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PubMed1992

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.

Adenine Nucleotides↗

PubMed1994

Nucleation of actin polymerization by gelsolin.

The time-course of assembly of actin with gelsolin was measured by the fluorescence increase of a fluorescent label covalently linked to actin. The actin concentrations ranged from values far below the critical concentration to values above the critical concentration of the pointed ends of actin filaments. If the concentration of actin was in the range of the critical monomer concentration (0.64 microM), the time-course of the concentration of actin assembled with gelsolin revealed a sigmoidal shape. At higher actin concentrations the time-course of association of actin with gelsolin approximated an exponential curve. The measured time-courses of assembly were quantitatively interpreted by kinetic rate equations. A poor fit was obtained if two actin molecules were assumed to bind to gelsolin to form a 1:2 gelsolin-actin complex and subsequently further actin molecules were assumed to polymerize onto the 1:2 gelsolin-actin complex toward the pointed end. A considerably better agreement between calculated and measured time-courses was achieved if additional creation of actin filaments by fast fragmentation of newly formed actin filaments by not yet consumed gelsolin was assumed to occur. This suggests that both polymerization of actin onto gelsolin and fragmentation of actin filaments contribute to formation of new actin filaments by gelsolin. Furthermore it could be demonstrated that below the critical monomer concentration appreciable amounts of actin are incorporated into gelsolin-actin oligomers.

Actins↗

PubMed1992

Monomer and excimer fluorescence of horse plasma gelsolin labelled with N-(1-pyrenyl)iodoacetamide.

Horse plasma gelsolin was labelled with the sulfhydryl-specific fluorescent reagent N-(1-pyrenyl)iodoacetamide. The level of incorporation of probe was 1.6 +/- 0.3 mol pyrene/mol gelsolin. The circular dichroism spectrum of pyrenyl-gelsolin and its ability to interact with muscle actin were not different from that found for unmodified gelsolin. The emission from pyrenyl-gelsolin was dominated by a broad emission band centred near 483 nm, characteristic of the presence of pyrene excimers. Analysis of excitation spectra for the monomer and excimer-type fluorescence suggested that ground-state interactions may occur between adjacent pyrenes in the gelsolin structure. In the case either of excimer formation or of ground-state pyrene-pyrene interactions in doubly labelled gelsolin molecules, the modified Cys residues must be in close proximity in the folded protein structure. Thermal denaturation of gelsolin could be monitored by observing the decrease in excimer emission that accompanied heating and unfolding of the tertiary structure. While heat treatment alone did not eliminate excimer fluorescence, digestion of gelsolin with chymotrypsin completely abolished such emission. Also, pyrenyl-gelsolin prepared and studied in 6 M guanidine-HCl exhibited fluorescence characteristic of pyrene monomers exclusively.

Actins↗

PubMed1987

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.

Actins↗

PubMed1992

Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.

The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.

Animals↗

PubMed1988

Functional comparison of villin and gelsolin. Effects of Ca2+, KCl, and polyphosphoinositides.

A family of homologous actin-binding proteins sever and cap actin filaments and accelerate actin filament assembly. The functions of two of these proteins, villin and gelsolin, and of their proteolytically derived actin binding domains were compared directly by measuring their effects, under various ionic conditions, on the rates and extents of polymerization of pyrene-labeled actin. In 1 mM Ca2+ and 150 mM KCl, villin and gelsolin have similar severing and polymerization-accelerating properties. Decreasing [Ca2+] to 25 microM greatly reduces severing by villin but not gelsolin. Decreasing [KCl] from 150 to 10 mM at 25 microM Ca2+ increases severing by villin, but not gelsolin, over 10-fold. The C-terminal half domains of both proteins have Ca2+-sensitive actin monomer-binding properties, but neither severs filaments nor accelerates polymerization. The N-terminal halves of villin and gelsolin contain all the filament-severing activity of the intact proteins. Severing by gelsolin's N-terminal half is Ca2+-independent, but that of villin has the same Ca2+ requirement as intact villin. The difference in Ca2+ sensitivity extends to 14-kDa N-terminal fragments which bind actin monomers and filament ends, requiring Ca2+ in the case of villin but not gelsolin. Severing of filaments by villin and its N-terminal half is shown to be inhibited by phosphatidylinositol 4,5-bisphosphate, as shown previously for gelsolin (Janmey, P.A., and Stossel, T.P. (1987) Nature 325, 362-364). The functional similarities of villin and gelsolin correlate with known structural features, and the greater functional dependence of villin on Ca2+ compared to gelsolin is traced to differences in their N-terminal domains.

Actins↗

PubMed1999

Cell motility as a prognostic factor in Stage I nonsmall cell lung carcinoma: the role of gelsolin expression.

BACKGROUND: Tumor cell motility is an important characteristic that facilitates the multistep process of tumor metastasis. Rac, ABP-280, and gelsolin are proteins that interact with actin and are important in cell motility. METHODS: The authors studied a cohort of 229 Stage I nonsmall cell lung carcinoma (NSCLC) patients who had a minimum of 3 years follow-up and had been previously analyzed for 22 clinical, pathologic, and molecular features, of which 9 had been found to provide significant prognostic information in a Cox proportional hazards model. Tumor sections were stained by the avidin-biotin complex method using monoclonal antibodies against rac, ABP-280, and gelsolin. RESULTS: In a pilot analysis of over 50 patients each, rac and ABP-280 were found to be moderately-to-highly expressed in the majority of tumors and to provide no prognostic information. Gelsolin expression was more variable and appeared to be negatively correlated with survival in the pilot population. In the larger 229-patient population, high focal gelsolin expression was seen in 32 tumors (14%) and conferred the highest relative risk (4.04) of cancer recurrence among all factors tested, compared with tumors that had no or low gelsolin expression. Moderate focal gelsolin expression, seen in 46 patients (20%), also conferred a significant risk of cancer recurrence, with a relative risk of 2.26 compared with tumors that had no or low gelsolin expression. Consideration of average gelsolin expression and of overall survival yielded similar results. CONCLUSIONS: Gelsolin expression appears to be a significant prognostic factor for cancer recurrence in cases of Stage I NSCLC.

Carcinoma, Non-Small-Cell Lung↗

PubMed2000

VASP protects actin filaments from gelsolin: an in vitro study with implications for platelet actin reorganizations.

An initial step in platelet shape change is disassembly of actin filaments, which are then reorganized into new actin structures, including filopodia and lamellipodia. This disassembly is thought to be mediated primarily by gelsolin, an abundant actin filament-severing protein in platelets. Shape change is inhibited by VASP, another abundant actin-binding protein. Paradoxically, in vitro VASP enhances formation of actin filaments and bundles them, activities that would be expected to increase shape change, not inhibit it. We hypothesized that VASP might inhibit shape change by stabilizing filaments and preventing their disassembly by gelsolin. Such activity would explain VASP's known physiological role. Here, we test this hypothesis in vitro using either purified recombinant or endogenous platelet VASP by fluorescence microscopy and biochemical assays. VASP inhibited gelsolin's ability to disassemble actin filaments in a dose-dependent fashion. Inhibition was detectable at the low VASP:actin ratio found inside the platelet (1:40 VASP:actin). Gelsolin bound to VASP-actin filaments at least as well as to actin alone. VASP inhibited gelsolin-induced nucleation at higher concentrations (1:5 VASP:actin ratios). VASP's affinity for actin (K(d) approximately 0.07 microM) and its ability to promote polymerization (1:20 VASP actin ratio) were greater with Ca(++)-actin than with Mg(++)-actin (K(d) approximately 1 microM and 1:1 VASP), regardless of the presence of gelsolin. By immunofluorescence, VASP and gelsolin co-localized in the filopodia and lamellipodia of platelets spreading on glass, suggesting that these in vitro interactions could take place within the cell as well. We conclude that VASP stabilizes actin filaments to the severing effects of gelsolin but does not inhibit gelsolin from binding to the filaments. These results suggest a new concept for actin dynamics inside cells: that bundling proteins protect the actin superstructure from disassembly by severing, thereby preserving the integrity of the cytoskeleton.

Actins↗

PubMed1993

Fluorescent responses of acrylodan-labeled plasma gelsolin.

Under nondenaturing conditions, 1 mol of horse plasma gelsolin reacts with 1.9 +/- 0.5 mol (mean +/- SD, n = 6) of the sulfhydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The degree of labeling in 6 M guanidine-HCl increases to about 3 mol of acrylodan per mole of gelsolin. Viscosity studies show that the modified gelsolin retains its ability to sever F-actin filaments. Circular dichroism spectra in the peptide bond absorption region are indistinguishable for labeled and unmodified gelsolin at room temperature. The thermal stability of gelsolin, as monitored by circular dichroism, is unaffected by reaction with acrylodan. While circular dichroism spectra of acrylodan-labeled gelsolin recorded at room temperature are not influenced significantly by Ca2+, fluorescence studies reveal a number of Ca(2+)-dependent changes in the protein. Ca2+ causes a decrease and red-shift in fluorescence emission, an increase in sensitivity to quenching by I-, and a decrease in polarization of the fluorescence of acrylodan-labeled gelsolin. Together, these changes in fluorescence properties indicate there to be an increased exposure of the label to the solvent when gelsolin binds Ca2+. Fluorescence polarization experiments in which acrylodan-labeled gelsolin is titrated with actin emphasize that Ca2+ is required for these two proteins to interact.

2-Naphthylamine↗

PubMed2002

Molecular mechanism of transcriptional repression of gelsolin in human breast cancer cells.

Loss of gelsolin, a tumor suppressor, is one of the most frequently occurring molecular defects in breast cancers of diverse etiologies and across at least three animal species: human, mouse, and rat. Our previous analysis of breast cancer cells demonstrated that the deficiency is not due to mutation of the gelsolin gene, but instead to epigenetic factors, including decreased transcription of the gene. The study described herein provides the first functional characterization of the human gelsolin promoter and reveals a mechanistic basis for the reduced gelsolin transcription. In reporter gene assays, the gelsolin promoter was less active in low-gelsolin-expressing breast cancer cells. A cis-element mediating this reduced promoter activity was defined as a 27-bp sequence located approximately 135 bp upstream of the transcription start site. Gel shift and supershift assays and Southwestern blotting analysis indicated that activating transcription factor-1 (ATF-1) and a protein of approximately 100 kDa may have cancer cell-specific DNA-binding activity to the 27-bp gelsolin cis-element. Although the ATF-1 protein was highly expressed in both benign and tumorigenic breast cells, its DNA-binding activity was selectively abundant in the cancer cells and correlated inversely with the gelsolin mRNA level. Thus, our results suggest a role for ATF-1 in gelsolin promoter silencing in contrast to its transactivating effect on various other promoters.

5' Flanking Region↗

PubMed1994

Distribution of gelsolin in mouse ovary.

The distribution of gelsolin, a calcium-dependent actin-modulating protein, and the expression of the corresponding gene, have been characterized with respect to morphogenetic processes in mouse ovary. Substantial amounts of gelsolin have been detected in the ovary and uterus of the mouse by immunoblot analysis. The similar relative ratio of mRNA of alpha-smooth muscle actin (alpha-SM actin) and gelsolin in the two organs suggests that expression of these two genes is coordinated at the transcriptional level. Immunofluorescence has demonstrated gelsolin predominantly in three types of cells in the ovary: (1) cells of the theca externa and stroma, (2) endothelial cells lining blood vessels, and (3) cells of the superficial epithelium of ovary. In the smooth-muscle-like cells of the theca externa, gelsolin appears tightly associated with the microfilamentous cytoskeleton, which is also rich in alpha-SM actin. The presence of gelsolin in myoid cells suggests that this protein, possibly by modulation of the activity of the actomyosin ATPase, plays a critical role in contractile and morphogenetic processes, e.g., during growth and maturation of the follicle or during ovulation. In cells of the endothelium, intracellular gelsolin is associated with the F-actin cytoskeleton around the nucleus. The circumferential belt lining the lateral cell membranes in cells of the superficial epithelium at the ovarian surface is also rich in gelsolin. Our observations indicate that the function of gelsolin as a calcium- and phospholipid-dependent modulator of actin assemblies is pivotal for the regulation of the dynamic alterations of the actin cytoskeleton in the superficial epithelium when cells become attenuated and retract their microvilli during growth of the follicle.

Actins↗

PubMed2001

Changes in gelsolin expression during ascidian metamorphosis.

Gelsolin is an actin regulatory protein that is expressed in a wide variety of tissues and is especially abundant in muscle and blood cells. The role of gelsolin during structural reorganization of the body, such as during metamorphosis or regeneration, is poorly understood. We analyzed changes in gelsolin expression during ascidian embryogenesis and metamorphosis using nucleic acid probes and a monoclonal antibody (AS23) specific for ascidian gelsolin; our results indicated that gelsolin is maternally provided and that its de novo gene transcription is initiated during the neurula stage. In the larva, gelsolin was detectable in specific types of nerve cells, i.e. the adhesive papillae, motor neurons and epidermal sensory neurons. During metamorphosis, the expression of gelsolin changes markedly: the expression is suppressed in nerve tissues after tail resorption but is induced in mesodermal tissues. Gelsolin accumulated in mesenchyme cells until the onset of tail resorption, and following tail resorption, these cells migrated to the tunic and differentiated into tunic cells with many fine processes. Migration of the mesenchyme cells into the tunic was completely inhibited by treatment with cytochalasin B. Gelsolin was colocalized with actin in tunic cells, suggesting that it is involved in the rearrangement of actin filaments during cell locomotion or morphogenesis.

Animals↗

PubMed2004

Abundant corneal gelsolin in Zebrafish and the 'four-eyed' fish, Anableps anableps: possible analogy with multifunctional lens crystallins.

The cornea accumulates high proportions (can be up to 50%) of taxon-specific, water-soluble, cytoplasmic proteins (often enzymes) that have been considered analogous to the multifunctional lens crystallins. We have shown that gelsolin (an actin-severing protein) is the major water-soluble corneal protein of the zebrafish (Danio rerio) and the 'four-eyed' fish (Anableps anableps). Each Anableps eye contains one lens, an aquatic ventral cornea with an epithelium comprising 5-7 cell layers, and an air-exposed flatter dorsal cornea with an epithelium comprising >20 cell layers and appreciably enriched with glycogen. Gelsolin accounts for 38 and 21% of the dorsal and ventral cornea, respectively, suggesting that the abundance of gelsolin in the cornea is not incompatible with its function in air. The thicker, glycogen-enriched, air-exposed dorsal cornea may protect against UV irradiation and desiccation. Gelsolin comprises approximately 50% of the 5 cell-layer thick aquatic corneal epithelium of zebrafish. Reported zebrafish ESTs have indicated the presence of a second gelsolin gene in this species. We show by RT-PCR that the abundant corneal gelsolin (also expressed weakly in lens) (C/L-gelsolin) is also expressed in early development and differs from a ubiquitously expressed gelsolin (U-gelsolin) that is not specialized for cornea. Microinjection tests showed that overexpression of C/L-gelsolin dorsalizes the embryo and can lead to axis duplication, while interruption of C/L-gelsolin expression with a specific morpholino oligonucleotide ventralizes the embryo and interferes with brain and eye development. The evidence that C/L-gelsolin participates in the bone morphogenetic protein (BMP)/Smad dorsal-ventral signaling pathway is reviewed. Finally, we speculate that soluble C/L-gelsolin:actin complexes in the cornea may be analogous to soluble alphaA:alphaB-crystallin complexes in the lens. Together, our data are consistent with an analogy between the abundance of gelsolin in fish corneas and taxon-specific multifunctional crystallins in lenses.

Animals↗

PubMed2005

Mechanisms of gelsolin-dependent and -independent EGF-stimulated cell motility in a human lung epithelial cell line.

Acquisition of motility is an important step in malignant progression of tumor cells and involves dynamic changes in actin filament architecture orchestrated by many actin binding proteins. A role for the actin-binding protein gelsolin has been demonstrated in fibroblast motility. In this report, we investigated the role of gelsolin in bronchial epithelial cell motility. The non-tumorigenic bronchial epithelial cell line, NL20 migrated towards EGF in a modified Boyden chamber cell motility assay. However, the tumorigenic NL20-TA cell line derived from the NL20 cells and lacking gelsolin, did not migrate towards EGF. Ectopic expression of gelsolin in NL20-TA cells restored the EGF response, while motility of NL20-TA derived cells towards serum, PDGF, and fibronectin was independent of gelsolin expression. PI3-kinase inhibition failed to block EGF-stimulated motility in gelsolin transfected NL20-TA cells. Furthermore, EGF stimulated a motility response in cells lacking gelsolin in the presence of fibronectin or fibrinogen that was blocked with PI3-kinase inhibition. Thus, EGF-stimulated motility in NL20 cells and its derivatives are gelsolin dependent and PI3-kinase independent, while fibronectin and fibrinogen enhances EGF-stimulated motility through a pathway independent of gelsolin and dependent upon PI3-kinase.

Antibodies, Monoclonal↗

PubMed1998

Purification and functional characterization of an 85-kDa gelsolin from the ascidians Microcosmus sulcatus and Phallusia mammilata.

From the pharyngeal baskets of the ascidians Microcosmus sulcatus and Phallusia mammilata we have purified an 85-kDa protein that is characterized as a member of the gelsolin family. These proteins from both species show the same behaviour in functional assays. The ascidian gelsolin binds two actin monomers in a highly cooperative manner. This complex formation is Ca(2+)-dependent, but not completely reversible, as on removal of Ca2+ one actin monomer dissociates leaving a 1:1 complex between gelsolin and G-actin. The properties of F-actin severing and G-actin nucleation depend on the presence of free Ca2+ in a micromolar range, with half maximum activation at about 3 x 10(-6) M. The protein becomes inactivated when Ca2+ concentrations of 0.5 mM are exceeded. Fragmentation of F-actin by the ascidian gelsolin is comparably fast to that of vertebrate gelsolin. A steady state of actin fragmentation is reached within 2-4 s. Promotion of G-actin nucleation is also comparable to that of vertebrate gelsolin. Regarding functional aspects, the ascidian gelsolin is more closely related to vertebrate gelsolin than to an arthropod gelsolin from crayfish tail muscle.

Actins↗

PubMed1985

Covalent complexes formed between plasma gelsolin and actin with a zero-length cross-linking compound.

Actin and plasma gelsolin were covalently cross-linked with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Two major intermolecularly linked products were identified on polyacrylamide gels. By use of 14C-labeled actin and 125I-labeled gelsolin, these were shown to be the 1:1 and 2:1 complexes of actin with gelsolin, respectively. The higher molecular weight complex predominated under all conditions tested including the presence and absence of Ca2+. In titration experiments in which actin at different concentrations was reacted with a fixed concentration of gelsolin, end points were obtained for the formation of both cross-linked species at about two actins per gelsolin, implying that a 2:1 noncovalent complex is cross-linked. In 0.1 mM Ca2+, the extent of cross-linking was independent of protein concentration down to 50 nM gelsolin. At low Ca2+ concentrations (less than 10(-8)M), the extent of cross-linking was very much reduced at micromolar gelsolin and fell to zero at about 100 nM gelsolin. The binding of actin to gelsolin to give a cross-linkable complex is therefore very strong at 0.1 mM Ca2+ but much weaker at low Ca2+ concentrations.

Actins↗

PubMed1998

Equus caballus gelsolin--cDNA sequence and protein structural implications.

We have generated and characterized the cDNA from equine smooth muscle that encodes gelsolin, an actin-modulating protein. Overlapping cDNA clones synthesized by the reverse transcriptase/polymerase chain reaction and clones isolated from a horse genomic library provided the complete primary structure for the intracellular isoform of gelsolin, while cDNA complemented with protein sequence data produced the full-length primary transcript of the gelsolin isoform found circulating in equine plasma. The deduced amino acid sequences of the intracellular and secreted versions of equine gelsolin infer polypeptides of 731 and 755 residues with apparent molecular masses of 80.7 kDa and 83.2 kDa, respectively. Multiple sequence alignment analysis of equine, human, porcine, and murine orthologs of gelsolin demonstrates prominent similarities among all of these proteins, with the horse and human molecules exhibiting the largest degree of likeness with respect to polypeptide length and overall sequence composition. Both horse and human plasma gelsolins are comprised of 755 amino acids with 94% of the residues identical, while the degree of sequence identity in the shorter (731 residues) cytoplasmic gelsolins is 95%. Analysis of the sequences and structures of the six related domains that comprise gelsolin emphasizes the strong correlation that exists between primary structural conservation among mammalian gelsolins and maintenance of the three-dimensional domain fold characteristic of members of this protein family.

Amino Acid Sequence↗

PubMed1999

Differential phospholipase D activation by bradykinin and sphingosine 1-phosphate in NIH 3T3 fibroblasts overexpressing gelsolin.

Gelsolin, an actin-binding protein, shows a strong ability to bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here we showed in in vitro experiments that gelsolin inhibited recombinant phospholipase D1 (PLD1) and PLD2 activities but not the oleate-dependent PLD and that this inhibition was not reversed by increasing PIP(2) concentration. To investigate the role of gelsolin in agonist-mediated PLD activation, we used NIH 3T3 fibroblasts stably transfected with the cDNA for human cytosolic gelsolin. Gelsolin overexpression suppressed bradykinin-induced activation of phospholipase C (PLC) and PLD. On the other hand, sphingosine 1-phosphate (S1P)-induced PLD activation could not be modified by gelsolin overexpression, whereas PLC activation was suppressed. PLD activation by phorbol myristate acetate or Ca(2+) ionophore A23187 was not affected by gelsolin overexpression. Stimulation of control cells with either bradykinin or S1P caused translocation of protein kinase C (PKC) to the membranes. Translocation of PKC-alpha and PKC-beta1 but not PKC-epsilon was reduced in gelsolin-overexpressed cells, whereas phosphorylation of mitogen-activated protein kinase was not changed. S1P-induced PLC activation and mitogen-activated protein kinase phosphorylation were sensitive to pertussis toxin, but PLD response was insensitive to such treatment, suggesting that S1P induced PLD activation via certain G protein distinct from G(i) for PLC and mitogen-activated protein kinase pathway. Our results suggest that gelsolin modulates bradykinin-mediated PLD activation via suppression of PLC and PKC activities but did not affect S1P-mediated PLD activation.

3T3 Cells↗