&Toxin (perfringolysin 0), secreted by Clostridium perfringens, shares with other known thiol-activated toxins a conserved undecapeptide, ECTGLAWEWWR, located in the C-terminal region of the protein and containing the unique cysteine of the molecule. Single and double amino acid substitutions were created in the 8-toxin molecule to investigate the role of individual tryptophan residues in the lytic activity of &toxin. Wild-type and mutant &toxins were overproduced in Escherichia coli by means of a T7 RNA polymerase/promoter system and purified. The relative hemolytic activities of four mutant toxins, each with a Trp to Phe substitution outside the common Cys-containing region, were more than 60% that of wild-type 8-toxin. In contrast, mutant toxins with Phe replacements within the Cys-containing region (at Trp436, Trp438 or Trp439) showed significantly reduced hemolytic and erythrocyte-membranebinding activities. The largest reduction in binding affinity, more than 100-fold, was observed for Trp438 mutant toxins. However, the mutants retain binding specificity for cholesterol and the ability to form arcshaped and ring-shaped structures on membranes. These results indicate that the low hemolytic activities of these mutant toxins can be ascribed, at least in part, to reduced binding activities. With respect to protease susceptibility and far-ultraviolet circular-dichroism spectra, only the W436-F mutant toxin, showed any considerable difference from wild-type toxin in secondary or higher-order structures, indicating that Trp436 is essential for maintenance of toxin structure.

Key Points• Clot retraction of sphingomyelin-rich raftdepleted platelets from sphingomyelin synthase knockout mouse is delayed.• Translocation of fibrin to sphingomyelin-rich rafts in platelet membrane is induced by thrombin in the presence of FXIII crosslinking activity.Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen g-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunitdeficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-b-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-aIIbb3-myosin complex is formed as a primary axis to promote platelet contraction. (Blood. 2013;122(19):3340-3348) IntroductionMembrane rafts are dynamic assemblies of sphingolipids, cholesterol, and proteins that can be stabilized into platforms involved in the regulation of a number of vital cellular processes. 1 The important role of rafts at the cell surface may be their function in signal transduction. A number of studies provide considerable evidence that rafts are integral to the regulation of immune and neuronal signaling. Membrane rafts are also involved in hemostasis and thrombosis. Among blood cells, platelets are critical for maintaining the integrity of the blood coagulation system. Platelet rafts are critical membrane domains in physiological responses such as adhesion and aggregation. 2 The localization of the adhesion receptor glycoprotein (GP)Ib-IX-V complex to membrane rafts is required for platelet adhesion to the vessel wall by binding the von Willebrand factor. 3 Membrane rafts are also required for platelet aggregation via the collagen receptor GPVI, 4 the adenosine 59-diphosphate (ADP) receptor P2Y12, 5 the Fcg receptor FcgRIIa, 6 and the C-type lectinlike receptor CLEC-2.7 Detergent-resistant membrane (DRM) rafts of platelets show round vesicles of heterogeneous sizes ranging from 20 to 500 nm, which are enriched in CD36 (GPIV). 8,9 Recent reports have demonstrated that membrane rafts are ...

No abstract

Neurons require Ca2+-dependent gene transcription for their activity-dependent survival, the mechanisms of which have not been fully elucidated yet. Here, we demonstrate that a novel primary response gene, alivin 1 (ali1), is an activity-dependent gene and promotes survival of neurons. Sequence analyses reveal that rat, mouse, and human Ali1 proteins contain seven leucine-rich repeats, one IgC2-like loop and a transmembrane domain, and display homology to Kek and Trk families. Expression of ali1 mRNA in cultured cerebellar granule neurons is rigidly regulated by KCl and/or NMDA concentrations in the culture medium and tightly correlated to depolarization-dependent survival and/or NMDA-dependent survival of the granule neuron. ali1 mRNA expression was regulated at the transcriptional step by the Ca2+ influx through voltage-dependent L-type Ca2+ channels when the cells were stimulated by 25 mm KCl. Expression of ali1 mRNA in cultured cortical neurons was inhibited when their spontaneous electrical activity was blocked by tetrodotoxin. Thus, the expression is neuronal activity dependent. Overexpression of Ali1 in cerebellar granule neurons inhibited apoptosis that was induced by the medium containing 5 mm KCl. The addition of anti-Ali1 antiserum or the soluble putative extracellular Ali1 domain to the 25 mm KCl-supported culture inhibited the survival of the granule neuron. These results suggest that expression of ali1 promotes depolarization-dependent survival of the granule neuron. Mouse ali1 was mapped to a locus approximately 55.3 cM from the centromere on chromosome 15 that is syntenic to positional candidate loci for familial Alzheimer's disease type 5 and Parkinson's disease 8 on human chromosome 12.

Perfringolysin O (theta-toxin) is a cholesterol-binding and pore-forming toxin that shares with other thiol-activated cytolysins a highly conserved sequence, ECTGLAWEWWR (residues 430-440), near the C-terminus. To understand the membrane-insertion and pore-forming mechanisms of the toxin, we evaluated the contribution of each Trp to the toxin conformation during its interaction with liposomal membranes. Circular dichroism (CD) spectra of Trp mutant toxins indicated that only Trp436 has a significant effect on the secondary structure, and that Trp436, Trp438, and Trp439 make large contributions to near-UV CD spectra. Quenching the intrinsic Trp fluorescence of the wild-type and mutant toxins with brominated lecithin/cholesterol liposomes revealed that Trp438 and probably Trp436, but not Trp439, contributes to toxin insertion into the liposomal membrane. Near-UV CD spectra of the membrane-associated mutant toxins indicated that both Trp438 and Trp439 are required for the CD peak shift from 292 to 300 nm, a signal related to theta-toxin oligomerization and/or pore formation, suggesting a conformational change around Trp438 and Trp439 in these processes.