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PubMed1984

Platelet activation induces the formation of a stable gelsolin-actin complex from monomeric gelsolin.

We have studied the interactions between gelsolin and actin in crude extracts from activated and unactivated platelets and in mixtures of purified platelet gelsolin and muscle actin. Extracts were prepared using 10 mM EGTA from human platelets treated either with 100 microM aspirin and 2.5 mM tetracaine to retard activation or with the calcium ionophore A23187 to effect activation. The extracts were fractionated by gel filtration on Sephadex G-150 or by sedimentation on sucrose gradients and then analyzed using anti-gelsolin immunoblots and actin filament nucleation assays. The nucleation activity in both extracts was associated with gelsolin. The activity in the extracts from unactivated platelets sedimented with an S value of 5.2 and had an Mr = 90,000. The activity in the extracts prepared with EGTA from activated platelets sedimented at 6.8 S and had an Mr = 130,000. We have shown previously that the Mr = 130,000 species is an EGTA-stable binary complex of one actin and one gelsolin. Transient exposure of the extracts from unactivated platelets to 100 microM Ca2+ and subsequent fractionation in EGTA-containing buffers demonstrated that the formation of the binary complex occurs in the presence of Ca2+. Fractionation in the presence of 100 microM Ca2+ demonstrated higher order complexes including a ternary complex with a sedimentation constant of 8.2 S and an Mr = 165,000. Sedimentation and gel filtration experiments using purified platelet gelsolin and rabbit skeletal muscle actin demonstrated that formation of the EGTA-stable binary complex required Ca2+. At least one additional actin is bound to the binary complex in the presence of Ca2+, but is not sufficiently stable to be purified when EGTA is added. The results suggest that gelsolin exists either as a monomer or perhaps as a weak complex with actin in unactivated platelets but complexes tightly with actin during the transient Ca2+ rise that occurs during activation.

Actins↗
PubMed1995

Haplotype analysis in gelsolin-related amyloidosis reveals independent origin of identical mutation (G654A) of gelsolin in Finland and Japan.

Familial amyloidosis, Finnish type (FAF) (gelsolin-related amyloidosis) is an autosomal dominant form of systemic amyloidosis characterized by corneal lattice dystrophy and peripheral polyneuropathy. The accumulating protein in FAF consists of fragments of gelsolin, an actin-modulating protein. The gelsolin mutation G654A has been found in both Finnish and Japanese patients. To study the origin of the gelsolin mutation in these patients we performed haplotype analysis in 10 Finnish and 2 Japanese FAF families. Poymorphic DNA markers GSN, D9S103, AFMa061xd9, and AFMa139xb9 revealed a uniform disease haplotype in all the disease-associated chromosomes of the Finnish FAF families, which was different from the one observed in the Japanese families. The present results and the previously detected gelsolin mutation G654T in Czech and Danish FAF patients suggest that nucleotide 654 may represent a mutation hot spot in the gelsolin gene. The DNA markers studied here will be useful in future genealogical analyses of FAF.

Amyloid Neuropathies↗
PubMed2001

Binding of gelsolin domain 2 to actin. An actin interface distinct from that of gelsolin domain 1 and from ADF/cofilin.

It is generally assumed that of the six domains that comprise gelsolin, domain 2 is primarily responsible for the initial contact with the actin filament that will ultimately result in the filament being severed. Other actin-binding regions within domains 1 and 4 are involved in gelsolin's severing and subsequent capping activity. The overall fold of all gelsolin repeated domains are similar to the actin depolymerizing factor (ADF)/cofilin family of actin-binding proteins and it has been proposed that there is a similarity in the actin-binding interface. Gelsolin domains 1 and 4 bind G-actin in a similar manner and compete with each other, whereas domain 2 binds F-actin at physiological salt concentrations, and does not compete with domain 1. Here we investigate the domain 2 : actin interface and compare this to our recent studies of the cofilin : actin interface. We conclude that important differences exist between the interfaces of actin with gelsolin domains 1 and 2, and with ADF/cofilin. We present a model for F-actin binding of domain 2 with respect to the F-actin severing and capping activity of the whole gelsolin molecule.

Actin Depolymerizing Factors↗
PubMed1997

Characterization of gelsolin truncates that inhibit actin depolymerization by severing activity of gelsolin and cofilin.

Gelsolin is a calcium-activated actin-binding protein with six subdomains. The N-terminal (G1) domain is essential for actin-filament-severing activity while other domains within G2-3 position the protein on the filament side allowing G1 to sever. In order to generate reagents capable of competitively inhibiting endogenous gelsolin and, potentially, other actin filament regulatory protein, we expressed several truncates of gelsolin in Escherichia coli, and analyzed how they affected the in vitro activity of two different actin-binding proteins, gelsolin and cofilin. A Ca2+-sensitive truncate containing G2-6 inhibited the F-actin-depolymerizing activities of both gelsolin and cofilin, while a G2-3 truncate was less effective. Using two independent assays, our results support the idea that gelsolin truncates inhibit actin filament severing and do not markedly affect actin subunit dissociation kinetics. Cosedimentation assays in the presence of calcium demonstrate that the G2-6 truncate binds to F-actin more strongly than the G2-3 truncate consistent with a protection mechanism by conformational change of F-actin and/or competitive binding to actin filaments which depends upon the presence of actin filament binding domains.

Actin Depolymerizing Factors↗
PubMed1991

Gelsolin-related amyloidosis. Identification of the amyloid protein in Finnish hereditary amyloidosis as a fragment of variant gelsolin.

The Finnish type of familial amyloidosis is a systemic disease characterized by progressive cranial neuropathy, corneal lattice dystrophy, and distal sensimotor neuropathy. Amyloid fibrils were isolated from the kidney and heart of a patient with Finnish amyloidosis. After solubilization, the amyloid proteins were fractionated by gel filtration and purified by reverse-phase HPLC. Complete amino acid sequence analyses show that the two amyloid components obtained are fragments of gelsolin, an actin-modulating protein occurring in plasma and the cytoskeleton. The larger component represents residues 173-243 and the minor component residues 173-225, respectively, of mature gelsolin. When compared with the predicted primary structure of human gelsolin a single amino acid substitution is present in amyloid: at position 15 of the amyloid proteins an asparagine is found instead of an aspartic acid residue at the corresponding position (187) in gelsolin. Antibodies to a dodecapeptide of the amyloidogenic region of gelsolin specifically stain the tissue amyloid deposits in Finnish hereditary amyloidosis. The results show that the amyloid subunit protein in Finnish hereditary amyloidosis represents a new type of amyloid that is derived from an actin filament-binding region of a variant gelsolin molecule by limited proteolysis.

Amino Acid Sequence↗
PubMed1994

Gelsolin displaces phalloidin from actin filaments. A new fluorescence method shows that both Ca2+ and Mg2+ affect the rate at which gelsolin severs F-actin.

We describe an assay for measuring both the extent and kinetics of the severing of F-actin, based on the enhanced fluorescence emission of tetramethylrhodamine-phalloidin bound to F-actin. The enhanced fluorescence is lost after exposure to active gelsolin by displacement of the phalloidin from actin during severing. This assay requires small amounts of actin and gelsolin, can be used to measure reaction times ranging from 1 to 10(3) s, and does not require covalent modification of either protein. The rate of fluorescence loss is linearly related to the concentrations of both actin and gelsolin. However, the apparent rate constant of the reaction is highly dependent on the divalent cation concentration, varying between 10(4) and 10(6) M-1 s-1 when the [Ca2+] varies between 20 and 200 microM. Addition of Mg2+ increases the apparent rate constant at equivalent Ca2+ concentration. These results suggest that in vitro the rate-limiting step in the severing process is the activation of gelsolin by the binding of Ca2+ and Mg2+ to several low affinity (Kd approximately 100 microM) sites on gelsolin. While activation of gelsolin by Ca2+ is a slow process, the binding and severing of actin occurs at a rate approaching the diffusion limit.

Actins↗
PubMed1990

Gel electrophoresis of native gelsolin and gelsolin-actin complexes.

BHK gelsolin migrated on non-denaturing 8-25% polyacrylamide gels with an apparent molecular mass of 80 kDa. In the absence of Ca2+ no complex formation occurred between BHK gelsolin and actin. In the presence of Ca2+ two complex species were found: a ternary complex, GA2, of apparent molecular mass of 210 kDa at gelsolin:actin ratio of 1:2, and a novel quaternary complex, GA3, of apparent molecular mass of 247 kDa when actin was in excess. Both cytoplasmic and plasma gelsolin species form GA3 with skeletal muscle actin. No complexes larger than GA3 were observed. The formation of GA3 involves the binding of a third actin to the gelsolin molecule at the site previously assumed to be masked, rather than to the actin molecules already present in GA2. In preference to GA3, GA2 was incorporated into actin filaments stabilized with phalloidin. On chelation of free Ca2+, both GA2 and GA3 dissociated to form the EGTA stable binary complex (GA) with an apparent molecular mass of 140 kDa and free actin.

Actins↗
PubMed1992

The amino-terminal fragment of gelsolin is cross-linked to Cys-374 of actin in the EGTA-resistant actin-gelsolin complex.

It has been shown that the EGTA-resistant actin, one of the two actin molecules associated to gelsolin, can be predominantly cross-linked to gelsolin by benzophenone-4-maleimide (BPM), a photoaffinity-labeling reagent, which was conjugated to Cys-374 of actin prior to cross-linking (Doi, Y., Banba, M. and Vertut-Doï, A. (1991) Biochemistry 30, 5769-5777). When a chymotryptic digest of gelsolin containing the amino-terminal 15-kDa fragment was mixed with BPM-actin (42 kDa) and irradiated for cross-linking, a band of 58 kDa appeared on SDS-PAGE which was shown to contain actin molecule by using fluorescently labeled actin. The amino-terminal sequence of the 58-kDa complex was identical to that of gelsolin, confirming that the amino-terminal segment (residues 1-133) of pig plasma gelsolin lies closely to Cys-374 of actin in the EGTA-resistant complex.

Actins↗
PubMed1991

Cysteine-374 of actin resides at the gelsolin contact site in the EGTA-resistant actin-gelsolin complex.

The interaction of pig plasma gelsolin (G) and actin (A) was examined by using photoreactive 4-maleimidobenzophenone-actin (BPM-actin) in which BPM was previously conjugated to Cys-374 of actin through the maleimide moiety. In the presence of micromolar [Ca2+], the major cross-linked product observed after irradiation of the mixture of gelsolin (82 kDa) and actin (42 kDa) had an apparent molecular mass of 130 kDa although gelsolin predominantly existed in the form of an A2G complex (170 kDa). No cross-linked product was detected in the absence of Ca2+. BPM-actin itself did not give any cross-linked product. By use of fluorescent-labeled gelsolin, the cross-linked 130 kDa was shown to be an AG complex. The cross-linked complex was also formed from the A2G complex after removal of Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetraacetic acid (EGTA) followed by irradiation, indicating that it was the EGTA-resistant AG complex that was cross-linked. The results show that Cys-374 at the C-terminal segment of actin in the EGTA-resistant AG complex is 9-10 A apart from gelsolin. Furthermore, it was shown that the EGTA-resistant actin molecule once incorporated in the A2G complex did not exchange with free actin in the presence of Ca2+. This was also supported by the effect of phosphatidylinositol 4,5-bisphosphate, which did not dissociate the EGTA-resistant actin molecule from the A2G complex in the presence of Ca2+, but did after removal of Ca2+.

Actins↗
PubMed1994

Toward understanding the pathogenic mechanisms in gelsolin-related amyloidosis: in vitro expression reveals an abnormal gelsolin fragment.

Gelsolin-related amyloidosis, also called familial amyloidosis, Finnish type (FAF) is an autosomal dominantly inherited disorder characterized by progressive polyneuropathy and corneal lattice dystrophy. All the analyzed patients are found to carry a nucleotide substitution of A or T for G654 in their gelsolin gene, which at the protein level results in the conversion of the 187 amino acid residue, aspartic acid, to asparagine or tyrosine, respectively. In this study, we transfected mammalian mesenchymal COS-1 cells with a derivative of the expression vector pCD-X containing cDNA coding for the wild-type (D187) and mutant forms (N187 and Y187) of plasma gelsolin. Both disease-associated mutant forms of gelsolin were found to be abnormally processed, which led to the secretion of an aberrant 68 kDa gelsolin fragment into the culture media. This fragment most probably represents a carboxy-terminal part of the protein and contains the suggested amyloid-forming sequence. Initial data were also obtained for involvement of a metalloendoprotease in the pathologic processing. This aberrant proteolysis is likely to represent a crucial initiator step in the cascade resulting in amyloid accumulation in patients' tissues.

Amyloid Neuropathies↗
PubMed1987

pH-dependent rate of formation of the gelsolin-actin complex from gelsolin and monomeric actin.

The assembly of gelsolin with actin was followed by the increase of the fluorescence intensity of a fluorescence label bound to actin. The time course of the formation of the gelsolin-actin complex in the presence of micromolar [Ca2+] could be quantitatively interpreted by a model in which one actin molecule binds slowly to gelsolin in a rate-determining step and subsequently a second actin molecule is bound at least 40 times more rapidly. The rate of binding of the first actin molecule to gelsolin was found to be remarkably slow and to depend on the pH. The rate constants of formation of the gelsolin-actin complex range from 1.5 X 10(4) M-1 s-1 at pH 8 to 7 X 10(4) M-1 s-1 at pH 6.

Actins↗
PubMed1988

Nucleotide sequence of pig plasma gelsolin. Comparison of protein sequence with human gelsolin and other actin-severing proteins shows strong homologies and evidence for large internal repeats.

Pig plasma gelsolin (Mr = 81595; 739 residues) contains 704 identical residues out of a maximum 730 when compared to the cytoplasmic form of human gelsolin. The cDNA sequence also codes for a peptide of 33 residues N-terminal to the nine-residue plasma extension sequence previously reported: these 33 residues are highly homologous to the human signal peptide and plasma extension. Comparison of the gelsolin sequences with chicken brush border villin, severin from Dictyostelium discoideum and fragmin from Physarum polycephalum shows a strong evolutionary relationship between all these proteins. There are six large repeating segments in gelsolin and villin, and three similar segments in severin and fragmin. Although these multiple repeats cannot be related to any known function of these actin-severing proteins, this superfamily of proteins appears to have evolved from an ancestral sequence of 120 to 130 amino acid residues.

Animals↗
PubMed1986

Characterization of the Ca2+-induced conformational changes in gelsolin and identification of interaction regions between actin and gelsolin.

Serum gelsolin, a Ca2+-dependent protein regulating the length of actin filaments, undergoes conformational changes upon binding Ca2+. These were detected and analyzed by several approaches including ultraviolet difference spectroscopy, circular dichroism studies, analytical ultracentrifugation, thiol group titration, and limited proteolytic digestions. The effect of Ca2+ binding on the UV absorption difference spectrum and the near-UV circular dichroism spectrum was consistent with changes in the environments of tyrosine and phenylalanine residues. In the presence of Ca2+, the S0(20),w value decreased from 5.3 to 4.7. This latter result implies a transformation to a more asymmetric molecular shape. Gelsolin contained only two accessible thiol groups per mole of protein, one of which was titratable in the native protein; it was more accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the absence than in the presence of Ca2+. The limited digestion of gelsolin from serum and bovine aorta smooth muscle by two different proteases, chymotrypsin and trypsin, proceeded much faster in the presence of Ca2+ than in its absence with the production of three main fragments of about 40K, 32K, and 21K. This fragment mixture was found still able to shorten F-actin in a Ca2+-dependent manner; this severing activity was expressed by the isolated 40K peptide. Gelsolin was cross-linked to F- and G-actin by the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), generating a covalent 130K binary complex (actin1-gelsolin1) followed by a covalent 180K ternary complex (actin2-gelsolin1).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1993

Homozygous familial amyloidosis, Finnish type: demonstration of glomerular gelsolin-derived amyloid and non-amyloid tubular gelsolin.

The immunohistochemical findings in the kidneys of two sisters with homozygous familial amyloidosis of Finnish type, an autosomal dominant disorder of gelsolin catabolism, is described. The disease, manifesting with nephrotic syndrome and end-stage renal failure was characterized by heavy glomerular deposits of gelsolin-derived amyloid. Immunostaining also revealed the presence of gelsolin in the tubular epithelium that was Congo-red negative. It is concluded that homozygous Finnish amyloidosis leads to a severe nephropathy due to the deposition of fragments of mutant gelsolin.

Adult↗
PubMed1995

Identification of the trapped calcium in the gelsolin segment 1-actin complex: implications for the role of calcium in the control of gelsolin activity.

The X-ray structure of the complex of actin with gelsolin segment 1 revealed the presence of two calcium ions, one bound at an intramolecular site within segment 1 and the other bridging the segment directly to actin. Although earlier calcium binding studies at pH 8.0 revealed only a single calcium trapped in the complex (and also in the binary gelsolin-actin complex), it is here shown that two calcium ions are bound under the conditions of crystallization at physiological pH. Mutation of acidic residues in either actin or segment 1 involved in ligation of the intermolecular calcium ion resulted in loss of one of the bound calcium ions at pH < 7, but not at pH 8. Thus the calcium ion trapped in the segment 1-actin complex is that located at the intramolecular site. The implications of this for gelsolin function are discussed.

Actins↗
PubMed1996

Evidence for an actin binding helix in gelsolin segment 2; have homologous sequences in segments 1 and 2 of gelsolin evolved to divergent actin binding functions?

Gelsolin is built up of six homologous segments that perform different functions on actin. Segments 1 and 2, which are suggested to be highly similar in their overall folds, bind monomeric and filamentous actin respectively. A long alpha-helix in segment 1 forms the major contact site of this segment with actin. We show that sequence 197-226 of segment 2, equivalent to the region around the actin binding helix in segment 1, contains F-actin binding activity. Consequently, positionally similar parts of segment 1 and 2 are implicated in the actin contact and solvent exposed residues in these parts must have evolved differentially to meet their different actin binding properties.

Actins↗
PubMed1988

Circulating actin-gelsolin complexes following oleic acid-induced lung injury.

Plasma gelsolin is one of two extracellular proteins that bind actin, a major body protein, with high affinity. The authors performed a series of experiments to determine whether tissue injury leads to actin release and the formation of circulating actin-gelsolin complexes. Two functions of plasma gelsolin, filament-nucleating and filament-severing activity, were used to measure total and free gelsolin concentrations, respectively. Both gelsolin and gelsolin-actin complexes nucleate actin assembly, whereas only free gelsolin severs actin filaments. Therefore, nucleation reflects the total gelsolin concentration, severing, the free gelsolin concentration, and the difference, gelsolin-actin complexes. Injection of F-actin in the rat caused a reduction in the free, but not total, gelsolin levels, consistent with the formation of circulating actin-gelsolin complexes. Oleic acid (50 mg/kg) administered intravenously in rats, a treatment that causes acute hemorrhagic pulmonary necrosis, caused the free gelsolin concentration to fall to a greater extent than the total gelsolin concentration, which indicated the presence of circulating actin-gelsolin complexes. Lower doses (9-27 mg/kg) in rabbits caused a qualitatively similar but smaller change in the free gelsolin level. Plasma gelsolin was immunoprecipitated at times when actin-gelsolin complexes were present, as determined functionally, and bound actin was demonstrated by immunoblotting with an anti-actin antiserum. These studies show that considerable amounts of actin are released into the extracellular space during acute lung injury and that circulating actin-gelsolin complexes can be detected in the peripheral blood.

Actins↗