Human plasma gelsolin reversibly binds Mg-ATP in Ca(2+)-sensitive manner.

Gelsolin is a Ca(2+)-regulated actin-modulating protein found in a variety of cellular cytoplasm and also in blood plasma. Affinity separation of human plasma gelsolin was successfully accomplished by eluting the protein with a low concentration of nucleoside polyphosphate from immobilized Cibacron Blue F3GA (1, 2). This finding was followed by the demonstration that the protein had one class of ATP binding site with Kd = 2.8 x 10(-7) M, which saturated at an ATP/gelsolin ratio of 0.6 in the absence of Ca2+ (3). To obtain further information on the nucleotide binding properties of gelsolin, binding studies were done in the presence of EGTA with GTP, ADP, and GDP by equilibrium dialysis. Incubation of plasma gelsolin with GTP resulted in binding of 0.6 mol of GTP per mol of protein with a dissociation constant of 1.8 x 10(-6) M, indicating that ATP binds to gelsolin with higher affinity than GTP. Neither ADP nor GDP at up to 100 microM appreciably bound to gelsolin at a physiological salt concentration. Then, the effects of divalent metal ions on the ATP binding to plasma gelsolin were examined. Gelsolin bound to ATP with Kd = 2.4 x 10(-6) M in a solution containing 2 mM MgCl2, whereas micromolar free Ca2+ concentrations inhibited ATP binding. Furthermore, addition of Ca2+ rapidly reversed the preformed nucleotide binding to gelsolin, suggesting that Ca2+ binding to gelsolin leads to a conformational change which disrupts a nucleotide binding fold in the protein molecule.     

The structure of gelsolin bound to ATP

Calcium activation of the actin-modifying properties of gelsolin is sensitive to ATP. Here, we show that soaking calcium-free gelsolin crystals in ATP-containing media results in ATP occupying a site that spans the two pseudosymmetrical halves of the protein. ATP binding involves numerous polar and hydrophobic contacts and is identical for the two copies of gelsolin related by non-crystallographic symmetry within the crystal. The gamma-phosphate of ATP participates in several charge-charge interactions consistent with the preference of gelsolin for ATP, as a binding partner, over ADP. In addition, disruption of the ATP-binding site through Ca2+ activation of gelsolin reveals why ATP binds more tightly to the inactive molecule, and suggests how the binding of ATP may modulate the sensitivity of gelsolin to calcium ions. Similarities between the ATP and PIP2 interactions with the C-terminal half of gelsolin are evident from their overlapping binding sites and in that both molecules bind more tightly in the absence of calcium ions. We propose a model for how PIP2 may bind to calcium-free gelsolin based on the ATP-binding site.     

Co-operative binding of Ca2+ ions to the regulatory binding sites of gelsolin.

The rate of association of actin with gelsolin was measured at various Ca2+ and ATP concentrations. The fraction of Ca2+-activated gelsolin was determined by quantitative evaluation of the association rates thereby assuming that Ca2+-binding gelsolin associates with actin and Ca2+-free gelsolin does not. A plot of the fraction of Ca2+-activated gelsolin vs. the free Ca2+ concentration revealed a sigmoidal shape suggesting that co-operative binding of Ca2+ ions is required for activation of gelsolin. A good fit of the experimental data by calculated binding curves was obtained if two Ca2+ ions were assumed to bind to actin in a highly co-operative manner. ATP decreased the rate of association of gelsolin with actin and bound to gelsolin at a low affinity (Kd = 32 microm for Ca2+-free and Kd = 400 microm for Ca2+-activated gelsolin). In contrast, a 1 : 1 gelsolin-actin complex was found to be activated for association with actin by a single Ca2+ ion in a non-co-operative manner.     

Human plasma gelsolin binds to fibronectin

Human plasma gelsolin, a 93,000-dalton actin-binding protein binds to human plasma fibronectin. Qualitative data obtained from experiments employing quasi-elastic light scattering, sucrose gradient sedimentation, gel filtration chromatography, and fibronectin polymerization indicate that gelsolin and fibronectin form a complex in solution. Solid-phase binding studies show that both human plasma and rabbit macrophage gelsolin bind to immobilized fibronectin with a Kd of about 1 microM in a 1:1 complex. The ability of gelsolin to interact with actin was not affected by the presence of fibronectin. Fibronectin also increased the amount of gelsolin binding to fibrin clots. Binding of gelsolin to fibronectin may serve to localize plasma gelsolin in regions where fibronectin is deposited, such as inflammatory sites.     

Sequential binding of actin monomers to plasma gelsolin and its inhibition by vitamin D-binding protein

Functional studies that distinguish free from actin-bound gelsolin based on the ability of the former to sever actin filaments reveal that the binding of actin monomers to gelsolin is highly cooperative and can be prevented by prior incubation of actin with vitamin D-binding protein (DBP), even though the apparent affinity of gelsolin for actin is 50-fold greater than that of DBP. Measurements of actin binding by immunoprecipitation and pyrene-actin fluorescence establish that DBP-actin complexes do not bind to gelsolin and that DBP removes one of the actin monomers in a 2:1 actin-gelsolin complex. These studies may explain why DBP-actin complexes exist in blood plasma in vivo in the presence of free gelsolin and suggest that the interaction of gelsolin with actin in cells and plasma may be regulated in part by actin monomer binding proteins.     

Regulation of gelsolin to plant actin filaments and its distribution in pollen

The effect of plasma gelsolin on plant microfilaments and its localization in plant cells were investigated. The results by using ultracentrifugation and electron microscopy showed that plant microfilaments could be severed into shorter fragments by gelsolin in a Ca2+-dependent manner. By measuring the binding ability of plasma gelsolin to pollen actin using the method of immunoprecipitation, it was shown that pollen actin could bind gelsolin at a ratio of 2.0±0.21 in the presence of Ca2+. Addition of EGTA could disassociate the actin-gelsolin complexes, reducing the ratio to 1.2±0.23, and the addition of PIP2 could further reduce the ratio to 0.8±0.1. The results indicate that plant actin has similar binding properties with plasma gelsolin as that of animal actin. By Western blotting we identified the existence of gelsolin in lily pollen. The results of immunolo-calization of gelsolin in pollen and pollen tube showed that gelsolin was mainly localized at the germinal furrow in pollen grains and at the cytoplasm in pollen tube, especially in the tip region.     

Inactivation of endotoxin by human plasma gelsolin

Septic shock from bacterial endotoxin, triggered by the release of lipopolysaccharide (LPS) molecules from the outer wall of Gram-negative bacteria, is a major cause of human death for which there is no effective treatment once the complex inflammatory pathways stimulated by these small amphipathic molecules are activated. Here we report that plasma gelsolin, a highly conserved human protein, binds LPS from various bacteria with high affinity. Solid-phase binding assays, fluorescence measurements, and functional assays of actin depolymerizing effects show that gelsolin binds more tightly to LPS than it does to its other known lipid ligands, phosphatidylinositol 4,5-bisphosphate and lysophosphatidic acid. Gelsolin also competes with LPS-binding protein (LBP), a high-affinity carrier for LPS. One result of gelsolin-LPS binding is inhibition of the actin binding activity of gelsolin as well as the actin depolymerizing activity of blood serum. Simultaneously, effects of LPS on cellular functions, including cytoskeletal actin remodeling, and collagen-induced platelet activation by pathways independent of toll-like receptors (TLRs) are neutralized by gelsolin and by a peptide based on gelsolin residues 160-169 (GSN160-169) which comprise part of gelsolin's phosphoinositide binding site. Additionally, TLR-dependent NF-kappaB translocation in astrocytes appears to be blocked by gelsolin. These results show a strong effect of LPS on plasma gelsolin function and suggest that some effects of endotoxin in vivo may be mediated or inhibited by plasma gelsolin.     

Regulation of gelsolin to plant actin filaments and its distribution in pollen

The dynamics of the actin cytoskeleton depends upon the unique constellation of ac- tin-binding proteins (ABPs), as well as their spatial distribution and local activation. However, the identification and characterization of actin-binding proteins in plant cells are still limited. At pre- sent, only a few plant ABPs have been identified in plant tissues, including profilin, ADF/cofilin, fimbrin, villin and several myosins. Compared with that in animals, there is still a long way for us …     

Interaction of plasma gelsolin with G-actin and F-actin in the presence and absence of calcium ions

Plasma gelsolin formed a very tight 1:2 complex with G-actin in the presence of Ca2+, but no interaction between gelsolin and G-actin was detected in the presence of excess EGTA. However, the 1:2 complex dissociated into a 1:1 gelsolin:actin complex and monomeric actin when excess EGTA was added. Plasma gelsolin bound tightly to the barbed ends of actin filaments and also severed filaments in the presence of Ca2+ and bound weakly to the filament barbed end in the presence of EGTA. The 1:2 gelsolin-actin complex bound to the barbed ends of filaments but did not sever them. By blocking the barbed end of filaments with plasma gelsolin, we determined the critical concentration at the pointed end in 1 mM MgCl2 and 0.2 mM ATP to be 4 microM. The dissociation rate constant for ADP-G-actin from the pointed end was estimated to be about 0.4 s-1 and the association rate constant to be about 5 X 10(4) M-1 s-1. Finally, we obtained evidence that plasma gelsolin accelerates but does not bypass the nucleation step and, therefore, that the concentration of gelsolin does not directly determine the concentration of filaments polymerized in its presence. Thus, gelsolin-capped filaments may not provide an absolutely reliable method for determining the rate constant for the association of ATP-G-actin at the pointed ends of filaments, but a reasonable estimate would be 1 X 10(5) M-1 s-1 in 1 mM MgCl2 and 0.2 mM ATP.     

Association of gelsolin with actin filaments and cell membranes of macrophages and platelets

Recent evidence that polyphosphoinositides regulate the function of the actin-modulating protein gelsolin in vitro raises the possibility that gelsolin interacts with cell membranes. This paper reports ultrastructural immunohistochemical data revealing that gelsolin molecules localize with plasma and intracellular membranes, including rough endoplasmic reticulum, cortical vesicles and mitochondria of macrophages, and blood platelets. Anti-gelsolin gold also labeled the surface and interior of secondary lysosomes presumably representing plasma gelsolin ingested by these cells from the lung surface by endocytosis. Gelsolin molecules, visualized with colloidal gold in replicas of the cytoplasmic side of the substrate-adherent plasma membrane of mechanically unroofed and rapidly frozen and freeze-dried macrophages, associated with the ends of short actin filaments sitting on the cytoplasmic membrane surface. A generalized distribution of gelsolin molecules in thin sections of resting platelets rapidly became peripheral, and plasmalemma association increased following thrombin stimulation. At later times the distribution reverted to the cytoplasmic distribution of resting cells. These findings provide the first evidence for gelsolin binding to actin filament ends in cells and indicate that gelsolin functions in both cytoplasmic and membrane domains.     

The binary complex of pig plasma gelsolin with Mg2+-G-actin in ATP and ADP

Pig plasma gelsolin combined with Mg-G-actin at less than 10(-8) M Ca2+ to yield a binary complex. Complexes formed from G-actin with bound ATP or ADP. They contained approx. 1 mol of non-exchangeable nucleotide per mol of actin. ATP hydrolysis was not coupled to binary complex formation, but ATP in the complex hydrolysed very slowly. The nucleotide in the binary complex behaved like one of the two nucleotide molecules in the ternary complex (two actin monomers to one gelsolin), but the actin-gelsolin interaction was weaker in the binary complex.     

The actin filament-severing domain of plasma gelsolin

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.     

Binding of gelsolin, a secretory protein, to amyloid beta-protein.

Soluble amyloid beta-protein (Abeta) is normally present in the cerebrospinal fluid (CSF) and plasma. However, it is fibrillized and deposited as plaques in the brains of patients with Alzheimer's disease. Cerebrospinal fluid (CSF) contains several circulating proteins (apolipoprotein E, apolipoprotein J, and transthyretin) that bind to Abeta. We report here that gelsolin, a secretory protein, also binds to Abeta in a concentration-dependent manner. Under similar conditions, other proteins such as G-actin, protein kinase C, polyglutamic acid, and gelatin did not bind to Abeta. Solid phase binding assays showed two Abeta binding sites on gelsolin that have dissociation constants (Kd) of 1.38 and 2.55 microM. Abeta was found to co-immunoprecipitate along with gelsolin from the plasma, suggesting that gelsolin-Abeta complex exists under physiological conditions. The gelsolin-Abeta complex was sodium dodecyl sulfate (SDS)stable in the absence of reducing agent, but was dissociated when the SDS stop solution contained dithiothreitol (reducing agent). This study suggests that the function of secretory gelsolin in the CSF and plasma is to bind and sequester Abeta.     

Weak binding of divalent cations to plasma gelsolin

Calcium binding of swine plasma gelsolin was examined. When applied to ion-exchange chromatography, its elution volume was drastically altered depending on the free Ca2+ concentration of the medium. The presence of two classes of Ca2+ binding sites, high-affinity sites (Kd = 7 microM) and low-affinity sites (Kd = 1 mM), was suggested from the concentration dependence of the elution volume. The tight binding sites were specific for Ca2+. The weakly bound Ca2+ could be replaced by Mg2+ once the tight binding sites were occupied with Ca2+. The binding of metal ions was totally reversible. Circular dichroism measurement of plasma gelsolin indicated that most change in secondary structure was associated with Ca2+ binding to the high-affinity sites. Binding of Mg2+ to the low-affinity sites caused a secondary structural change different from that caused by Ca2+ bound to the high-affinity sites. Gel permeation chromatography exhibited a small change in Stokes radius with and without Ca2+. Microheterogeneity revealed by isoelectric focusing did not relate to the presence of two classes of Ca2+ binding sites. These results indicated that plasma gelsolin drastically altered its surface charge property due to binding of Ca2+ or Ca2+, Mg2+ with a concomitant conformational change.     

Affinity separation of human plasma gelsolin on Affi-Gel Blue

Human plasma gelsolin was specifically eluted with 1 mM adenosine 5'-triphosphate from an Affi-Gel Blue column. Since the ionic strength of sodium chloride required to elute the protein from the dye column was much higher than that of 1 mM adenosine 5'-triphosphate, the binding of plasma gelsolin with the dye-ligand appeared to be biospecific. Taking advantage of this affinity interaction, we have developed a revised purification method of human plasma gelsolin. The purification included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, Affi-Gel Blue chromatography, and Phenyl-Sepharose chromatography. The method allowed a reproducible purification of the protein to apparent homogeneity, producing a 331-fold purification with a yield of 6%.     

Formation of vitamin D-binding protein-actin and binary and ternary plasma gelsolin-actin complexes in human serum

After the addition of actin to serum, the binding of actin to serum actin-binding proteins was analyzed by the method of immunoblotting using monospecific antibodies against vitamin D-binding protein (DBP) (group-specific component, Gc), human skeletal actin and human plasma gelsolin. When increasing amounts of globular actin were added to serum, actin bound to DBP preferentially. After exhausting DBP, actin began to bind to plasma gelsolin. When equally increasing amounts of filamentous actin were added to serum, actin was bound to both plasma gelsolin and DBP, and then uncomplexed DBP removed one actin molecule from gelsolin-actin 1:2 complex, resulting in a gelsolin-actin 1:1 complex. These results support the theory that the actin-depolymerizing activity of serum is due to the concerted role of plasma gelsolin and DBP.     

Identification of critical functional and regulatory domains in gelsolin

Gelsolin can sever actin filaments, nucleate actin filament assembly, and cap the fast-growing end of actin filaments. These functions are activated by Ca2+ and inhibited by polyphosphoinositides (PPI). We report here studies designed to delineate critical domains within gelsolin by deletional mutagenesis, using COS cells to secrete truncated plasma gelsolin after DNA transfection. Deletion of 11% of gelsolin from the COOH terminus resulted in a major loss of its ability to promote the nucleation step in actin filament assembly, suggesting that a COOH-terminal domain is important in this function. In contrast, derivatives with deletion of 79% of the gelsolin sequence exhibited normal PPI-regulated actin filament-severing activity. Combined with previous results using proteolytic fragments, we deduce that an 11-amino acid sequence in the COOH terminus of the smallest severing gelsolin derivative identified here mediates PPI-regulated binding of gelsolin to the sides of actin filaments before severing. Deletion of only 3% of gelsolin at the COOH terminus, including a dicarboxylic acid sequence similar to that found on the NH2 terminus of actin, resulted in a loss of Ca2+-requirement for filament severing and monomer binding. Since these residues in actin have been implicated as potential binding sites for gelsolin, our results raise the possibility that the analogous sequence at the COOH terminus of gelsolin may act as a Ca2+-regulated pseudosubstrate. However, derivatives with deletion of 69-79% of the COOH-terminal residues of gelsolin exhibited normal Ca2+ regulation of severing activity, establishing the intrinsic Ca2+ regulation of the NH2-terminal region. One or both mechanisms of Ca2+ regulation may occur in members of the gelsolin family of actin-severing proteins.     

Molecular biology of gelsolin, a calcium-regulated actin filament severing protein

Gelsolin is a Ca2+-binding protein of mammalian leukocytes, platelets and other cells which has multiple and closely regulated powerful effects on actin. In the presence of micromolar Ca2+, gelsolin severs actin filaments, causing profound changes in the consistency of actin polymer networks. A variant of gelsolin containing a 25-amino acid extension at the NH2-terminus is present in plasma where it may be involved in the clearance of actin filaments released during tissue damage. Gelsolin has two sites which bind actin cooperatively. These sites have been localized using proteolytic cleavage and monoclonal antibody mapping techniques. The NH2-terminal half of the molecule contains a Ca2+-insensitive actin severing domain while the COOH-terminal half contains a Ca2+-sensitive actin binding domain which does not sever filaments. These data suggest that the NH2-terminal severing domain in intact gelsolin is influenced by the Ca2+-regulated COOH-terminal half of the molecule. The primary structure of gelsolin, deduced from human plasma gelsolin cDNA clones, supports the existence of actin binding domains and suggests that these may have arisen from a gene duplication event, and diverged subsequently to adopt their respective unique functions. The plasma and cytoplasmic forms of gelsolin are encoded by a single gene, and preliminary results indicate that separate mRNAs code for the two forms. Further application of molecular biological techniques will allow exploration into the structural basis for the multifunctionality of gelsolin, as well as the molecular basis for the genesis of the cytoplasmic and secreted forms of gelsolin.     

Loss of calcium sensitivity of plasma gelsolin is associated with the presence of calcium ions during preparation

Gelsolin is a calcium-dependent actin severing and capping protein. Calcium 'opens' the molecule to make actin binding sites accessible, but removal of calcium from the medium does not necessarily fully reverse this process. The calcium sensitivity of actin monomer binding and actin filament severing is here shown to vary considerably with the source of gelsolin and conditions of preparation. Plasma gelsolin undergoes irreversible loss of calcium sensitivity when prepared in the presence of calcium ions. This is not due solely to effects of bound calcium, because purified human plasma gelsolin expressed in E. coli and stored in calcium shows no comparable loss of calcium sensitivity when prepared or stored in calcium. These results suggest the presence of factors in plasma which, in the presence of calcium, promote an irreversible structural change in gelsolin resulting in permanent loss of calcium sensitivity.     

Gelsolin binding and cellular presentation of lysophosphatidic acid

Lysophosphatidic acid (LPA) in biological fluids binds to serum albumin and other proteins that enhance its effects on cellular functions. The actin-severing protein gelsolin binds LPA with an affinity (K(d) = 6 nm) similar to that of the G protein-coupled LPA receptors encoded by endothelial differentiation genes 2, 4, and 7 (Edg-2, -4, and -7 receptors) and greater than that of serum albumin (K(d) = 360 nm). At concentrations of 10% or less of that in plasma, which are observed in fluids of injured tissues, purified and recombinant gelsolin augment LPA stimulation of nuclear signals and protein synthesis in rat cardiac myocytes (RCMs) that express Edg-2 and -4 receptors. At concentrations of 20% or more of that in plasma, gelsolin suppresses LPA stimulation of RCMs. The lack of effect of gelsolin on RCM responses to monoclonal anti-Edg-4 receptor antibody plus a phorbol ester without LPA attests to its specificity for LPA delivery and the absence of post-receptor effects. Inhibition of gelsolin binding and cellular delivery of LPA by l-alpha-phosphatidylinositol-4,5-bisphosphate (PIP2) and peptides constituting the two PIP2 binding domains of gelsolin suggests competition between LPA and PIP2 for the same sites. Thus, delivery of LPA to RCMs is affinity-coupled to Edg receptors by gelsolin in a PIP2-regulated process.