S-100 protein binds to annexin II and p11, the heavy and light chains of calpactin I.

S-100 protein, a dimeric, Ca(2+)-binding protein of the EF-hand type, interacts with annexin II (p36, the heavy chain of the cytoskeletal protein complex, calpactin I), with p11 (the light and regulatory chain of calpactin I) and with the hetero-tetramer annexin II2-p11(2) (calpactin I) in a Ca(2+)-regulated way, but not with annexins I, V and VI. The interaction of S-100 protein with the above proteins was investigated by fluorescence spectroscopy using acrylodan-S-100 protein and acrylodan-annexin II and by cross-linking experiments using the bifunctional cross-linker disuccinimidyl suberate (DSS). S-100 protein binds with the highest affinity to annexin II (Kd approx. 0.4 microM) and with the lowest affinity to calpactin I (Kd approx. 10 microM), with a constant stoichiometry of about 2 mol of protein/S-100 dimer. Thus, S-100 protein could substitute for p11 in regulating the activities of annexin II in cells which do not express p11 and/or act synergistically with p11 in cells expressing both p11 and S-100. The binding of S-100 protein to p11 could reflect the natural tendency of S-100 subunits and p11 to dimerize. Chimeric p11-S-100 alpha and p11-S-100-beta proteins could therefore form in a Ca(2+)-regulated way. The interaction of S-100 protein with calpactin I appears of doubtful physiological importance, because of the low binding affinity, of the small extent of fluorescence changes induced by calpactin I in acrylodan-S-100 protein and of lack of DSS-induced complex formation between the two protein species.

Mechanism of S100 protein-dependent inhibition of glial fibrillary acidic protein (GFAP) polymerization.

S100 protein, a subfamily of Ca(2+)-binding proteins of the EF-hand type, was recently shown to bind to and to inhibit the polymerization of the glial fibrillary acidic protein (GFAP), the intermediate filament component of astroglial cells, in the presence of micromolar levels of Ca2+ (J. Biol. Chem. 268, 12669-12674). By a sedimentation assay and viscometry we show here that S100 protein interferes with the very early steps of GFAP polymerization (nucleation) and with the GFAP polymer growth, thereby retarding the onset of GFAP assembly, reducing the rate and the extent of GFAP assembly, and increasing the critical concentration of GFAP assembly. Moreover, S100 protein disassembles preformed glial filaments. All the above effects can be explained by sequestration of soluble GFAP by S100 protein, as also indicated by the stoichiometry of S100 protein binding to GFAP and of S100 protein effects on GFAP assembly. Our data suggest that S100 protein might serve the function of avoiding excess GFAP polymerization and might participate in remodeling of glial filaments following elevation of the intracellular free Ca2+ concentration. Also, our data lend support to the notion that intermediate filaments are dynamic cytoskeleton structures that assemble and disassemble, and to the existence of cytoplasmic factors implicated in the regulation of the state of assembly of intermediate filaments.

Calpactin I binds to the glial fibrillary acidic protein (GFAP) and cosediments with glial filaments in a Ca(2+)-dependent manner: implications for concerted regulatory effects of calpactin I and S100 protein on glial filaments.

Calpactin I, a heterotetrameric, cytoskeletal protein complex composed of two copies of annexin II cross-linked by two copies of p11, an S100-like protein, binds to the glial fibrillary acidic protein (GFAP) and cosediments with glial filaments (GF) in a Ca(2+)-dependent manner, apparently without affecting GFAP polymerization under the present experimental conditions. Cosedimentation of calpactin I with GF, which occurs at micromolar free Ca2+ concentrations, is proportional to the concentrations of both calpactin I and GFAP and does not occur under conditions where GFAP assembly is maximally inhibited by, e.g., S100 protein. Annexin II also cosediments with GF and binds to GFAP, although to much smaller extents. Other annexins, such as annexins I, V, and VI, or p11 do not bind to either GF or GFAP. Calpactin I and S100 protein bind to different sites on GFAP, as investigated by fluorescence spectroscopy using acrylodan-labeled GFAP. Calpactin I and S100 protein might act, in the presence of Ca2+, in a concerted manner to determine the number and topography of GF in differentiating and/or mature glial cells.

Time-resolved fluorescence of S-100a protein in the absence and presence of calcium and phospholipids.

We have used phase-modulation fluorescence lifetime measurements to study the single Trp residue of the Ca(2+)-binding protein S-100a. Trp fluorescence decay was not exponential for the protein irrespective of the absence or presence of Ca2+. Fluorescence decay was best described by Lorentzian lifetime distributions centered around two components (approx. 3 and 0.7 ns) for protein in absence of Ca2+ and one component (approx. 2.9 ns) for the protein in presence of 2 mM Ca2+. Similar studies were performed with S-100a interacting with cardiolipin, phosphatidylserine or egg phosphatidylcholine, both in absence and in presence of 2 mM Ca2+. Our data suggest that the conformation of the protein and its Ca(2+)-binding properties vary depending on the characteristics of charge and structure of phospholipids.

Novel isoforms of CaBP 33/37 (annexin V) from mammalian brain: structural and phosphorylation differences that suggest distinct biological roles.

Two calcium-dependent phospholipid- and membrane-binding proteins have been purified from bovine brain. These are termed CaBP33 and CaBP37. Complete sequence analysis has revealed that these two proteins are isoforms of annexin V. Despite an apparent difference of 4 kDa between the two proteins on SDS-PAGE, only two amino-acid substitutions were found. These are, in CaBP33, Ser-36 and Lys-125 and in CaBP37, Thr-36 and Glu-125. This corresponds to a mass difference of 15 Da. This was confirmed by electrospray mass spectrometric analysis. Both isoforms can be phosphorylated substoichiometrically in vitro by protein kinase C at residue Thr-22.

S-100 (alpha and beta) binding peptide (TRTK-12) blocks S-100/GFAP interaction: identification of a putative S-100 target epitope within the head domain of GFAP.

Alignment of previously characterized S-100 (alpha and beta)-binding peptides (J. Biol. Chem. 270, 14651-14658) has enabled the identification of a putative S-100 target epitope within the head domain of glial fibrillary acidic protein (GFAP). The capacity of a known peptide inhibitor of S-100 protein (TRTK-12), homologous to this region, to perturb the interaction of S-100 (alpha and beta) and GFAP (J. Biol. Chem 268, 12669-12674) was investigated. Fluorescence spectrophotometry and chemical cross-linking analyses determined TRTK-12 to disrupt S-100:GFAP interaction in a dose- and Ca(2+_dependent manner. TRTK-12 also inhibited S-100's ability to block GFAP assembly and to mediate disassembly of preformed glial filaments. Each of these events was strictly dependent upon the presence of calcium and inhibitory peptide, maximal inhibition occurring at a concentration of TRTK-12 equivalent to the molar amount of S-100 monomer present. Together with our recent report demonstrating TRTK-12 also blocks the interaction of S-100 protein with the actin capping protein, CapZ, these results suggest TRTK-12 functions as a pleiotropic inhibitor of S-100 function. Availability of a functional inhibitor of S-100 will assist the further characterization of S-100 protein function in vitro and in vivo. Moreover, this report provides additional evidence supportive of a role for S-100 as a multi-faceted regulator of cytoskeletal integrity.

Interaction of S-100b protein with cardiolipin vesicles as monitored by electron spin resonance, pyrene fluorescence and circular dichroism.

The interaction of S-100b protein with cardiolipin (CL) vesicles has been studied by electron spin resonance, pyrene fluorescence, and circular dichroism. Electron spin resonance and pyrene fluorescence data indicate that S-100b binds to the polar surface of vesicles Ca2+-independently. In the presence of Ca2+, S-100b potentiates the Ca2+-induced clustering of the polar headgroups of CL molecules and causes a further reduction in the Ca2+-dependent decrease in the lateral mobility of the pyrene inserted into the lipid bilayer, which points to an effect of the protein on the hydrophobic core of the lipid bilayer through a larger perturbation of its polar surface. Circular dichroism analyses indicate that CL vesicles cause a decrease in the alpha-helical content of S-100b, analogous to that produced by Ca2+ and that the effects of CL vesicles and of Ca2+ on the secondary structure of the protein are supra-additive. By this technique, we found that the affinity of Ca2+ for S-100b increases substantially in the presence of CL vesicles, even in the presence of physiologic concentrations of KCl, suggesting that once S-100b had interacted with CL vesicles it assumes a new conformation in which its Ca2+-binding properties are greatly enhanced. These results are discussed in relation to binding of S-100b proteins to natural membranes, and to a possible involvement of S-100b in the regulation of membrane structural organization.

S-100b protein regulates aggregation and fusion of cardiolipin vesicles.

We have recently shown that S-100b protein interacts with the polar surface of cardiolipin vesicles [6]. This interaction produces changes in the secondary structure of S-100b as well as changes in the structural organization of cardiolipin vesicles. We report here on the effects of S-100b on cardiolipin vesicles as investigated by turbidity, terbium-dipicolinate fluorescence and freeze-fracture. Experiments were carried out in the absence and in the presence of Ca2+. In the absence of Ca2+ (0.1 mM EDTA), S-100b favors the aggregation and fusion of vesicles to some extent. Under these conditions, electron microscope analyses reveal the presence of fused vesicles along with particles similar to those observed in protein reconstituted systems or to lipid particles observed during fusional processes. In the presence of Ca2+, S-100b counteracts the Ca2(+)-dependent tendency of vesicles to aggregate and fuse. Under these conditions, bilayer phases along with hexagonal phases can be observed by electron microscopy. The latter effects of S-100b are not due to chelation of Ca2+ because of the relative concentrations of S-100b and Ca2+ under our experimental conditions and since much larger concentrations of EDTA are required to produce the S-100b effects. We propose that the dimeric nature of S-100b plays a major role in these events. In the absence of Ca2+, the S-100b molecules probably cross-link adjacent vesicles, one subunit contacting one vesicle and the other subunit contacting another vesicle through electrostatic bonds. In the presence of Ca2+, due to the large changes occurring in the conformation of the protein (which loses about 52% of its alpha-helical content), S-100b associates strongly with the polar surface of individual vesicles, thus generating some kind of physical barrier to aggregation and fusion of vesicles.

Replicating myoblasts and fused myotubes express the calcium-regulated proteins S100A1 and S100B.

We investigated the expression and the subcellular localization of S100A1 and S100B, two Ca(2+)-binding proteins of the EF-hand type, in replicating myoblasts and fused myotubes. Northern blot and reverse transcriptase-polymerase chain reaction analyses revealed the presence of S100A1 mRNA and S100B mRNA respectively, in myoblasts. Immunofluorescence and immunogold electron microscopy were used to localize individual proteins in myoblasts and myotubes. In the present report we document that: (1) in replicating myoblasts S100B is localized to intracellular membranes, including Golgi membranes, vimentin intermediate filaments (IFs) and microtubule (MT) structures; (2) in the same cells S100A1 is found associated with intracellular membranes; (3) following treatment of replicating myoblasts with colchicine, a fraction of S100B remains colocalized with bundled and collapsed vimentin IFs, whereas another fraction follows the destiny of endoplasmic membranes; (4) under the same conditions S100A1, like a fraction of S100B, follows the collapse of the endoplasmic reticulum around the nucleus; and (5) in fused myotubes S100A1 is found diffusely in the cytoplasm, whereas S100B is mostly found associated with vimentin IFs. These data suggest that in the skeletal myogenic cell line used in the present study S100A1 and S100B might share binding sites on or close to intracellular membranes, but display a significant degree of target specificity with respect of IFs and MTs. The results of these analyses suggest that expression of S100B in skeletal muscle cells may be developmentally regulated and lend support to the possibility that S100B might regulate the MT and IF dynamics.

Time-resolved fluorescence of S-100a protein: effect of Ca2+, Mg2+ and unilamellar vesicles of egg phosphatidylcholine.

Phase-modulation fluorescence lifetime measurements were used to study the single Trp residue of the Ca(2+)-binding protein S-100a both in the absence and in the presence of Ca2+ and/or Mg2+. Trp fluorescence decay for the protein was satisfactorily described by Lorentzian lifetime distributions centered around two components (approximately 4 ns and 0.5 ns). Lifetime values were unchanged by 2 mM Ca2+, but the fractional intensity associated with longer lifetime increased up to 75%. In the presence of Mg2+, the Ca2+ induced increase of the fractional intensity associated with longer lifetime was only 57%. For the protein in buffer, about the 85% of the recovered anisotropy was associated to a rotational correlation time of 6.7 ns. After the addition of Ca2+, this value was increased to 16.08 ns. In the presence of Mg2+, Ca+2 increased the rotational correlation time to 33.75 ns. Similar studies were performed with S-100a interacting with egg phosphatidylcholine vesicles (SUV). Our data suggest that the conformation of the protein may be influenced by structural features of the lipidic membrane. Moreover, data obtained in the presence of Mg2+ indicate some interaction between lipids and S-100, likely mediated by this ion.

Cardiac S-100a0 protein: purification by a simple procedure and related immunocytochemical and immunochemical studies.

A simple procedure is described for the purification of the alpha alpha isoform of S-100 proteins (S-100a0) from porcine heart. Purification steps include the following: i) extraction of the tissue with a hypotonic medium containing EDTA; ii) ammonium sulfate fractionation (0-50%) of the extract; iii) Ca2+-dependent affinity chromatography of the supernatant obtained through the preceding step on phenyl-sepharose and elution of absorbed proteins through a two-chamber gradient of 1.0-0.0 mM CaCl2 and 0.0--1.0 mM EGTA, respectively; and iv) chromatography of the resultant S-100-containing fractions on Sephadex G-200. The yield is 20 mg S-100a0/kg porcine heart. The whole procedure takes five days and is highly reproducible. Data obtained from the phenyl-sepharose step suggest that the affinity of Ca2+ for S-100a0 increases by several orders of magnitude once the protein had interacted with that matrix. This observation is discussed in relation to the role of S-100 proteins in amplification of the Ca2+ signal. Immunocytochemical and immunoblotting analyses indicate that S-100a0 is exclusively found at the level of the sarcolemmal membranes, the membranes of the sarcoplasmic reticulum, the external mitochondrial membranes, and in the adjacent sarcoplasm. No evidence of S-100a0 being associated with the nuclei or with myofibrils has been obtained. Finally, the cardiac tissue does not contain the Triton X-100-extractable fraction of S-100 normally detected in the brain and in adipocytes. Our data suggest that S-100a0 behaves as a peripheral membrane protein in cardiac tissue.

Interaction of two brain annexins, CaBP33 and CaBP37, with membrane-skeleton proteins.

CaPB33 and CaPB37, two annexins purified from bovine brain, interact with a Triton X-100-resistant fraction (cytoskeleton) from bovine brain membranes in a Ca2(+)-dependent way in vitro. The binding is saturable with respect to the CaBP33-CaBP37 concentration, half-maximal binding occurring at approximately 15 micrograms of the CaBP33-CaBP37 mixture/ml. The binding of these two annexins to the crude cytoskeleton preparation as a function of free Ca2+ concentration is biphasic, with half-maximal binding at approximately 50 microM and approximately 400 microM free Ca2+ for the first and the second component, respectively. By an overlay technique, CaBP33 and CaBP37 bind to a set of low Mr polypeptides (10-20 kDa) in the crude cytoskeleton preparation, with formation of an 85-90 kDa complex as investigated in cross-linking experiments. No binding of the CaBP33-CaBP37 mixture to either G- or F-actin has been observed. Identification of the CaBP33-CaBP37-binding proteins in cytoskeletons would help elucidating the function(s) of these annexins in the brain.

Two novel brain proteins, CaBP33 and CaBP37, are calcium-dependent phospholipid- and membrane-binding proteins.

Two acidic Ca2(+)-binding proteins (CaBP33 and CaBP37) purified from bovine brain have been characterized in terms of immunological properties, heat-sensitivity, electrophoretic mobility, and Ca2(+)-dependent binding to negatively charged phospholipids and to brain membranes. They were induced to bind to membranes by homogenization of brain tissue in the presence of CaCl2. The membrane-bound CaBP33/CaBP37 mixture resisted extraction with detergents and was solubilized with high concentrations of EGTA/KCl. However, apparent Ca2(+)-independent binding of the two proteins to membranes seemed to occur as well. This latter fraction of membrane-bound CaBP33 and CaBP37 could be solubilized with Triton X-100, indicating that brain membranes normally contain the two proteins as intrinsic components.

Identification of S-100 proteins and S-100-binding proteins in a detergent-resistant EDTA/KCl-extractable fraction from bovine brain membranes.

The Triton X-100-resistant residue of brain membranes contains appreciable amounts of S-100 proteins. This fraction of S-100 can be solubilized by high concentrations of EDTA plus or minus high concentrations of KCl. Whereas KCl (0.6 M) extracts the detergent-resistant S-100, NaCl (1 M) does not. Endogenous Ca2+ is required and is sufficient for S-100 to remain associated with the detergent-resistant residue. However, 0.6 M KCl extracts a further fraction of Triton X-100-resistant S-100. In contrast, the Triton X-100-extractable fraction of S-100 resists the action of EDTA. These data suggest that Ca2+ regulates the extent of association of S-100 with Triton X-100-resistant components in brain membranes, whereas the association of S-100 with the lipid bilayer of brain membranes and/or with some intrinsic membrane proteins is less Ca2+-regulated. Several S-100-binding proteins are identified in the detergent-resistant residue of brain membranes by an overlay procedure.

Characterization of mammalian heart annexins with special reference to CaBP33 (annexin V).

Porcine heart was observed to express annexins V (CaBP33) and VI in large amounts, and annexins III and IV in much smaller amounts. Annexin V (CaBP33) in porcine heart was examined in detail by immunochemistry. Homogenization and further processing of heart in the presence of EGTA resulted in the recovery of annexin V (CaBP33) in the cytosolic fraction and in an EGTA-resistant, Triton X-100-soluble fraction from cardiac membranes. Including Ca2+ in the homogenization medium resulted in a significant decrease in the annexin V (CaBP33) content of the cytosolic fraction with concomitant increase in the content of this protein in myofibrils, mitochrondria, the sarcoplasmic reticulum and the sarcolemma. The amount of annexin V (CaBP33) in each of these subfractions depended on the free Ca2+ concentration in the homogenizing medium. At the lowest free Ca2+ concentration tested, 0.8 microM, only the sarcolemma appeared to contain bound annexin V (CaBP33). Membrane-bound annexins V (CaBP33) and VI partitioned in two fractions, one EGTA-resistant and Triton X-100-extractable, and one Triton X-100-resistant and EGTA-extractable. Altogether, these data suggest that annexins V and VI are involved in the regulation of membrane-related processes.

Membrane-bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins.

The distribution of annexin V isoforms (CaBP33 and CaBP37) and of annexin VI in bovine lung, heart, and brain subfractions was investigated with special reference to the fractions of these proteins which are membrane-bound. In addition to EGTA-extractable pools of the above proteins, membranes from lung, heart, and brain contain EGTA-resistant annexins V and VI which can be solubilized with detergents (Triton X-100 or Triton X-114). A strong base like Na2CO3, which is usually effective in extracting membrane proteins, only partially solubilizes the membrane-bound, EGTA-resistant annexins analyzed here. Also, only 50-60% of the Triton X-114-soluble annexins partition in the aqueous phase, the remaining fractions being recovered in the detergent-rich phase. Altogether, these findings suggest that, by an as yet unknown mechanism, following Ca(2+)-dependent association of annexin V isoforms and annexin VI with membranes, substantial fractions of these proteins remain bound to membranes in a Ca(2+)-independent way and behave like integral membrane proteins. These results further support the possibility that the above annexins might play a role in membrane trafficking and/or in the regulation of the structural organization of membranes.

S-100b protein regulates the activity of skeletal muscle adenylate cyclase in vitro.

We have investigated the effect of the b isoform of S-100 proteins on adenylate cyclase activity of rat skeletal muscle. S-100b inhibits the adenylate cyclase activity in the presence of Mg2+ (5.0-50 mM), while it activates the same enzyme in the presence of Ca2+ (0.1-1.0 mM) dose-dependently in both cases. S-100b counteracts the stimulatory effect of NaF on adenylate cyclase in the presence of Mg2+ and the inhibitory effect of RMI 12330 A in the presence of Ca2+.

S-100a0 protein stimulates Ca2+-induced Ca2+ release from isolated sarcoplasmic reticulum vesicles.

S-100a0 protein, the alpha alpha-isoform of the S-100 family, stimulates Ca2+-induced Ca2+ release from terminal cisternae isolated from rat skeletal muscle cells. The stimulatory effect of S-100a0 is maximal at approximately 5 microM S-100a0 and half maximal at approximately 0.1 microM S-100a0, at 1.8 microM free Ca2+ in the presence of 5 mM Mg2+ plus 0.1 M KCl. The effect of the protein on Ca2+-induced Ca2+ release is completely inhibited by the calcium release blocker, ruthenium red.

'Neuron-specific' protein gene product 9.5 (PGP 9.5) is also expressed in glioma cell lines and its expression depends on cellular growth state.

Protein gene product 9.5 (PGP 9.5), which in the normal nervous system is restricted to certain neurons, has been detected in two glioma cell lines, rat C6 and human GL15, by immunoblotting and immunocytochemistry. Its expression in these cells depends on the cellular growth state, being maximal between the first and second post-plating day. Only a faint PGP 9.5 immunoreactivity can be observed in glioma cells after the eleventh post-plating day, i.e. about one week after confluency has been reached. The present results suggest that PGP 9.5 in cultured glial cells is maximally expressed during the growth phase and that the protein could play a role during brain development in glial cells, in reactive gliosis, or in tumorigenesis of the glial lineage.

S-100a0 protein stimulates the basal (Mg2+-activated) adenylate cyclase activity associated with skeletal muscle membranes.

S-100a0 protein, the alpha alpha isoform of the S-100 family, stimulates basal (Mg2+-activated) adenylate cyclase (AC) activity associated with the sarcolemma, longitudinal tubules and terminal cisternae of rat skeletal muscle cells. The stimulatory effect of S-100a0 on AC activity is maximal around 5 microM S-100a0 and half-maximal around 0.2 microM S-100a0. Also, the stimulatory effect is greatest on the AC activity associated with the terminal cisternae than on the other membrane fractions studied. These data are discussed in relation to the subcellular localization of S-100a0 in muscle cells.