Bombina Maxima Skin Research Articles

The frog trefoil factor Bm-TFF2 activates human platelets via Gq and G12/13 signaling pathway

Bm-TFF2 is an amphibian trefoil factor purified from the Bombina maxima skin secretion that is highly toxic to mammals. We previously reported that Bm-TFF2 activates human platelets via protease-activated receptor 1. In this study, for a better understanding of platelet activation induced by Bm-TFF2, we used affinity chromatography and pharmacological inhibitors to investigate the downstream signaling pathway. Using Bm-TFF2-affinity chromatography, Gq was specifically eluted from the Bm-TFF2-coulped column. Pharmacological inhibitors such as U73122, Xestospongin C, BAPTA-AM and Gö6976 can significantly inhibit Bm-TFF2-induced platelet aggregation. These results suggested that Gq activation and the downstream PLCβ-IP3 receptor-cytoplasmic Ca2+-PKC signaling pathway is crucial for Bm-TFF2 to stimulate platelet aggregation. Furthermore, Bm-TFF2 induced strong platelet shape change at the concentrations of 5nM, in which the Ca2+ mobilization of the platelets stimulated was not detectable. The p160ROCK inhibitorY27632 totally inhibited the shape change, indicating that Bm-TFF2 may activate the G12/13 pathway which leads to the activation of RhoA-p160ROCK. In conclusion, Bm-TFF2 induced platelet activation mainly via the Gq and G12/13 signaling pathway. This study on the signaling pathway of Bm-TFF2 stimulation may help us understand the toxicity of B. maxima skin secretion to the human platelets.

Establishment, characterization and immortalization of a fibroblast cell line from the Chinese red belly toad Bombina maxima skin

The skin of the amphibian Bombina maxima is rich in biologically active proteins and peptides, most of which have mammalian analogues. The physiological functions of most of the mammalian analogues are still unknown. Thus, Bombina maxima skin may be a promising model to reveal the physiological role of these proteins and peptides because of their large capacity for secretion. To investigate the physiological role of these proteins and peptides in vitro, a fibroblast cell line was successfully established from Bombina maxima tadpole skin. The cell line grew to form a monolayer with cells of a uniform shape and abundant rough endoplasmic reticulum, which are typical characteristics of fibroblasts. Further identification at a molecular level revealed that they strongly expressed the fibroblast marker protein vimentin. The chromosome number of these cells is 2n=28, and most of them were diploid. Growth property analysis showed that they grew well for 14 passages. However, cells showed decreased proliferative ability after passage 15. Thus, we tried to immortalize the cells through the overexpression of SV40 T antigen. After selecting by G418, cells stably expressed SV40 large T antigen and showed enhanced proliferative ability and increased telomerase activity. Signal transduction analysis revealed functional p42 mitogen-activated protein (MAP) kinase in immortalized Bombina maxima dermal fibroblasts. Primary fibroblast cells and the immortalized fibroblast cells from Bombina maxima cultured in the present study can be used to investigate the physiological role of Bombina maxima skin-secreted proteins and peptides. In addition, the methods for primary cell culturing and cell immortalization will be useful for culturing and immortalizing cells from other types of amphibians.

βγ-CAT, a non-lens betagamma-crystallin and trefoil factor complex, induces calcium-dependent platelet apoptosis

In recent years, it has been reported that apoptosis may occur in platelets and play a role in the clearance of effete platelets. βγ-CAT, a newly identified non-lens βγ-crystallin and trefoil factor complex from frog Bombina maxima skin secretions, caused several in vivo toxic effects on mammals. Through determined haematological parameters of rabbits, it has been found that βγ-CAT significantly reduced the number of platelets in a time-dependent manner. Here, in order to explore the effect of βγ-CAT on platelets, washed platelets were incubated with various concentrations of βγ-CAT for 30 minutes. We found that βγ-CAT induced several apoptosis events in human platelets, including caspase-3 activation, phosphatidylserine (PS) exposure, depolarisation of mitochondrial inner transmembrane potential (ΔΨm), cytochrome c release and strong expression of pro-apoptotic Bax and Bak proteins. However, βγ-CAT did not significantly induce platelet activation as detected by P-selectin surface expression, GPIIb/IIIa activation and platelet aggregation. In addition, we observed that βγ-CAT-induced PS exposure and ΔΨm depolarisation in platelets are Ca2+-dependent. Taken together, βγ-CAT can induce Ca2+-dependent platelet apoptosis but does not cause platelet activation.

Bm-TFF2, a toad trefoil factor, promotes cell migration, survival and wound healing

Toad skin is naked and continually confronted by various injurious factors. Constant skin renewal and repairs occur frequently. However, the mechanisms of the renewal and repair have not clearly elucidated. In our previous work, a trefoil factor (TFF), Bm-TFF2, has been purified from the Bombina maxima skin and characterized as a platelet agonist. The mRNA of TFFs in toad skin was up-regulated greatly during the metamorphosis, indicating a pivotal role of TFFs in amphibian skin. Here, we presented the effects of Bm-TFF2 on the cell migration, apoptosis and proliferation. Bm-TFF2 bound to epithelial cells and showed strong cell motility activity. At the concentrations of 1–100 nM, Bm-TFF2-induced migration of human epithelial AGS and HT-29 cells, and rat intestinal epithelial IEC-6 cell lines. The in vitro wound healing assay also verified the activity of Bm-TFF2. Bm-TFF2 could also inhibit cell apoptosis induced by ceramide and sodium butyrate. The cell migration-promoting activity was abolished by MEK1 inhibitors, U0126 and PD98059, suggesting that ERK1/2 activation is crucial for Bm-TFF2 to stimulate cell migration. Taken together, Bm-TFF2 promoted wound healing by stimulating cell migration via MAPK pathway and preventing cell apoptosis. The potent biological activity of Bm-TFF2 makes it a useful molecular tool for further studies of structure–function relationship of the related human TFFs.

A novel annexin A2 protein with platelet aggregation-inhibiting activity from amphibian Bombina maxima skin

Annexin A2 is a unique member of annexin family with multi-functions in membrane physiology, implicated in inflammation and cancer progression. mRNA of Annexin A2 is abundant in the skin of some amphibians. However, no annexin A2 protein has been isolated and characterized from amphibian skin. In this report, a novel annexin A2 protein with apparent molecular weight of 33 kDa and named Bm-ANXA2, was purified from frog Bombina maxima skin, which is highly toxic to mammals, by a combination of ion exchange and gel filtration chromatography. A full-length cDNA encoding the protein was obtained from the cDNA library constructed from the frog skin. Sequence analysis indicates that Bm-ANXA2 shares 89% and 80% amino acid sequence identities with those of Xenopus and human annexin A2, respectively. Different from other annexin A2 proteins, the N-terminal 26 amino acids of Bm-ANXA2 were truncated. Bm-ANXA2 dose-dependently inhibited human platelet aggregation stimulated by various agonists in a Ca 2+-dependent manner. It bound to activated platelets and significantly inhibited α IIbβ 3 activation and α-granular secretion. This is the first report that an annexin A2 protein possesses platelet aggregation-inhibiting activity, providing novel clues in the illustration of pathophysiological roles of annexin A2 proteins.

Melanoma cell growth inhibition by βγ-CAT, which is a novel non-lens betagamma-crystallin and trefoil factor complex from frog Bombina maxima skin

In vertebrates, non-lens βγ-crystallins are widely expressed in various tissues but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive. βγ-CAT, which is responsible for the potent hemolytic activity and lethal toxicity on mice of frog Bombina maxima skin secretions, is the first example of a non-lens βγ-crystallin and trefoil factor complex. Its α- and β-subunits show significant sequence homology to human Absent In Melanoma 1 and trefoil factors, respectively. Here, we showed that βγ-CAT triggered two types of cellular responses in human melanoma cells. On one hand, the protein (25–200 pM) was able to stimulate cell migration in melanoma A375 cells. On the other hand, it inhibited cell proliferation by delaying S-G2/M cell phase transition. Blockade of the cell cycle was associated with increased p21/WAF1 expression, and reduced amounts of Cdc2 and Cdc25C. βγ-CAT also reduced Cdc2 function by increasing the level of inactivated phospho-Cdc2. In addition, the expression of JunB, a well-known cell proliferation inhibitor, was significantly up-regulated. These results provide the first evidence of an anti-proliferative role for a non-lens βγ-crystallin member and action mechanism via association with a trefoil factor.

βγ-CAT, a non-lens βγ-crystallin and trefoil factor complex from amphibian skin secretions, caused endothelium-dependent myocardial depression in isolated rabbit hearts

Previous in vivo study demonstrated that βγ-CAT, a newly identified non-lens βγ-crystallin and trefoil factor complex from frog Bombina maxima skin secretions, possessed potent lethal toxicity on mammals resulted from hypotension and cardiorespiratory arrest. However, the mechanism of cardiac dysfunction induced by the protein is unknown. Here, we report that βγ-CAT, with dosages of 0.8–3.0 nM, elicited an acute negative inotropic effect in isolated rabbit heart Langendorff preparations, which mimicked acute heart failure. In addition, the effect of βγ-CAT on the hearts was mediated by endothelium-dependent coronary vasoconstriction ( P < 0.01, compared between endothelium-intact and removal hearts). After βγ-CAT (3.0 nM) treatment, the positive signal of tumor necrosis factor-α (TNF-α) was detected mainly around the endothelial cell layer as detected by in situ indirect immunofluorescence, indicating that the release of TNF-α occurred. At the same time, a rapid TNF-α release was detected in primary cultured rabbit endocardial endothelial cells (REECs) treated with βγ-CAT. After addition of βγ-CAT (3.0 nM) for 10 min and 30 min, the TNF-α levels were increased to 57.33 ± 3.22 pg/ml and 60.00 ± 5.35 pg/ml ( P < 0.05, compared with the control values of 21.67 ± 3.45 pg/ml and 33.70 ± 6.24 pg/ml, respectively). At high concentrations, βγ-CAT interfered with the cell viability of REECs (CC 50 about 25 nM). Taken together, βγ-CAT was able to induce acute myocardial depression and the toxic effect might be partially explained by the release of TNF-α. The finding provides new information to understand the patho-physiological roles of non-lens βγ-crystallins and trefoil factors.

Acute toxicity of βγ-CAT, a naturally existing non-lens βγ-crystallin and trefoil factor complex from frog Bombina maxima skin secretions

In vertebrates, non-lens βγ-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive. βγ-CAT is the first example of a naturally existing multifunctional protein complex of a non-lens βγ-crystallin and a trefoil factor from frog Bombina maxima skin secretions. Here we report the investigation of its in vivo toxic effects on mice and rabbits. The LD 50 values of βγ-CAT on mice were determined to be 0.4 mg/kg and 20 μg/kg under intraperitoneal (i.p.) and intravenous (i.v.) injection, respectively. The mice subcutaneously injected with βγ-CAT (6 μg/g body weight) showed strong hyperaemia of subcutaneous capillary vessel, but no hemorrhagic spots were observed. Intravenous injection of βγ-CAT in rabbits (8–22 μg/kg body weight) caused a rapidly hypotensive effect and followed with cardiovascular collapse. Injection with βγ-CAT (22 μg/kg, i.v.) significantly decreased hematocrit ( P < 0.05) and mean corpuscular volume ( P < 0.05) of the rabbits in 5 min. At the same time, the counts of platelets and white blood cells were significantly decreased ( P < 0.05), while the blood levels of glucose, lactate dehydrogenase and serum glutamic–oxaloacetic transaminase were significantly increased ( P < 0.05). Furthermore, serials of tissues edema and damages were also observed. These results indicate that βγ-CAT rapidly caused several in vivo toxic effects on mammals and its lethal toxic potency was mainly contributed by hypotension and cardiovascular collapse, providing new clues for the understanding of the patho-physiological roles of non-lens βγ-crystallins and trefoil factors.

A novel non-lens betagamma-crystallin and trefoil factor complex from amphibian skin and its functional implications.

BackgroundIn vertebrates, non-lens βγ-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.Principal FindingsA naturally existing 72-kDa complex of non-lens βγ-crystallin (α-subunit) and trefoil factor (β-subunit), named βγ-CAT, was identified from frog Bombina maxima skin secretions. Its α-subunit and β-subunit (containing three trefoil factor domains), with a non-covalently linked form of αβ2, show significant sequence homology to ep37 proteins, a group of non-lens βγ-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. βγ-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. βγ-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K+ efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa) were detected by antibodies against the α-subunit with Western blotting. Furthermore, βγ-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of βγ-CAT (25–50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC50 10 nM) and apoptosis. βγ-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited βγ-CAT induced vacuole formation, significantly inhibited βγ-CAT induced cell detachment, suggesting that βγ-CAT endocytosis is important for its activities.Conclusions/SignificanceThese findings illustrate novel cellular functions of non-lens βγ-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.

Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B.maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1:1 molar ratio. The equilibrium dissociation constants (KD) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys53-Cys62), which comprises the scissile bond Arg58(P1)-His59(P1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

Bm-TFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions

In mammals, trefoil factor family (TFF) proteins are involved in mucosal maintenance and repair, and they are also implicated in tumor suppression and cancer progression. A novel two domain TFF protein from frog Bombina maxima skin secretions (Bm-TFF2) has been purified and cloned. It activated human platelets in a dose-dependent manner and activation of integrin α IIbβ 3 was involved. Aspirin and apyrase did not largely reduce platelet response to Bm-TFF2 (a 30% inhibition), indicating that the aggregation is not substantially dependent on ADP and thromboxane A2 autocrine feedback. Elimination of external Ca 2+ with EGTA did not influence the platelet aggregation induced by Bm-TFF2, meanwhile a strong calcium signal (cytoplasmic Ca 2+ release) was detected, suggesting that activation of phospholipase C (PLC) is involved. Subsequent immunoblotting revealed that, unlike in platelets activated by stejnulxin (a glycoprotein VI agonist), PLCγ2 was not phosphorylated in platelets activated by Bm-TFF2. FITC-labeled Bm-TFF2 bound to platelet membranes. Bm-TFF2 is the first TFF protein reported to possess human platelet activation activity.

Maximins S, a novel group of antimicrobial peptides from toad Bombina maxima

Amphibian skin secretions are rich in antimicrobial peptides acting as important components of innate defense system against invading microorganisms. A novel type of peptide, designated as maximin S, was deduced by random sequencing of 793 clones from a constructed Bombina maxima skin cDNA library. The putative primary structures of maximin S peptides can be grouped into five species, in which maximin S1 has 14 amino acid residues and the rest of maximin S peptides (S2–S5) all have 18 amino acid residues. Unlike most of the amphibian antimicrobial peptides so far identified, the newly characterized four maximin S precursors are composed of maximin S1 and different combinations of tandem repeated maximin S2–S5 linked by internal peptides. Except maximin S1, the predicted secondary structures of maximin S2–S5 show a similar amphipathic α-helical structure. MALDI-TOF mass spectrometry analysis of partially isolated skin secretions of the toad indicates that most of the deduced maximin S peptides are expressed. Two deduced maximin S peptides (S1, S4) were synthesized and their antimicrobial activities were tested. Maximin S4 only had an antibiotic activity against mycoplasma and had no antibacterial or antifungal activity toward tested strains. Maximin S1 had no activity under the same conditions.