David A. Hessinger

Isolation and partial characterization of a hemolytic and toxic protein from the nematocyst venom of the Portuguese man-of-war, Physalia physalis

Nematocysts isolated from the stinging tentacles of the Atlantic Portuguese Man-of-War (Physalia physalis) possess a potent venom composed of several proteins. A hemolytic protein lethal to mice has been isolated from this nematocyst venom. This protein, physalitoxin, appears to be responsible for both the venoms hemolytic and lethal activities. The hemolysin has a molecular weight of approx. 240 000, a sedimentation coefficient of 7.8 S, and is rod-like in shape with a calculated axial ratio of about 1 : 10. It appears to be composed of three subunits of unequal size, each of which is glycosylated. Two of these subunits seem to have pKi values near 8.2 and the third near 5.5. Physalitoxin comprises about 28% of the total nematocyst venom protein. It is 10.6% carbohydrate by weight and represents the major glycoprotein of the venom. Physalitoxin is inactivated by concanavalin A and this inactivation can be blocked with alpha-methyl-mannoside. The inactivation by concanavalin A is temperature-dependent about 12 degrees C and the hemolytic activity of untreated venom is temperature-dependent below 12 degrees C. Physalitoxin is the first hemolytic toxin from a cnidarian to be purified directly from isolated nematocysts.

Purification and partial characterization of the phospholipase A2 and co-lytic factor from sea anemone (Aiptasia pallida) nematocyst venom.

Functional nematocysts of one specific morphological class, the penetrant microbasic mastigophores, were isolated from the sea anemone, Aiptasia pallida. These nematocysts contain a multicomponent venom composed of several proteins, including those with neurotoxic, hemolytic, and lethal activities. Hemolytic activity is produced by at least three synergistic venom proteins. One of these proteins is identified as a phospholipase A2 (EC 3.1.1.4) which exists in two isozymic forms, alpha and beta, with molecular weights of 45,000 and 43,000, respectively. The beta isozyme has been purified to homogeneity. It is a single-chained glycoprotein with an isoelectric point (pI) of 8.8 and represents 70% of the phospholipase activity of the venom. The activity of the beta isozyme is relatively labile and is inactivated by 3.5 M urea or by heating at 45 degrees C. It is most stable at pH 4.0 and loses 50% of its activity at pH values below 3.5 and above 8.0. A second venom protein has also been purified. It is essential for the hemolytic activity of the venom and is termed co-lytic factor (CLF). It is a monomeric glycoprotein having a pI of 4.5. CLF has a molecular weight of approximately 98,000, a sedimentation coefficient of 4.8 S, and is prolate in shape, having a frictional ratio of about 1.6. CLF constitutes about 1.25% of the total venom protein and is assayed by reversing fatty acid inhibition of the venom hemolysis activity.

Receptors for N-acetylated sugars may stimulate adenylate cyclase to sensitize and tune mechanoreceptors involved in triggering nematocyst discharge☆

In fishing tentacles of sea anemones, cnidocyte/supporting cell complexes (CSCCs) trigger the discharge of nematocysts following stimulation by swimming prey of specific mechanoreceptors and chemoreceptors located on the supporting cells. Two types of mechanoreceptors have been identified: a contact-sensitive mechanoreceptor (CSM), and a vibration-sensitive mechanoreceptor (VSM). The CSMs become predisposed to initiate nematocyst discharge into static (i.e., nonvibrating) test probes in the presence of submicromolar free and conjugated N-acetylated sugars, a process referred to as sensitization. In seawater, the VSMs cause maximal discharge in response to test probes vibrating at 30, 50-55, and 75 Hz, whereas in the presence of submicromolar N-acetylated sugars the VSMs cause maximal discharge into test probes vibrating at 5, 15, 30, and 40 Hz, a process referred to as tuning. Tuning of the VSMs is accompanied by elongation of the stereocilium bundles comprising the VSMs. We report that dibutyryl cyclic-AMP sensitizes CSMs and tunes VSMs to the lower frequencies of 5, 15, 30, and 40 Hz, while cyclic-AMP has no such effects. Endogenous adenylate cyclase activity at the apical plasma membrane of the supporting cells is detectable by cytochemical methods in the presence of N-acetylated sugars but not in seawater alone. By activating adenylate cyclase with L858051, an analogue of forskolin, or by activating the stimulatory form of G proteins (Gs) with cholera toxin, CSCCs are induced to sensitize CSMs and to tune VSMs to the lower frequencies of 5, 15, 30, and 40 Hz. Caged GTP-gamma S also sensitizes CSMs but tunes VSMs to 5, 15, 30, 40, 55, 65, and 75 Hz, suggesting that VSM tuning may be regulated both by Gs and inhibitory G-proteins. Together, these results implicate cAMP as the second messenger for activated supporting cell chemoreceptors involved in sensitizing the CSMs and tuning the VSMs to lower frequencies.

Apparent membrane pore-formation by portuguese Man-of-war (Physalia physalis) venom in intact cultured cells

Intracellular, ratiometric microfluorimetry with fura-2 reveals that low doses of Portuguese Man-of-war (Physalia physalis) venom cause a linear increase in intracellular calcium accumulation by cultured L-929 cells. The influx of calcium is preceded by a lag period that is relatively independent of venom concentration, except at very low concentrations. Electron micrographs of negatively stained preparations of membranes from venom-treated L-929 and GH(4)C(1) cells exhibit 10-80 nm diameter lesions. The number and diameter of these lesions correlate with venom concentration. The venom forms lesions in GH(4)C(1) cells at much lower concentrations than in L-929 cells. Osmotic protectants such as sucrose and polyethylene glycol (PEG), reduce the extent of lactate dehydrogenase (LDH) release from venom-treated cells with the higher molecular weight PEG causing a greater inhibition of LDH release than sucrose. These results imply that Man-of-war venom produces pore-like structures in the membranes of target cells, which leads to colloid osmotic swelling with subsequent release of intracellular proteins and cell lysis.

Portuguese Man-of-war (Physalia physalis) venom induces calcium influx into cells by permeabilizing plasma membranes

Portuguese Man-of-war (Physalia physalis) nematocyst venom dose-dependently stimulates calcium (45Ca(2+)) influx into L-929, GH(4)C(1), FRL, and embryonic chick heart cells. Venom-induced calcium influx is not blocked by ouabain, vanadate, nor organic calcium channel blockers, but is blocked by transition metal cations, such as lanthanum and zinc. Venom-induced calcium influx is accompanied in a dose-dependent manner by the release of intracellular lactate dehydrogenase, indicating a loss in plasma membrane integrity and cytolysis. Concentrations of zinc that block 45Ca(2+) influx also block lactate dehydrogenase release. Lanthanum, which also blocks 45Ca(2+) uptake, does not neutralize the cytolytic activity of the venom, but rather inhibits the venoms cytolytic action at the level of the target cell plasma membrane. Our findings indicate that Man-of-war venom causes an influx of calcium into several different cells types, not just those of the cardiovascular system, and this influx likely occurs by permeabilizing the plasma membranes of cells.

Cnidocytes and adjacent supporting cells form receptor-effector complexes in anemone tentacles.

Cnidocytes, the stinging cells of enidarians, discharge enidae (intracellular capsules containing eversible tubules) in response to physical contact combined with the stimulation of specific chemoreceptors. These receptors, occurring in at least two classes, bind N-acetylated sugars and certain amino-compounds, respectively (Thorington and Hessinger, 1988). Colloidal gold coated with bovine submaxillary mucin (mucin-gold) binds exclusively at the surface of the supporting cells which surround enidocytes (Watson and Hessinger, 1986). We now find that mucin-gold sensitizes enidocytes to discharge nematocysts in a dose-dependent manner. In addition, we find that the number of mucin-gold particles appearing at the surface of supporting cells changes over time, and that such changes correlate with the time-course of enidocyte responsiveness. Thus, the discharge of nematocysts by enidocytes may be regulated by the number of receptor-ligand complexes at the surface of the adjacent supporting cells. We conclude that enidocytes and supporting cells constitute receptor-effector complexes. Subsequent to binding at the cell surface, mucin-gold is endocytosed (Watson and Hessinger, 1987a). Multivesicular bodies seem to dispose of the endocytosed mucin-gold at the cell surface rather than via lysosomes. This novel route appears to be the major pathway by which endocytosed mucingold is removed from supporting cells.

Receptor-mediated endocytosis of a chemoreceptor involved in triggering the discharge of cnidae in a sea anemone tentacle

Collodial gold coated with the glycoprotein, bovine submaxillary mucin (BSM-gold), was used to localize chemoreceptors known to be involved in triggering the discharge of cnidae in sea anemones. BSM-gold binds exclusively at the apical surface of the supporting cell, the cell adjacent to the cnidocyte (Watson and Hessinger, 1986). Subsequent to binding, BSM-gold is internalized into endosomes and then translocated to multivesicular bodies (MVBs) and lysosomes. At cold temperature (4 degrees C), BSM-gold appears in endosomes near the surface of the cell but not in endosomes located more medially in the cell, nor in MVBs or lysosomes. The kinetics and sequence of intracellular translocation of BSM-gold were studied by fixing animals at various intervals following incubation in BSM-gold. Unlike that for supporting cells adjacent to non-cnidocytes, the amount of gold at the surface of supporting cells adjacent to penetrant cnidocytes does not seem to change despite considerable internalization of the mucin-probe. Apparently, free receptors replace receptor-ligand complexes in a one-for-one fashion in these cells.

Cnidocil apparatus: sensory receptor of Physalia nematocytes

The cnidocil apparatus, a cluster of subcellular structures at the external surface of the nematocytes of Physalia physalis , was examined by scanning and transmission electron microscopy. The cnidocil apparatus consists of a modified cilium or cnidocil surrounded by 15 to 21 stereocilia. The stereocilia contain closely packed, longitudinally arranged microfilaments. The cnidocil contains numerous singlet and doublet microtubules that lack the classical 9 + 2 microtubule pattern characteristic of most cilia and flagella. The basal body of the cnidocil does, however, maintain the usual circle of nine triplets of microtubules. It is proposed that the cnidocil of the nematocyte is truly the sensory receptor for mechanical and chemical stimuli that elicit nematocyst discharge.

Cellular basis for tentacle adherence in the Portuguese man-of-war (Physalia physalis)

The fishing tentacles of Physalia physalis (Portuguese man-of-war) adhere to prey and human victims by the penetration of a barbed tubule connected to an intracellular nematocyst. The nematocyst is surrounded by a fibrillar system of microtubules and microfilaments that terminate in hemidesmosomal processes which anchor the nematocyst to the acellular mesoglea of the tentacle.

Evidence for calcium channels involved in regulating nematocyst discharge

In the tentacles of sea anemones, nematocyst discharge is regulated by cnidocyte/supporting cell complexes (CSCCs) of which three functional types have been identified: A, B and C. Type A CSCCs respond to contact by vibrating targets. Types B and C CSCCs respond to contact by static targets. Whereas type C CSCCs respond to contact alone, type B CSCCs require that surface chemoreceptors bind ligands before becoming responsive. Reducing Ca2+ levels in artificial seawater to below 1 mM inhibits discharge from each type of CSCC. The calcium channel inhibitors, nifedipine or verapamil, selectively inhibit discharge from type B CSCCs. The calcium channel activator, Bay K-8644, mimics the biphasic dose response of type B CSCCs to natural chemosensitizers such as N-acetylated sugars. Discharge from type A CSCCs is unaffected by inhibitors of L-type calcium channels, but is selectively inhibited by the aminoglycoside antibiotics, gentamicin and streptomycin. While each type of CSCC requires extracellular calcium, the calcium channels employed may vary according to CSCC type.