previously (46). Briefly, the final solutions contained 40 µM TNS and 8 µM protein in buffer A (mouse S100A16) or B (human S100A16). The Ca2+ and Zn2+ titrations were carried out on 6-8 µM metal-free protein in buffer A or B.

Thiol Reactivity—The metal dependence of the single thiol in human S100A16 and of the four thiols in mouse S100A16 was monitored after the protein samples had been thoroughly reduced by overnight incubation at room temperature with 100 mM dithiothreitol in the presence or absence of 8 M urea. The reducing agent and urea were removed by gel filtration on Sephadex G-25 equilibrated in buffer A, which was freshly degassed and saturated with nitrogen. The kinetics of the thiols was initiated by mixing 12.5 µM mouse S100A16 in buffer A or 25 µM human S100A16 in buffer B with 5,5'-dithiobis(2-nitrobenzoic acid) at 100 µM final concentration, and the absorption was continuously monitored at 412 nm (47). The pseudo first order rate constants and t1/2 values were extracted by standard procedures.

Quantitative PCR Analysis—Two-month-old mouse brain RNA was extracted using TRIzol reagent (Invitrogen) and treated with DNase (Roche Diagnostics) for 30 min at 37 °C. S100 mRNAs, converted to cDNA, were quantified by real-time PCR using the ABI/PRISM 7700 sequence detection (Applied Biosystems). Analyses were performed using primers, internal fluorescence probes specific to each S100 mRNA, and the TaqMan Gene Expression assays kit (Applied Biosystems). PCR contained 0.4 µM primers and 0.2 µM TaqMan MGB probe and consisted of a 10-min denaturation step at 95 °C followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Mix of unlabeled primers and TaqMan MGB probes were obtained from Applied Biosystems (Assays-on-demandTM Gene Expression Products). Normalized value for S100 mRNAs expression in each sample was calculated as the relative quantity of S100 divided by the relative quantity of S100B.

RNA Probe Design and Labeling—Two different antisense mouse S100A16 probes corresponding either to the open reading frame (ORF) or to the 3'-untranslated region (3'UTR) sequence of the gene were obtained by PCR using the mouse S100A16 cDNA clone as template (IMAGE consortium, ID IMAGp998J199175).The primers used for the amplification of mouse S100A16-ORF and mouse S100A16-3'UTR were, respectively, 5'-GGAATTCCATATGGCTGACTGCTATAC-3'/5'-CGGGATCCTAGCTGCTGCCCTGC-3' and 5'-ACCTCATCCGCCAACAGGAG-3'/5'-CACTACCACAGCTGCCACAG-3'. Each PCR product was cloned into a pCRII-TOPO vector (Invitrogen). Antisense digoxigenin (DIG)-labeled RNA probes (or control sense probe) were obtained following the manufacturer's protocol (Roche Diagnostics). DIG-labeled RNA was diluted to a concentration of 50 ng/µl, aliquoted, and stored at -80 °C.

In Situ Hybridization—Briefly, 7- to 9-week-old C57/BL6 mice were perfused intracardially with 250 ml of cold 4% paraformaldehyde solution in 0.1 M phosphate buffer, pH 7.4. The brains were removed, post-fixed for 4 h, placed in 20% sucrose overnight, and stored at -80 °C until use. Slices were obtained on a freezing microtome at 20-µm thickness. Sections were treated with proteinase K (0.5 µg/ml), washed in phosphate-buffered saline, acetylated, washed in distilled water and pre-hybridized for 4 h at 56 °C. Each slide was then hybridized overnight in hybridization buffer containing 200 ng/ml DIG-labeled cRNA. Slides were washed under stringent conditions (56 °C, 2x SSC, 2x 50% formamide, 0.2x SSC, 50% formamide, 0.1x SSC). The in situ signal was visualized using alkaline phosphatase conjugated to anti-digoxigenin antibody (Roche Diagnostics) diluted 1:2000 in phosphate-buffered saline, 1% blocking reagent, and developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate (Roche Diagnostics). Sections were examined and photographed using a Zeiss Axioskop light microscope (Zeiss, Germany).

Production and Purification of Polyclonal Antibodies—Antisera against human recombinant S100A16 were produced in rabbits by DakoCytomation (Glostrup, Denmark). The titers of the antisera were determined using 1 µg of recombinant protein on Western blots as described by Ilg et al. (12). An aliquot of antiserum was affinity-purified by using a HiTrap protein A column (Amersham Biosciences) following the manufacturer's protocol. This antibody (hS100A16 Ab) was used for further studies.

Western Blot Analysis—Total cell extracts were prepared as described previously (12). Nuclear protein fractions were obtained from extracts of U373 MG cells by centrifugation at 1000 x g, 10 min at 4 °C.

Total mouse brain tissue, cortical, hippocampal, and cerebellar regions were lysed in 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1% Triton-X supplemented with Complete Proteinase Inhibitor Mixture (Roche Diagnostics). The lysates were passed through a 23-gauge needle and centrifuged at 13,000 x g at 4 °C for 15 min, and supernatants were analyzed. Subcellular fractionation (nuclei, cytosol, and membrane) from mouse cortex were performed as described in Ref. 48.

Protein concentration of the lysates was measured by use of the BCA Protein Assay Reagent (Pierce). Per sample, 20-50 µg of total protein were separated with 10% SDS-PAGE and blotted onto nitrocellulose (Schleicher and Schuell, Dassel, Germany). The membrane was incubated for 1 h with hS100A16 (1:5,000), with the mouse monoclonal human anti-beta-tubulin (1:10,000) (Sigma) or with anti-nuclear laminin (1:1,000, Abcam, Cambridge, UK). The blot was incubated with anti-IgG conjugated with peroxidase (1:10,000, Sigma). The bands were detected using ECL solution (Amersham Biosciences).

Cell Cultures and Transient Transfections—SKN-LE and SKN-BE neuroblastoma (a gift from Dr. A. Fontana, University Hospital, Zurich) and U87 MG and U373 MG glioblastoma (ATCC, Manassas, VA) cells were cultivated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. SHSY-5Y (ATCC) neuroblastoma cells were maintained in RPMI supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin.

U87 MG and U373 MG cells were transfected with human S100A16-GFP construct using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions and incubated for 16 h to allow gene expression. Cells were stimulated from 1 to 30 min with 1 µM thapsigargin (Sigma), 1 µM ionomycin (Sigma) in medium supplemented with 1.5 mM CaCl2, 2 mM EGTA, or BAPTA-AM.

Immunocytochemistry—For GFP fluorescence and immunostaining, U87 MG and U373 MG cell lines were transferred into 24-well plates. The stainings were performed as described (12). For double immunofluorescence staining, hS100A16 Ab and the mouse monoclonal anti-human nucleolin (Abcam) were diluted at 1:1000. The secondary fluorescein isothiocyanateconjugated antibody and the secondary Cy3-conjugated antibody were used at a dilution of 1:500. The coverslips were mounted on glass slides and embedded in Mowiol (Hoechst, Frankfurt, Germany). The fluorescence signals were visualized using a Leica SP2 confocal laser microscope (Leica, Germany) or a Zeiss Axioskop light microscope (Zeiss, Germany).


Figure 1
View larger version (40K):
[in this window]
[in a new window]

  		FIGURE 1.
Expression and purification of human and mouse recombinant S100A16. A, SDS-PAGE of protein extracts of human S100A16 transformed Escherichia coli BL21 culture before (lane 1) and after induction with isopropyl 1-thio-beta-D-galactopyranoside (lane 2). Human (Lane 3) and mouse (Lane 6) His-tagged S100A16 proteins were purified on a nickel-nitrilotriacetic acidagarose column. Purified human (lane 4) and mouse (lane 7) S100A16 protein after His tag removal runs as a monomer. S100A16 dimers where present even after treatment with 250 mM dithiothreitol (lane 5 and 8). B, mass spectrum of recombinant human S100A16 protein. The figure shows the mass spectrum indicating the molecular mass of monomers, dimers, trimers, and even tetramers.

 
For immunostaining of tissue sections, 7- to 9-week-old C57/BL6 mice were perfused intracardially with 250 ml of cold 4% paraformaldehyde solution in 0.1 M phosphate buffer, pH 7.4, containing 1% picric acid. The brains were removed, post-fixed for 4 h, placed in 20% sucrose overnight, and stored at -80 °C until use. Coronal sections (20 µm) were pretreated (10 min at 90 °C in citrate buffer) and incubated with hS100A16 Ab (1:1000) and with a chicken anti-mouse Gfap antibody (1:2,000, Abcam) overnight at 4 °C, with secondary fluorophore-conjugated antibodies for 2 h at room temperature, counterstained with 4',6-diamidino-2-phenylindole (1:2000, Sigma) and mounted in eukit mounting media (Merck, Darmstadt, Germany). Sections were imaged by using a Zeiss Axioskop light microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Purification of Human and Mouse Recombinant S100A16—Human and mouse recombinant S100A16 proteins were expressed in BL-21 bacteria and purified. Expression of recombinant mouse and human S100A16 was facilitated by the presence of a His tag at the N-terminal end (Fig. 1A, lanes 1 and 2). The human and the mouse His-tagged proteins were purified by passing the bacterial crude lysate onto a nickel-nitrilotriacetic acid column (Fig. 1A, lanes 3 and 6). About 10 mg of each protein could be expressed in 500 ml of LB medium. After cleavage of the His tag and thrombin removal using a benzamidine FF column, purity and concentration of both human and mouse S100A16, were examined by SDS gel electrophoresis (Fig. 1A, lanes 4 and 7). S100A16 proteins migrate in two bands of ~12 and 24 kDa for the human protein and 15 and 30 kDa for the mouse S100A16 (Fig. 1A, lanes 4 and 7) suggesting that each protein can form covalent dimers as it was already reported for most of the other members of this family (34-38). Furthermore, even harsh treatment with 250 mM dithiothreitol could not completely reduce either the human or the mouse dimers to the monomeric form (Fig. 1A, lanes 5 and 8). The correct synthesis and molecular mass of both recombinant proteins were verified by MALDI-TOF mass spectrometry (matrix-assisted laser desorption/ionization-time of flight). The spectra show a peak with a molecular mass of 11951 Da for the human recombinant protein (Fig. 1B) and 14464 Da for the mouse recombinant protein (data not shown), corresponding to the theoretically calculated molecular masses plus one additional serine and one additional glycine. In addition, higher order oligomers, namely dimers (23884 Da), trimers (35805 Da), and even tetramers (47706 Da) were also observed in both spectra as shown for human S100A16 (Fig. 1B).

Biophysical Characterization—The recombinant human S100A16 appeared to be poorly soluble compared with the mouse protein, likely due to its more basic character (pI 6.3 versus pI 5.6, respectively). The additional QQE(C/S) repeats present in the C-terminal part of mouse S100A16 may also account for the improved solubility of the mouse protein. In physiological conditions, the mouse S100A16 is soluble to up to 5-10 mg/ml protein, whereas the human S100A16 is only soluble to ~0.1 mg/ml. Higher concentrations of human S100A16 can only be kept in solution of high ionic strength, i.e. in the presence of 0.5 M KCl. The onset of human S100A16 precipitation takes several hours, which allows performing short lasting experiments with clear solutions. The addition of Zn2+ (>200 µM) to both proteins facilitates their precipitation.

Direct Cation Binding Studies—Flow dialysis experiments on apo mouse S100A16 (50-90 µM) in buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl) yielded a simple isotherm (Fig. 2, red) with saturation at one Ca2+ per monomer and a Kd of ~750 µM and nH close to 1. Thus, the affinity for Ca2+ is very low and there is only one binding site per monomer. Similar experiments on human S100A16 in buffer B (50 mM Tris-HCl, pH 7.5, 500 mM KCl) yielded also one Ca2+-binding site per monomer (Fig. 2, blue) with a Kd of 430 µM and nH ~1. In the presence of 100 µM Zn2+ the two proteins precipitate at the protein concentrations employed. Because this may alter the diffusion properties of the membrane, Ca2+ titrations in the presence of Zn2+ were not performed. The main difference between the S100A16 proteins and other S100 proteins is the presence of only one functional Ca2+-binding site (per monomer) in S100A16. This is predicted from the S100A16 amino acids sequences, which both show a Glu to Ser substitution in the critical Z position of the N-terminal EF-hand. The human protein differs from the mouse one mainly in its higher affinity for Ca2+, i.e. 430 µM, which approximates the value in several other S100 proteins (39). Direct Zn2+ binding measurements were performed with mouse S100A16 by equilibrium gel filtration at low Zn2+ concentration (30 µM) to minimize protein precipitation. In these conditions ~0.47 atom of Zn2+ was bound per monomer. This suggests the presence of one Zn2+-binding site in the monomer with a Kd of ~25 µM (see below). At higher free Zn2+ concentrations the protein remains precipitated on the Sephadex G-25 column and could only be eluted with EGTA.


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 2.
Ca2+-binding to the S100A16 proteins. Direct Ca2+ binding was monitored by flow dialysis at 25 °C in 50 mM Tris-HCl, pH 7.5, 150 mM KCl (buffer A) for mouse S100A16 (red) and in 50 mM Tris-HCl, pH 7.5, 500 mM KCl (buffer B) for human S100A16 (blue). The protein concentration is 50 (circles) or 90 µM (rectangles). The solid lines are the theoretical isotherm calculated with the Adair equation for one site.

 


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 3.
Conformational changes measured by intrinsic Trp fluorescence. A, mouse S100A16 in buffer A. B, human S100A16 in buffer A. The color code is as follows: blue, apo; red,Ca2+; green,Zn2+; dotted red,Ca2+ plus Zn2+; black, denatured (4 M guanidine-HCl). C, titration of apo human S100A16 in buffer A with Ca2+.

 
Trp Fluorescence—Both mouse S100A16 and human S100A16 contain a single Trp-80 located in the second helix of the C-terminal EF-hand. In the native apo proteins the Trp is well protected from the solvent, because addition of 4 M guanidine-HCl leads to a red shift from 331 to 351 nm and a slight decrease of the fluorescence intensity (Fig. 3). In mouse S100A16 in buffer A the Trp fluorescence intensity is not affected by Ca2+ up to 2 mM, but there isa4nmred shift of the maximum. Zn2+ (40 µM) increases the fluorescence slightly (1.1-fold) without a wavelength shift (Fig. 3A), but strongly increases the light scattering measured at 285-290 nm, meaning that the protein aggregates (Fig. 3A). This protein precipitation starts at 10 µM Zn2+ and is very marked at 250 µM.Ca2+ amplifies the extent of Zn2+-induced aggregation (Fig. 3A, red dotted versus green solid line).

Upon addition of 2 mM Ca2+ to human S100A16 in buffer A, there was very limited aggregation (Fig. 3B, red solid line in the 285-290 nm region) and no precipitation, but the fluorescence intensity decreased to half its initial value (Fig. 3B, red arrow). This step was titrated at 331 nm (Fig. 3B, red arrow) and yielded a Kd of 220 µM in buffer A (Fig. 3C) and of 180 µM in buffer B (not shown). In human S100A16 (12 µM) in buffer A, Zn2+ provoked a mild decrease of the Trp fluorescence again accompanied with protein aggregation (Fig. 3B). In conclusion, Trp-80 in the C-terminal EF-hand is quite strongly influenced by the C-terminal poly QQE(C/S) sequence, which is present in mouse but not in human. Whereas Ca2+ binding to this EF-hand lowers the fluorescence in human, the C-tail prevents this phenomenon in mouse. This segment of 21 amino acids, which has equal propensity to form coil and helix, may interact intramolecularly with the Ca2+-binding core.

Interaction with the Hydrophobic Probe TNS—Metal-free mouse S100A16 in buffer A enhances the fluorescence of the hydrophobic probe TNS 5-fold with a maximum at 426 nm (Fig. 4A). Upon further addition of up to 2 mM Ca2+ this fluorescence emission does not change (red versus blue solid line), suggesting that Ca2+ binding does not induce a hydrophobic surface. In contrast, binding of Zn2+ leads to a 2-fold increase in fluorescence intensity over that of the apo form (green versus blue solid line). The signal change is complete at 250 µM Zn2+ and the [Zn2+]0.5 is estimated at 25 µM, both in the presence and absence of 2 mM Ca2+ (Fig. 4C) This value is in agreement with the direct Zn2+-binding data for mouse S100A16. Note that the presence of Ca2+ does not modify the interaction of mouse S100A16 with Zn2+. The Zn2+ effect is mostly reversible upon addition of 0.35 M KCl corresponding to buffer B (Fig. 4A, green dotted line), which solubilizes (visual observation) the Zn2+-induced protein precipitate, suggesting that the aggregation creates hydrophobic sites. Cu2+ up to 100 µM does not affect the TNS fluorescence enhancement by mouse S100A16 (data not shown) and, indirectly, Cu2+ has also no effect on the Zn2+ enhancement, suggesting that Cu2+ does not interact with the protein.

Metal-free human S100A16 in buffer A enhances the basic TNS fluorescence 3-fold with a maximum at 428 nm. One mM Ca2+ induces a 1.7-fold increase in fluorescence enhancement (Fig. 4C, red arrow), and the fluorescence change was monitored at 435 nm; the estimated [Ca2+]0.5 is 150 µM, and the curve displays pronounced positive allostery with nH equal to 1.7 (Fig. 4D). Zn2+ addition to apo human S100A16 promotes a 2.1-fold enhancement and a [Zn2+] of 25 µM (Fig. 4C). Again, Cu2+ has no effect, neither on the fluorescence of the apostate, Ca2+-, or Zn2+-, or Ca2+/Zn2+-state. The difference between the mouse and the human protein in Ca2+-dependent hydrophobic exposure suggests that the extended C terminus in the mouse neutralizes the hydrophobic patch.


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 4.
Ca2+-dependent exposure of hydrophobic side chains. Hydrophobic exposure was monitored by the fluorescence change of TNS at 25 °C. A, mouse S100A16 in buffer A. B, human S100A16 in buffer A. The color code is as follows: black, TNS alone; blue, plus apo protein; green, 1 mM Zn2+; red, 1 mM Ca2+; red, dashed, and dotted, 1 mM CaCl2 plus ZnCl2; dotted green,Zn2+ in the presence of buffer B; dotted blue, apoprotein in buffer B. C, the titration at 425 nm of apo protein with Zn2+: apo mouse S100A16 (red), Ca2+-loaded mouse S100A16 (green), and apo human S100A16 (blue). D, the fluorescence titration at 425 nm of apo human S100A16 with Ca2+ (red arrow in Fig. 4B).

 
Conformational Changes Measured by Thiol Reactivity—Mouse S100A16 possesses four Cys residues, one at position 4 from the N-terminal end, one in the middle of the C-terminal EF-hand, and two in the unusual C-terminal tail containing the QQE(C/S) repeats. After extensive reduction only two thiols were instantaneously and completely titrated in both the apoprotein and in the Ca2+-saturated form of mouse S100A16. In the presence of high concentrations of Zn2+ only one of these two thiols reacted instantaneously, whereas the second was partly protected. Half-maximal protection of the latter thiol showed an estimated [Zn2+]0.5 of 170 µM (see Table 1). The two remaining Cys were either oxidized to a disulfide bridge or in a thiol form, therefore inaccessible to 5,5'-dithiobis(2-nitrobenzoic acid). We estimate that the non-reactive thiols in mouse S100A16 are located in the C-terminal tail. Human S100A16 has only one Cys residue in position 4, and this thiol reacted instantaneously in apo and Ca2+ form and was protected by Zn2+ in a similar way as the Zn2+-sensitive thiol in mouse S100A16. It is thus likely that in mouse S100A16 also the Zn2+-sensitive thiol is Cys-4.


View this table:
[in this window]
[in a new window]

  		TABLE 1
Thiol reactivity in mouse and human S100A16

 
S100A16 mRNA Expression and Distribution in Mouse Brain—Previous studies reported low S100A16 gene expression in human brain (2). We first compared the relative expression of S100A16 and other S100 protein genes to the expression of S100B, a brain-specific calcium-binding protein, using real-time PCR assay. Total RNA was extracted from adult mouse brain and mRNA quantification revealed a 10-fold lower expression of S100A16 compared with S100B (Fig. 5A). Furthermore, S100A16 transcript levels were also below those of S100A1, S100A6, and S100A13, whereas S100A16 mRNA was ten times more abundant than S100A4 (Fig. 5A).


Figure 5
View larger version (111K):
[in this window]
[in a new window]

  		FIGURE 5.
A, relative quantification of multiple S100 mRNAs in mouse brain. S100A16 transcripts were ten times less abundant compared with S100B. In situ hybridization analysis of S100A16 mRNA distribution in mouse brain (B-D). B, the figures show positive labeled coronal sections of the parietal (Par), occipital (Oc), and temporal cortex (Te), the hippocampus-dentate gyrus complex (C), and cerebellum (D). Darker areas correspond to hybridization signals. Arrowheads in C and D indicate labeling corresponding to cells surrounding, neurons of the pyramidal cell layer (Py) of CA2 field (CA2), and Purkinje neurons (P). CL 1-6, layers 1-6 of cerebral cortex; CA1-3, Ammon's horn fields 1-3; DG, dentate gyrus; CbM, molecular layer; CbG, granular layer. Scale bars = 200 µm; 50 µmin D.

 
The expression pattern of S100A16 in adult mouse brain was also investigated by in situ hybridization. To analyze the S100A16 mRNA distribution, two specific probes corresponding to the ORF and to the 3'UTR were generated. The different types of anesthesia, tissue preparation, or specific S100A16 antisense probes did not influence the appearance of the staining pattern. Moreover, sections hybridized with sense probes did not reveal any positive signal (data not shown). In adult mouse brain, expression of S100A16 gene was observed in the cerebral cortex at all levels of the mouse brain (Fig. 5B). We found an intense staining throughout layer I to layer VI (Cl 1-6) with no particular enrichment in any layer, in the parietal (Par), occipital (Oc), and temporal cortex (Te). Moreover, lower, but unambiguous S100A16 mRNA levels were detected in cells resident in cortical layer 1 (Cl 1). In the hippocampal formation (Fig. 5C), hybridization signal was detected in the Ammon's horn fields 2 (CA2) and 3 (CA3) and in the hilus of the dentate gyrus (DG). Higher magnification imaging revealed S100A16 mRNA labeling in cell bodies present around neurons of the pyramidal cells layer. In the cerebellum, hybridization performed at the cellular level, showed S100A16 expression in cells that surround Purkinje cells (Fig. 5D). These results indicate that S100A16 gene is widely expressed in the cortical regions, whereas its expression is restricted to discrete regions and specific cell types in both the hippocampus and the cerebellum.

Characterization of hS100A16 Ab—The human recombinant protein was used to produce specific rabbit polyclonal antisera. Affinity-purified polyclonal anti-human S100A16 antibodies (hS100A16 Ab) were first tested for their cross-reactivity against other S100 proteins by Western blot analysis. hS100A16 Ab recognized specifically the corresponding antigen (Fig. 6) and did not cross-react with the other family members. Furthermore, hS100A16 Ab also recognized the mouse S100A16 recombinant protein (Fig. 6). In addition to the monomer, hS100A16 Ab recognized two additional protein bands of ~22 kDa and 35 kDa, which correspond to the dimeric and trimeric forms of human and mouse recombinant S100A16 (Fig. 6) in agreement with the MS data presented in Fig. 1B.

Expression and Immunolocalization of S100A16 in Mouse Brain—To examine S100A16 protein expression, 50 µg of total protein extracts from different brain regions were analyzed by Western blot analysis. As shown in Fig. 7A, hS100A16 Ab detected a single protein band in cortical, hippocampal, and cerebellar extracts, with a relative molecular mass of ~15 kDa, consistent with the theoretically calculated molecular weight, and our MS analysis. Immunoblotting of the 20-µg membrane, cytosolic, and nuclear fractions of cortical extract revealed the presence of S100A16 protein in the cytoplasm and a high amount in the nucleus of cells (Fig. 7A, Cy and Nu).

We next performed immunohistochemistry using hS100A16 Ab to evaluate the distribution and the intracellular localization of S100A16 in mouse brain. The expression of S100A16 protein was relatively prominent in layers 1-6 of all cortical regions (Fig. 7B). To further investigate cell type specificity of S100A16 protein expression in the cortex, double-label immunofluorescence staining was performed. S100A16 labeling was mainly associated with nuclei and somata of cells positive for Gfap, an astrocyte marker (Fig. 7C). In the cerebellum (Fig. 7D), we found prominent staining at the level of Purkinje neurons. However, higher resolution images of co-immunostaining revealed labeling in somata and neurites of cells co-expressing Gfap (Fig. 7D, right panel). Moreover, only a few cells in the molecular and in the granular layer were also positive for S100A16 (Fig. 7D). In the hippocampal formation (Fig. 7, E-G), hS100A16 Ab consistently labeled cells in the molecular cell layer of the CA2-CA3 subfields (Fig. 7E). A more prominent staining was detected in the polymorphic cell layer of the dentate gyrus (Fig. 7F), where S100A16 was present in cell bodies and neurites spreading into the granular cell layer. In this brain structure, S100A16 and Gfap staining overlap (Fig. 7G). There was virtually no labeling of dentate granule cells (Fig. 7F) or pyramidal neurons in CA1-CA3 regions (Fig. 7E). Altogether, these data confirm in situ hybridization observation and provide first evidence for the occurrence of S100A16 protein in nuclei, somata, and neurites of astrocytes in the adult mouse brain.


Figure 6
View larger version (79K):
[in this window]
[in a new window]

  		FIGURE 6.
Specificity of hS100A16 Ab. hS100A16 Ab was tested against other human S100 proteins and mouse S100A16 protein. Western blot analysis against indicated recombinant proteins (2 µg each) revealed the specificity of the antibody and cross-reaction with the mouse S100A16 protein. The proteins were blotted onto nitrocellulose membrane and stained with Ponceau S as a loading control (bottom), and immunoreactivity was visualized using horseradish peroxidase-coupled antibody.

 


Figure 7
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 7.
S100A16 protein levels in different parts of the mouse brain. A, top, Western blot analysis using hS100A16 Ab revealed different levels of the protein in the cortex (Cx), hippocampus (Hip), and cerebellum (cer). Immunoblotting of subcellular fractions of cortical tissue in membrane (Mb), cytoplasmic (Cy), and nuclear (Nu) extracts (A, bottom). Immunolocalization of S100A16 in mouse brain. Coronal sections were immunostained with hS100A16 Ab. DNA is stained with 4',6-diamidino-2-phenylindole (blue). B, in cortex, S100A16-positive cells (red) were uniformly distributed throughout cortical layers 1-6 (Cl 1-6). C, in cortical layer 2, S100A16 (green), was detected in the cytoplasm and nuclei (arrowheads) of cells co-expressing Gfap (red). D, in cerebellum, intense S100A16 staining (red) was observable in the vicinity of Purkinje cells and in single cells of the molecular layer (CbM) and granular layer (CbG). S100A16 labeling was not observed in cerebellar white matter (CbW). E-G, in the hippocampal formation, S100A16 immunoreactivity was mainly localized in the molecular layer (HmoL) of the CA2 field (CA2) and in the polymorphic cell layer (Po) of the dentate gyrus (F). G, at higher magnification, endogenous S100A16 (green) was distributed throughout neurites. Note the absence of in pyramidal neurons (Py) and in granule cell neurons (Gr). C, D (right), and G, coronal sections were double immunolabeled with specific antibody for S100A16 (green) and for Gfap (red). The astrocyte-specific protein Gfap stains the S100A16 immunopositive cells in cortex (C), cerebellum (D, right) and dentate gyrus (G). Scale bars = 100 µm; 10 µmin C, D (right), and G.

 
Intracellular Localization of S100A16 in Human U373 MG and U87 MG Glioblastoma Cells—Western blot analysis, using hS100A16 Ab, revealed a single band of ~12-kDa in total protein extract of human U87 MG and U373 MG glioblastoma cell lines (Fig. 8A). A previous study identified S100A16 protein in nucleolar fraction of HeLa cells (49). Because S100 protein expression and subcellular localization might differ between each member of the family and between different tissues and cell types, we examined intracellular distribution of endogenous S100A16 in both glioblastoma cell lines as well as in HeLa cells. Cells were fixed and co-stained with hS100A16 Ab and with anti-nucleolin, a nucleoli marker.

We found intense staining in the nucleus of U373 MG (Fig. 8, B and C, arrowheads) and U87 MG cells (data not shown). Confocal microscopy revealed a clear punctated peripheral cytoplasmic S100A16-staining in both U373 MG (Fig. 8, B-D, arrows) and U87 MG cells (data not shown). Moreover, S100A16 nuclear labeling overlaps with nucleolin staining, defining submicron-sized subdomains within the nucleolus in glioblastoma cells (Fig. 8, E-G) and in HeLa cells (Fig. 8I), in accordance with previous MS data (49).

To confirm S100A16 subcellular localization, glioblastoma cells were transfected with a GFP-S100A16 construct. The exogenous fusion protein exhibited similar intracellular distribution and was also found to co-localize with nucleolin within the nuclei of cells (Fig. 8H). To determine the specificity of the staining, we carried out peptide competition assays. Preincubation of hS100A16 Ab with the recombinant antigen resulted in the complete loss of immunoreactivity (Fig. 8J). These results indicate that S100A16 exhibits a particular and overlapping subcellular distribution in human glioblastoma cells and HeLa cells. Consistently, S100A16 was found in nucleus and cytoplasm of astrocytes in vivo (Fig. 7).


Figure 8
View larger version (57K):
[in this window]
[in a new window]

  		FIGURE 8.
Expression of S100A16 in different human cell lines. A, Western blot analysis of whole cell extract of neuroblastoma cells (SKN-LE, SKN-BE, and SHSY-5Y) and glioblastoma cells (U87 MG and U373 MG) using hS100A16 Ab. B-G, immunostaining of S100A16 in human glioblastoma U373 MG cells. Cells were fixed and double stained for endogenous S100A16 protein and nucleolin. DNA is stained with 4',6-diamidino-2-phenylindole (blue). Intracellular localization of S100A16 was visualized by confocal microscopy after indirect immunofluorescent staining. B-D, nuclear (arrowheads) and cytoplasmic (arrows) localization of endogenous S100A16. E-H, endogenous S100A16 and exogenous GFP-S100A16 fusion protein co-localize with nucleolin in the nucleus (Merge). I, HeLa cells were stained for endogenous S100A16 and nucleolin. J, preincubation of hS100A16 Ab with the recombinant protein resulted in the loss of staining. Scale bar = 10 µm.

 
Ca2+-dependent Nuclear Export/Import of S100A16 in U373 MG Cells—S100 proteins have been reported to relocate within the cells in response to [Ca2+]i oscillation. Therefore, we investigated Ca2+-dependent translocation of S100A16 in U373 MG cells exposed to either ionomycin, a Ca2+ ionophore supplemented with extracellular Ca2+, or thapsigargin, an endoplasmic reticulum Ca2+-ATPase inhibitor. Cells were treated for different periods of time, fixed, and co-stained for hS100A16 Ab and anti-nucleolin. Overall, in presence of 1.5 mM or 5 mM extracellular Ca2+, the response of U373 MG cells to both drugs was similar. No immediate cytotoxicity was noted in U373 MG cells during the observation periods in response to the different treatments. Both ionomycin and thapsigargin treatments resulted in increased S100A16 fluorescence in both the nucleoplasm and perinuclear area (Fig. 9, A and B). Higher magnification images revealed that a fraction of S100A16 was still associated with the nucleoli after 5 min of treatment (Fig. 9A, bottom). Up to 10 min of exposure to 1 µM ionomycin resulted in the total loss of S100A16 immunoreactivity within nucleoli (Fig. 9, B and C, bottom), and a punctate cytoplasmic S100A16-staining clearly appeared after 15 min of treatment (Fig. 9C).

Ca2+-dependent cytoplasmic translocation was then confirmed using the S100A16-GFP fusion protein. As shown in Fig. 9G, 15 min after ionomycin addition fluorescence was only observed in the cytoplasm. In contrast, addition of a membranepermeable Ca2+ chelator, EGTA or BAPTA-AM, induced S100A16 perinuclear accumulation (Fig. 9, D and E) and nuclear entry (Fig. 9, E and F). Typically, intense nuclear staining and loss of S100A16 immunoreactivity in the cytoplasm were observed 30 min after EGTA addition (Fig. 9F). Furthermore, an intense S100A16 fluorescence was observed within nucleoli 20-30 min after treatment (Fig. 9, E and F, bottom). Western blot analysis using nuclear extracts of U373 MG cells confirmed the Ca2+-dependent nuclear export/import of S100A16 (Fig. 9H). Together, these results indicate that [Ca2+]i regulates S100A16 subcellular localization; i.e. 1) a high intracellular Ca2+ level induces S100A16 nucleolar exit and nucleocytoplasmic transport, whereas 2) lowering intracellular Ca2+ concentration leads to S100A16 nuclear translocation and accumulation within specific region of nucleoli.


Figure 9
View larger version (71K):
[in this window]
[in a new window]

  		FIGURE 9.
Ca2+-dependent nucleocytoplasmic translocation of S100A16. U373 MG cells were fixed and double stained for endogenous S100A16 protein (red) and nucleolin (green), DNA is stained with 4',6-diamidino-2-phenylindole (blue). A-C, cells were treated with 1 µM ionomycin for 5 min (A), 10 min (B), and 15 min (C). A-C, bottom, higher magnification images of the boxed areas in A-C. Note that S100A16 is found within and around nucleolin-positive domains (A, bottom). 10 min after ionomycin addition, S100A16 is present in the nucleus in areas that exclude nucleolin (B, bottom, arrowheads). D-F, cells were treated with 2 mM EGTA for 10 min (D), 20 min (E), and 30 min (F). G, U373 MG cells transiently transfected with the GFP-S100A16 construct and stimulated for 20 min with ionomycin. H, Western blot analysis of nuclear extracts, at various times after increasing (1.5 mM Ca2+ plus 1 µM Ionomycin) or decreasing (plus 2 mM EGTA) intracellular Ca2+ level. On exposure to high Ca2+, S100A16 was exported from nuclei. In opposite, S100A16 was transferred to nuclei in response to low Ca2+. Scale bar = 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study firmly establishes that both human and mouse S100A16 proteins have only one functional Ca2+-binding site located in the C-terminal EF-hand, as was already expected from the sequences (2). The C-terminal EF-hand is canonical and should bind Ca2+, whereas the N-terminal EF-hand diverging from the corresponding motif in most S100 proteins, should not bind Ca2+. Comparatively, the S100-specific EF-hand of S100A7 displays the same Glu to Ser substitution in position Z (50) and does not bind Ca2+. Surprisingly, the affinity of the C-terminal EF-hand for Ca2+ in mouse S100A16 is very low; the Ca2+ affinity of this site in human S100A16 is 2-fold higher. Because high ionic strength buffer can depress the Ca2+ affinity 2- to 3-fold, we speculate that at physiological ionic strength the Ca2+ affinity of human S100A16 would be higher and would correspond to a Kd of 100-200 µM, i.e. very similar to that of many other S100 proteins. Indeed, the dissociation constant for Ca2+ measured by the Trp fluorescence changes in buffer A was 220 µM (Fig. 4D). The weak binding of Ca2+ to the C-terminal EF-hand of the mouse S100A16 may be due to the unusual C-tail, which is located in the dimer, close to the EF-hand II of the neighboring subunit (based on the three-dimensional structures of other S100 proteins). Ca2+ binding to the mouse protein either 1) does not lead to the exposure of a hydrophobic patch as it usually occurs in many other S100 proteins, with the notable exception of S100A13 (13) or 2) leads to the formation of a hydrophobic patch, which is then occupied by the C-tail. Surprisingly, Ca2+ binding to human S100A16 leads to an enhancement of the TNS fluorescence and thus to hydrophobic exposure. The major difference in the C-terminal EF-hand loops of the two proteins is a Cys in position Y in mouse S100A16 only. Possibly, the very short C-tail of human S100A16 is not capable of neutralizing the hydrophobic patch and thus leaves room for TNS binding.

In both proteins, Zn2+ binding leads to enhancement of the TNS fluorescence, and one should consider the question if this represents the solvent exposure of a hydrophobic surface. An independent way to prove hydrophobic exposure on the surface in Ca2+-binding proteins consists of phenyl-Sepharose chromatography. Unfortunately, this experiment could not be performed, because after binding of the Zn2+-saturated protein to the surface, Zn2+ sequestration to restore the apo form did not allow elution of the protein. Either the apoprotein is hydrophobic enough to stick to the column, or EGTA does not efficiently remove Zn2+ from the protein.

Human S100A16 favors TNS fluorescence enhancement upon binding of Ca2+ or Zn2+ and binding of both cations leads to additive fluorescence enhancement, thus indicating that Ca2+ and Zn2+ do not bind to the same sites and that the hydrophobic patches are different. We favor the idea that the Ca2+-dependent patch is entirely made up of elements within each subunit, i.e. the hydrophobic residues in helix 3 and 4 and in the linker 3-4. These are thus independent patches in each dimer, and likely these surfaces are also instrumental for the binding of two natural target peptides (36, 51). In contrast, the Zn2+-dependent patch may result from oligomerization with exposure of hydrophobicity at the interfaces of the oligomers. Two arguments point in this direction: 1) the kinetic effect in the fluorescence experiments with TNS and 2) EGTA cannot easily reverse the effect of Zn2+. In several S100 proteins with resolved three-dimensional structure, such as S100A7 and S100B, the two Zn2+ sites are located at the ends of the antiparallel helices IV and IV' in the dimer. A possible explanation is the exposure of hydrophobic sites upon Ca2+ binding.

S100A16 has previously been reported in a wide spectrum of adult human tissues including brain (2). Among the S100 protein examined, the relative expression of S100A16 was low in adult mouse brain (Fig. 5A) as previously described in the human adult brain (2). However, S100A16 transcript levels were ten times higher when compared with S100A4 mRNA levels, a S100 protein involved in neuronal differentiation as well as in the response of astrocytes to degeneration of myelinated axons (52, 53). Moreover, as reported for other members of the S100 protein family, brain expression gradually decreases with age (54). Indeed, S100 proteins exhibit specific spatiotemporal patterns of expression in agreement with the different roles they play during brain development (S100B, S100A6, S100A4, S100A5, and S100A13), or in cell cycle (S100B and S100A4) (54-57). In the present study, the expression and distribution of S100A16 was examined in the adult mouse cortex, hippocampus, and cerebellum using in situ hybridization and immunohistochemistry. Our results showed that expression of S100A16 mRNA and protein is restricted to cells of the molecular layer in the CA2 and CA3 field of the hippocampus (Figs. 5C and 7E). An S100A16 positive signal was stronger in the polymorphic layer of the dentate gyrus, throughout the whole cortex and in cells located in the vicinity of Purkinje neurons in the cerebellum (Fig. 7, B, D, and F).

At the cellular level, in situ hybridization revealed an S100A16 mRNA expression in cells of the cortical layer I and of white matter structures suggesting that the S100A16 gene is expressed in the glia cell population. In contrast, neuronal cell bodies were unreactive for S100A16 mRNA and protein, as indicated by the lack of positive signal noted for the Purkinje cells of the cerebellum and for the pyramidal and the granule cell layer of the hippocampus. Furthermore, in the brain regions investigated, our immunohistochemical data confirmed that S100A16 protein is specifically expressed in reactive astrocytes, as indicated by the co-localization with Gfap (Fig. 7C, D, and G). Consistently, S100A16 cDNA sequence was initially isolated from a human astrocytoma cDNA library (2), and hS100A16 Ab revealed the presence of the S100A16 only in glioblastoma cell lines, whereas the protein was not observed in neuroblastoma cell lines (Fig. 8A). Taken together, these observations indicate that S100A16 is widely distributed throughout the adult mouse brain and predominantly expressed within specific astrocyte populations. This brain distribution partially overlaps that of the previously studied S100B and S100A13, also expressed in glia cells of the temporal cortex and the hippocampus, indicating a possible functional cooperation between these proteins.

At the subcellular level, S100A16 immunoreactivity was found to be mainly associated with the nucleus of protoplasmic-like astrocytes in the cortex (Fig. 7, A and C), whereas the protein was found to be uniformly distributed along the neurites of fibrous-like astrocytes in the hippocampus (Fig. 7G). Consistently, endogenous S100A16 and exogenous GFP-S100A16 fusion protein showed identical subnuclear distribution in cells (Fig. 8). Double-immunofluorescence experiments combined with an integrated co-localization analysis demonstrated that S100A16 is strongly associated with the nucleolus (Fig. 8, E and F), suggesting a possible role for the S100A16 protein in ribonucleoprotein complex processing, gene silencing, or cell cycle progression. Interestingly, nuclear presence of S100B, and S100A4 was found to correlate with differentiation and migration of glial cells (56-58). This set of data is in good agreement with our in vivo findings and with recent proteomic experiments that identified S100A16 in nucleolus of cells by MS (59, 60).

Ca2+ regulates a wide range of cytoplasmic and nuclear events in a spatial and temporal manner. Intracellular translocation of S100 protein in response to [Ca2+]i variation has been reported to play an important role in the regulation of signaling complexes activating specific cellular pathways (14, 33). In the present study, an increase in [Ca2+]i stimulated the export of endogenous S100A16 from the nucleolus to the cytoplasm (Fig. 9). [Ca2+]i increase appears to be critical for S100A16 relocation. Our biochemical data indicate that, upon Ca2+ binding, S100A16 undergoes conformational changes leading to the exposure of hydrophobic patches (Figs. 3 and 4) implicated in S100 protein translocation (27). As nuclear and cytoplasmic Ca2+ signaling are regulated independently (61), S100A16 Ca2+-dependent translocation might be regulated by the recently described nucleoplasmic reticulum, which terminates near the nucleoli and is responsible for the release of free Ca2+ into localized subnuclear regions (62). In contrast, opposite Ca2+-dependent translocation was described for other members of the EF-hand protein family. Indeed, S100B, S100A11, and calmodulin translocate from the cytosol to the nucleus in response to Ca2+ stimulation. However, in the present study, the decrease in [Ca2+]i resulted in the nuclear import of S100A16 and its accumulation within the nucleoli, re-establishing the initial situation (Figs. 8E and 9). Like other members of the family, S100A16 lacks the canonical nuclear localization signal. Nuclear import might then require interaction with transporter proteins as it was described for S100A11 (63) or might occur via alternative facilitated-diffusion pathways as observed for calmodulin (64). Furthermore, phosphorylation has been reported to be essential for S100A11 nuclear import (49, 63). Interestingly, a recent study identifies phosphorylated S100A16 protein in nucleoli of HeLa cells (49), indicating a possible function of phosphorylation in S100A16 nuclear import. However, further studies are required to elucidate the roles of S100A16 in the nucleolus of cells, and the origin and functional significance of S100A16 Ca2+-dependent nuclear export.


   FOOTNOTES
 
* This work was supported by the National Centers of Competence in Research (NCCR) on Neural Plasticity and Repair and the Swiss National Science Foundation (Grant 3100A0-101970). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Steinwiesstrasse 75, 8032 Zurich, Switzerland. Tel.: 41-(44)-266-7541; Fax: 41-(44)-266-7169; E-mail: claus.heizmann{at}kispi.unizh.ch.

2 The abbreviations used are: GFAP, glial fibrillary acidic protein; TNS, 2-p-toluidinylnaphtalene 6-sulfonate; GFP, green fluorescent protein; NLS, nuclear localization signal; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester); ORF, open reading frame; UTR, untranslated region; DIG, digoxigenin; Ab, antibody; MS, mass spectrometry. Back

3 Nomenclature (see Ref. 3) used was updated in Marenholz, I., Lovering, R. C., and Heizmann, C. W., Biochim Biophys Acta (2006) Vol. 1763, in press. Back


   ACKNOWLEDGMENTS
 
We are indebted to Drs. R. Lang, D. P. Wolfer, E. Leclerc, D. Boller, and A. Galichet for continuous support. We thank H. Winter (DakoCytomation) for the help in producing the antibodies, P. Kleinert for the MS analyses, and Dr. M. Gruetter for providing the pET20-nHisT vector.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Heizmann, C. W. (2005) J. Pediatrics 147, 731-738[CrossRef][Medline] [Order article via Infotrieve]
  2. Marenholz, I., and Heizmann, C. W. (2004) Biochem. Biophys. Res. Commun. 313, 237-244[CrossRef][Medline] [Order article via Infotrieve]
  3. Marenholz, I., Heizmann, C. W., and Fritz, G. (2004) Biochem. Biophys. Res. Commun. 322, 1111-1122[CrossRef][Medline] [Order article via Infotrieve]
  4. Weterman, M. A., Wilbrink, M., Dijkhuizen, T., van den Berg, E., and Geurts van Kessel, A. (1996) Hum. Genet. 98, 16-21[CrossRef][Medline] [Order article via Infotrieve]
  5. Gendler, S. J., Cohen, E. P., Craston, A., Duhig, T., Johnstone, G., and Barnes, D. (1990) Int. J. Cancer 45, 431-435[Medline] [Order article via Infotrieve]
  6. Nilsson, M., Meza-Zepeda, L. A., Mertens, F., Forus, A., Myklebost, O., and Mandahl, N. (2004) Int. J. Cancer 109, 363-369[CrossRef][Medline] [Order article via Infotrieve]
  7. Boni, R., Burg, G., Doguoglu, A., Ilg, E. C., Schafer, B. W., Muller, B., and Heizmann, C. W. (1997) Br. J. Dermatol. 137, 39-43[CrossRef][Medline] [Order article via Infotrieve]
  8. Kizawa, K., Toyoda, M., Ito, M., and Morohashi, M. (2005) Br. J. Dermatol. 152, 314-320[CrossRef][Medline] [Order article via Infotrieve]
  9. Nacken, W., Roth, J., Sorg, C., and Kerkhoff, C. (2003) Microsc. Res. Tech. 60, 569-580[CrossRef][Medline] [Order article via Infotrieve]
  10. Adami, C., Sorci, G., Blasi, E., Agneletti, A. L., Bistoni, F., and Donato, R. (2001) Glia 33, 131-142[CrossRef][Medline] [Order article via Infotrieve]
  11. Glenney, J. R., Jr., Kindy, M. S., and Zokas, L. (1989) J. Cell Biol. 108, 569-578[Abstract/Free Full Text]
  12. Ilg, E. C., Schafer, B. W., and Heizmann, C. W. (1996) Int. J. Cancer 68, 325-332[CrossRef][Medline] [Order article via Infotrieve]
  13. Ridinger, K., Schafer, B. W., Durussel, I., Cox, J. A., and Heizmann, C. W. (2000) J. Biol. Chem. 275, 8686-8694[Abstract/Free Full Text]
  14. Donato, R. (2003) Microsc. Res. Tech. 60, 540-551[CrossRef][Medline] [Order article via Infotrieve]
  15. Sheu, F. S., Huang, F. L., and Huang, K. P. (1995) Arch. Biochem. Biophys. 316, 335-342[CrossRef][Medline] [Order article via Infotrieve]
  16. Baudier, J., Mochly-Rosen, D., Newton, A., Lee, S. H., Koshland, D. E., Jr., and Cole, R. D. (1987) Biochemistry 26, 2886-2893[CrossRef][Medline] [Order article via Infotrieve]
  17. Baudier, J., and Cole, R. D. (1988) J. Biol. Chem. 263, 5876-5883[Abstract/Free Full Text]
  18. Ziegler, D. R., Innocente, C. E., Leal, R. B., Rodnight, R., and Goncalves, C. A. (1998) Neurochem. Res. 23, 1259-1263[CrossRef][Medline] [Order article via Infotrieve]
  19. Fernandez-Fernandez, M. R., Veprintsev, D. B., and Fersht, A. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4735-4740[Abstract/Free Full Text]
  20. Markowitz, J., Rustandi, R. R., Varney, K. M., Wilder, P. T., Udan, R., Wu, S. L., Horrocks, W. D., and Weber, D. J. (2005) Biochemistry 44, 7305-7314[CrossRef][Medline] [Order article via Infotrieve]
  21. Delphin, C., Ronjat, M., Deloulme, J. C., Garin, G., Debussche, L., Higashimoto, Y., Sakaguchi, K., and Baudier, J. (1999) J. Biol. Chem. 274, 10539-10544[Abstract/Free Full Text]
  22. Wilder, P. T., Rustandi, R. R., Drohat, A. C., and Weber, D. J. (1998) Protein Sci. 7, 794-798[Medline] [Order article via Infotrieve]
  23. Svenningsson, P., Chergui, K., Rachleff, I., Flajolet, M., Zhang, X., El Yacoubi, M., Vaugeois, J. M., Nomikos, G. G., and Greengard, P. (2006) Science 311, 77-80[Abstract/Free Full Text]
  24. Rammes, A., Roth, J., Goebeler, M., Klempt, M., Hartmann, M., and Sorg, C. (1997) J. Biol. Chem. 272, 9496-9502[Abstract/Free Full Text]
  25. Mandinova, A., Atar, D., Schafer, B. W., Spiess, M., Aebi, U., and Heizmann, C. W. (1998) J. Cell Sci. 111, 2043-2054
  26. Davey, G. E., Murmann, P., and Heizmann, C. W. (2001) J. Biol. Chem. 276, 30819-30826[Abstract/Free Full Text]
  27. Hsieh, H. L., Schafer, B. W., Cox, J. A., and Heizmann, C. W. (2002) J. Cell Sci. 115, 3149-3158[Abstract/Free Full Text]
  28. Whitaker-Azmitia, P. M., and Azmitia, E. C. (1994) Perspect. Dev. Neurobiol. 2, 233-238[Medline] [Order article via Infotrieve]
  29. Kiryushko, D., Novitskaya, V., Soroka, V., Klingelhofer, J., Lukanidin, E., Berezin, V., and Bock, E. (2006) Mol. Cell Biol. 26, 3625-3638[Abstract/Free Full Text]
  30. Nishiyama, H., Knopfel, T., Endo, S., and Itohara, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 4037-4042[Abstract/Free Full Text]
  31. Tramontina, F., Tramontina, A. C., Souza, D. F., Leite, M. C., Gottfried, C., Souza, D. O., Wofchuk, S. T., and Goncalves, C. A. (2006) Cell Mol. Neurobiol. 26, 81-86[Medline] [Order article via Infotrieve]
  32. Van Eldik, L. J., and Wainwright, M. S. (2003) Restor. Neurol. Neurosci. 21, 97-108[Medline] [Order article via Infotrieve]
  33. Leclerc, E., Sturchler, E., and Heizmann, C.W. (2007) in Handbook of Neurochemistry and Molecular Neurobiology (Mikoshiba, K., ed) Springer Publishers, New York, in press
  34. Nelson, M. R., and Chazin, W. J. (1998) Biometals 11, 297-318[CrossRef][Medline] [Order article via Infotrieve]
  35. Zimmer, D. B., Wright Sadosky, P., and Weber, D. J. (2003) Microsc. Res. Tech. 60, 552-559[CrossRef][Medline] [Order article via Infotrieve]
  36. Rustandi, R. R., Baldisseri, D. M., and Weber, D. J. (2000) Nat. Struct. Biol. 7, 570-574[CrossRef][Medline] [Order article via Infotrieve]
  37. Bhattacharya, S., Bunick, C. G., and Chazin, W. J. (2004) Biochim. Biophys. Acta 1742, 69-79[Medline] [Order article via Infotrieve]
  38. Fritz, G., and Heizmann, C.W. (2004) in (Messerschmidt, A., Bode., W., and Cygler, M., eds) pp. 529-540, Handbook of Metalloproteins, Vol. 3, John Wiley & Sons, New York
  39. Heizmann, C. W., and Cox, J. A. (1998) Biometals 11, 383-397[CrossRef][Medline] [Order article via Infotrieve]
  40. Drohat, A. C., Amburgey, J. C., Abildgaard, F., Starich, M. R., Baldisseri, D., and Weber, D. J. (1996) Biochemistry 3, 11577-11588
  41. Drohat, A. C., Baldisseri, D. M., Rustandi, R. R., and Weber, D. J. (1998) Biochemistry 37, 2729-2740[CrossRef][Medline] [Order article via Infotrieve]
  42. Schafer, B. W., Fritschy, J. M., Murmann, P., Troxler, H., Durussel, I., Heizmann, C. W., and Cox, J. A. (2000) J. Biol. Chem. 275, 30623-30630[Abstract/Free Full Text]
  43. Nishikawa, T., Lee, I. S., Shiraishi, N., Ishikawa, T., Ohta, Y., and Nishikimi, M. (1997) J. Biol. Chem. 272, 23037-23041[Abstract/Free Full Text]
  44. Colowick, S. P., and Womack, F. C. (1969) J. Biol. Chem. 244, 774-777[Abstract/Free Full Text]
  45. Cox, J. A. (1996) in Guidbook to the Calcium Binding Protein (Celio, M. R., Pauls, T., and Schwaller, B., ed) pp. 1-12, Oxford University Press, Oxford
  46. Durussel, I., Mehul, B., Bernard, D., Schmidt, R., and Cox, J. A. (2002) Biochemistry 41, 5439-5448[CrossRef][Medline] [Order article via Infotrieve]
  47. Riddles, P. W., Schnitzerling, H. J., and Davey, P. A. (1983) Anal. Biochem. 132, 105-109[CrossRef][Medline] [Order article via Infotrieve]
  48. Pascale, A., Gusev, P. A., Amadio, M., Dottorini, T., Govoni, S., Alkon, D. L., and Quattrone, A. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 1217-1222[Abstract/Free Full Text]
  49. Beausoleil, S. A., Jedrychowski, M., Schwartz, D., Elias, J. E., Villen, J., Li, J., Cohn, M. A., Cantley, L. C., and Gygi, S. P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 12130-12135[Abstract/Free Full Text]
  50. Brodersen, D. E., Nyborg, J., and Kjeldgaard, M. (1999) Biochemistry 38, 1695-1704[CrossRef][Medline] [Order article via Infotrieve]
  51. Bhattacharya, S., Large, E., Heizmann, C. W., Hemmings, B., and Chazin, W. J. (2003) Biochemistry 42, 14416-14426[CrossRef][Medline] [Order article via Infotrieve]
  52. Kozlova, E. N., and Lukanidin, E. (1999) Glia 27, 249-258[CrossRef][Medline] [Order article via Infotrieve]
  53. Novitskaya, V., Grigorian, M., Kriajevska, M., Tarabykina, S., Bronstein, I., Berezin, V., Bock, E., and Lukanidin, E. (2000) J. Biol. Chem. 275, 41278-41286[Abstract/Free Full Text]
  54. Chan, W. Y., Xia, C. L., Dong, D. C., Heizmann, C. W., and Yew, D. T. (2003) Microsc. Res. Tech. 60, 600-613[CrossRef][Medline] [Order article via Infotrieve]
  55. Tiu, S. C., Chan, W. Y., Heizmann, C. W., Schafer, B. W., Shu, S. Y., and Yew, D. T. (2000) Brain Res. Dev. Brain Res. 119, 159-168[Medline] [Order article via Infotrieve]
  56. Deloulme, J. C., Raponi, E., Gentil, B. J., Bertacchi, N., Marks, A., Labourdette, G., and Baudier, J. (2004) Mol. Cell Neurosci. 27, 453-465[CrossRef][Medline] [Order article via Infotrieve]
  57. Takenaga, K., and Kozlova, E. N. (2006) Glia 53, 313-321[CrossRef][Medline] [Order article via Infotrieve]
  58. Olson, M. O., and Dundr, M. (2005) Histochem. Cell Biol. 123, 203-216[CrossRef][Medline] [Order article via Infotrieve]
  59. Scherl, A., Coute, Y., Deon, C., Calle, A., Kindbeiter, K., Sanchez, J. C., Greco, A., Hochstrasser, D., and Diaz, J. J. (2002) Mol. Biol. Cell 13, 4100-4109[Abstract/Free Full Text]
  60. Andersen, J. S., Lam, Y. W., Leung, A. K., Ong, S. E., Lyon, C. E., Lamond, A. I., and Mann, M. (2005) Nature 433, 77-83[CrossRef][Medline] [Order article via Infotrieve]
  61. Leite, M. F., Thrower, E. C., Echevarria, W., Koulen, P., Hirata, K., Bennett, A. M., Ehrlich, B. E., and Nathanson, M. H. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2975-2980[Abstract/Free Full Text]
  62. Echevarria, W., Leite, M. F., Guerra, M. T., Zipfel, W. R., and Nathanson, M. H. (2003) Nat. Cell Biol. 5, 440-446[CrossRef][Medline] [Order article via Infotrieve]
  63. Sakaguchi, M., Miyazaki, M., Takaishi, M., Sakaguchi, Y., Makino, E., Kataoka, N., Yamada, H., Namba, M., and Huh, N. H. (2003) J. Cell Biol. 163, 825-835[Abstract/Free Full Text]
  64. Thorogate, R., and Torok, K. (2004) J. Cell Sci. 117, 5923-5936[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/50/38905    most recent
M605798200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sturchler, E.
Right arrow Articles by Heizmann, C. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sturchler, E.
Right arrow Articles by Heizmann, C. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement