Origin, Chemistry, and Mechanisms of Action of a Repellent

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P. Lazarovici , N. Primor , J. Gennaro , J. Fox , Y. Shai , P. I. Lelkes , C. G. Caratsch , G. Raghunathan , H. R. Guy , Y. L. Shih , and C. Edwards 7

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Section on Growth Factors, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 Osborn Laboratories of Marine Sciences, New York Aquarium, New York, NY 11224 Department of Biology, New York University, New York, NY 10003 Department of Microbiology, School of Medicine, University of Virginia, Charlottesville, VA 22308 Molecular, Cellular and Nutritional Endocrinology Branch, National Institutes of Health, Bethesda, MD 20892 Laboratory of Cell Biology and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Department of Pharmacology, University of Zurich, 8006 Zurich, Switzerland Laboratory of Mathematical Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 2

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Pardaxin, a marine neurotoxic polypeptide isolated from the secretion of the flatfish Pardachirus marmoratus or synthesized by the solid phase method, is a single chain, acidic, amphipathic polypeptide with the sequence: NH -G-F-F-A-L-I-P-K-I-I-S-S-P-L-F-K-T-L-L-S-A-V-G-S-A-L-S-S-S-G-G-Q-E. Pardaxin is secreted together with a family of steroid aminoglycosides by pairs of cylindrical, acinar glands through secretory ducts to the ocean water. Pardaxin repels sharks and is toxic to marine organisms at 10 -10 M. The target of pardaxin is the gills and the pharynx of aquatic animals. It is suggested that pardaxin interference with the ionic transport of the gill epithelium (10 -10 M) is the main reason for its toxic effects and that its presynaptic activity (10 M) is responsible for the initiation of the escape behavioral reflex underlying its repellent action. On a molecular level pardaxin forms voltage-dependent, cation- and anion-permeable pores at low concentrations (10 -10 M) and causes cytolysis at higher concentrations (10 -10 M). The ionic selectivity properties of pardaxin channels in artificial 2

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Current address: Department of Pharmacology and Experimental Therapeutics, School Pharmacy, The Hebrew University, P.O. Box 1172,91010 Jerusalem, Israel 0097-6156/90/0418-0347S06.00/0 o 1990 American Chemical Society

Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY

membranes suggest ion binding sites in the channel. Models of the pardaxin pore favor an antiparallel oligomer of the helical segments with a narrow, negatively charged entrance due to the carboxy terminal groups. Pardaxin seems to be a suitable tool to investigate the molecular structures underlying channel selectivity and voltage dependence, and the relationship between channel activity, cytotoxicity and repellency to marine organisms.

Moses Sole — Source of Shark Repellents Certain ichthyocrinotoxic fish secrete toxic compounds that repel their predators. Among them, the Red Sea Moses Sole, Pardachirus marmoratus (Figure la,b) exudes a fluid from specialized glands (Figure 1) into the surrounding ocean water. This secretion repels sharks, and it may function as a naturally occurring weapon of defense against shark predation (1,2). The principle factors in the secretion, a polypeptide and a family of steroidaminoglycosides, responsible for both its toxicity and noxious effects in sharks, were isolated, identified, and named pardaxin (5-6) and mosesins (5,7), respectively. The secretion of Pardachirus pavoninus, Peacock Sole, in the Western Pacific, presumably contains similar factors: a family of steroid monoglycosides (pavonins) (7) and ichthyotoxic peptides (8). The Morphology of the Moses Sole Toxins Secretory Apparatus toxins are secreted by a double row of small cylindrical simple acinar glands (Figure lb), one located epaxially and the other hypaxially between each of the fin rays from head to tail (Figure lb). The ventral member of the pair of glands releases its secretion from a pore located on the ventral side of the fish, more peripheral (lateral) to that of its dorsal partner (Figure lc). Each cylinder contains a central channel (Figure lc,f) which is part of the secretory duct and is lined with interdigitating epithelial cells of the type found in glands in which water is withdrawn from the secretion before it is released (Figure lc-e). The outside periphery of the cylinder houses a prominent capillary network and the secretory acini lined by thin secretory cells which surround the acinus, a compartment filled with the secretion. Together these pairs of secretory units comprise an appreciable portion of the body mass of this small flatfish. A light microscope image through a longitudinally sectioned pair of toxin secreting glands shows acini in each gland, although the epaxial gland contains more secreted material than the hypaxial one (Figure lc). TTie secretory duct lies within the plane of the fin ray (the midaxial plane) whose articulation can be seen in the left of the image, which separates both glands. An electron micrograph of one acinus shows a large mass of stored secretory material and, adjacent to this, is the thin cytoplasm of the almost flat secretory cell (Figure le). The secretion is released into the acinar pool from the secretory cell as globules (Figure ld,e), some of which can be seen elevating the plasma membrane. The cytoplasm of an epithelial cell (Figure le) contains mitochondria, smooth endoplasmic reticulum, and an extensive and dense network of intermediate filaments, probably keratin, but lacks secretory granules. The acinus is surrounded by a system of satellite cells (cells with pleomorphic nuclei) applied closely to them with no intervening amorphous basement material. Instead, both amorphous and reticular components of the basement material are peripheral to the satellite cell. An electron micrograph of the epithelium lining the secretory duct shows irregular rounded apical elevations which protrude into the duct space and the complexity of the interdigitations along their lateral boundaries (Figure If). These structures

Pardachirus marmoratus


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Presynaptic Excitatory, Ionophore Polypeptide 349

Figure 1. Pardachirus marmoratus fish and the toxin secretory glands. a,b - Lateral views o f the the gland openings.

fish.

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A r r o w s indicate white secretion around

c - Photomicrograph (20 x ) o f the two toxin secreting glands i n sagittal section. T h e glands (g), are right o f the ray articulation (a) and are filled w i t h secretion. T h e clear space w i t h i n each gland lies i n the secretory duct(s) (d) which end out o f the image to the right. Continued on next page.


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Figure 1. Continued. Pardachirus marmoratus fish and the morphology o f the toxin secretory glands. d - A n electron micrograph (30,000x) o f the glandular secretory epithelium and a p o r t i o n o f o n e acinus. Peripheral to the area o f moderately electron dense material (sc) w h i c h is the toxic secretion(s), is the t h i n secretory epithelial cell (fsc). Several secretory vesicles (bse) can be seen o n its surface, some separated from the contents o f the acinar p o o l o n l y by the thickness o f the plasma membrane. e - A n electron micrograph (80,000x) showing the secretory cell cytoplasm i n detail. T h e fibrillar nature o f the cytoplasmic matrix is visible as well as the smooth (agranular) appearance o f the endoplasmic membranes. M i t o c h o n d r i a (m) can be seen and a p o r t i o n o f o n e o f the plasma membrane elevations w i t h i n which is material o f almost identical granularity and electron density to that i n the acinar p o o l . N o secretory granules are visible i n the cytoplasm. f - This electron micrograph (20,000x) is an image o f the cells l i n i n g the secretory duct (d). T h e i r rounded apical elevations (ae) project into the secretory space and come into contact w i t h the secretion.



Presynaptic Excitatory, Ionophore Polypeptide 351

possess a p e c u l i a r e l e c t r o n density subadjacent t o t h e p l a s m a m e m b r a n e a n d under that a c y t o p l a s m r i c h i n e l e c t r o n dense particles w i t h e n d o p l a s m i c membranes. T h e o t h e r most c o n s p i c u o u s structural feature o f these cells is t h e c o m p l e x n a t u r e o f t h e i r i n t e r d i g i t a t i o n i n t h e r e g i o n o f their lateral boundaries. M a n y mitochondria w i t h p r o m i n e n t cristae are present i n t h e basal regions o f s o m e duct cells. T h i s e n t i r e c o n s t e l l a t i o n o f structures suggests energy p r o d u c t i o n t o s u p p o r t active i o n transport processes.

Pardaxin Sequence, Secondary Structure Predictions, and Modeling in Water Solutions T h e m a i n t o x i c p o r e f o r m i n g c o m p o n e n t o f P. marmoratus secretion, n a m e d pardaxin, was isolated by l i q u i d c o l u m n c h r o m a t o g r a p h y (5). O r i g i n a l l y t w o t o x i c (5) polypeptides, P a r d a x i n I a n d II, were isolated. H o w e v e r , their p r i m a r y sequences have been f o u n d to be identical (6); therefore, the two c o m p o n e n t s most probably represent different aggregates o f o n e polypeptide. T h i s f i n d i n g is i n contrast t o t h e secretion o f P. pavonicus, w h i c h contains three toxic polypeptides (8). P a r d a x i n is a single c h a i n , acidic, a m p h i p a t h i c , h y d r o p h o b i c polypeptide, c o m p o s e d o f 33 a m i n o acids a n d w i t h a mass a r o u n d 3500 daltons (5,6). T h e p r i m a r y sequence is (6) ra -Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Ile-Phe-Lys-ThrLeu-Leu-Ser-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-Gly-Gly-Gln-Glu-COOH. 2

T h e h y d r o p h o b i c m o m e n t p l o t o f pardaxin residue values, based o n t h e c o n sensus h y d r o p h o b i c i t y scale o f E i s e n b e r g (11), indicates that most o f t h e p o i n t s fall o n o r near t h e peptide's surface region, b u t that those f o r its h i g h l y h y d r o p h i l i c carboxy t e r m i n a l l i e i n t h e g l o b u l a r region. T h i s p r o p e r t y is typical o f surfaceseeking, a m p h i p h i l i c proteins w h i c h have large h e l i c a l h y d r o p h o b i c moments, such as m e l i t t i n , Staphylococcus aureus delta t o x i n , a n d o t h e r cytolysins (11). P o s s i b l e secondary structures o f pardaxin (6) were predicted by t h e D e l p h i p r o gram (12) using decision constants ( D H A = -75, D H E = 50) that favor a helices over extended segments a n d by t h e A m p h i p r o g r a m that identified possible a m p h i p a t h i c a helices a n d p strands ( G u y , u n p u b l i s h e d data). D e l p h i predicted that pardaxin segments 1-8 a n d 16-25 were a helices a n d t h e remainder m a y be c o i l s o r turns. A m p h i predicted that segments 1-12 a n d 13-28 c o u l d f o r m a m p h i p a t h i c a helices. P a r d a x i n is p r e d o m i n a n t l y f o u n d i n different o l i g o m e r i c forms i n aqueous s o l u tions (5,6). P a r d a x i n binds deoxycholate, as does m e l i t t i n , a n d most probably dissociates f r o m a tetramer to a m o n o m e r u p o n interaction w i t h detergent micelles ( u n p u b l i s h e d data). T h i s possibility is strongly s u p p o r t e d by t h e results o f crosslinki n g experiments (10), by the s p o n t a n e o u s aggregation seen w i t h gel electrophoresis (5), a n d by t h e greater susceptibility o f pardaxin o l i g o m e r s t o proteolysis i n t h e presence o f deoxycholate ( u n p u b l i s h e d data). T h e molecular mechanism o f the reversible o l i g o m e r i z a t i o n o f pardaxin is unclear a n d requires further experimentat i o n . T h e m e c h a n i s m m a y be t h e same as that responsible f o r t h e aggregation o f typical integral m e m b r a n e proteins i n aqueous buffers i n t h e absence o f l i p i d o r detergents. A p p r o x i m a t e three d i m e n s i o n a l models o f pardaxin aggregates were developed using c o m p u t e r graphics. T h e s e models were then used as starting points for energy refinement calculations to o b t a i n m o r e precise models. I n t h e models presented here ( F i g u r e 2) pardaxin h a d two a h e l i c a l segments, t h e N - h e l i x a n d t h e C - h e l i x c o m p r i s e d o f residues 2-10 a n d 13-27. These segments were assigned h e l i c a l structures for t h e f o l l o w i n g reasons: • P r o l i n e is frequently f o u n d i n t h e first p o s i t i o n o f a helices a n d P r o - 1 3 is i m m e d i a t e l y f o l l o w e d by residues that are frequently observed i n a helices,


352


Figure 2. Opposite. C o m p u t e r graphic views o f pardaxin tetramers and pardaxin channel model. (Top left) T h e front center portions o f the green and yellow monomers are the l o n g amphipathic a helices that cross at an angle o f 1 5 ° predicted by "3-4 ridges-intogrooves" helix packing, as i n the channel model. Helices o f the green and yellow dimer cross those o f the blue and purple dimer by an angle o f 40 ° , consistent with "4-4 ridges-into-grooves" helix packing. This places the two-residue-wide hydrophobic strip o n each dimer next to each other. T h e N-helices o f each m o n o m e r fit into hydrophobic grooves and interact with the hydrophobic strips o n each side o f the tetramer. (Top right) T h e tetramer viewed from the top. T h e transition segment between the N - h e l i x and the C-helix is shown i n yellow and blue and the C-terminus is shown i n green and purple. T h e hydrophobic strips are i n the center and the N-helices are o n each side. T h e tetramer has 2-fold symmetry about each o f the three axes. (Middle left) Side view o f one-half (six monomers) o f the channel as seen from inside the channel. M o n o m e r s with the N - t e r m i n i o n top are white, those with the N termini o n the b o t t o m are green. P o l a r side chain atoms are colored red for negatively charged carboxyls, blue for positively charged amines, p i n k for hydroxyls and oxygen o f amides, and cyan for nitrogen o f amides. N-helices are horizontal and form an outer ring around the entrance o f the channel; C-helices are vertical and form the walls o f the pore. Side chains o f G l n - 3 2 near the C-terminus extend toward the center o f the pore to form narrow i o n selective regions near each entrance. (Middle right) T o p view o f channel entrance formed by residues 1-16 (white outer segments) and residues 31-33 (green inner segments). N o t e central ring formed by amide groups o f G l n - 3 2 side chains (pink and light blue) and salt bridges between carboxy groups o f G l u - 3 3 (red) and amine groups (dark blue). (Bottom) Central region o f the channel formed by residues 17-30. C-helices with N termini p o i n t i n g out o f the page are white, those with N - t e r m i n i p o i n t i n g i n are green. Hydroxyl groups are pink. T h e hexagonal opening is about 11 & angstrom & o n each side.


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Figure 2. See caption p. 352.


353

354


• i n i t i a l m o d e l s i n d i c a t e d that adjacent a helices pack w e l l w h e n t h e C - h e l i x extends to residue 29, and

next

to

each

other

• t h e segment 11-13 (Ser-Ser-Pro) is quite likely n o t to be i n a helical c o n f o r m a t i o n a n d t h e residues that precede a p r o l i n e are n o t usually an a helical conformation. T h e i n i t i a l C - h e l i x was created by a p r o g r a m that generated a helices w i t h the b a c k b o n e structure and side c h a i n c o n f o r m a t i o n s most c o m m o n l y observed for a helices i n crystal structures ( C o r n e t t e , G u y and M a r g a l i t , u n p u b l i s h e d data). This p r o g r a m was n o t a p p r o p r i a t e for an a helix that contains an i n t e r i o r p r o l i n e . T h e i n i t i a l b a c k b o n e structure, the N - h e l i x , was m o d e l e d from t h e a h e l i c a l segment 120-128 i n lactate dehydrogenase (13) w i t h the sequence P H E - l y s - p h e - / / e - I L E - P R O a s n - I L E - F f l / i n w h i c h most residues were i d e n t i c a l o r s i m i l a r to those o f the pardaxin segment 2-10 as indicated by capitals and u n d e r l i n i n g . T h e r e m a i n i n g C t e r m i n u s residues were assigned p h i and psi b a c k b o n e t o r s i o n angles c o m m o n l y observed i n r a n d o m c o i l segments and that extended t h e G l u - 3 3 carboxyl groups i n t o a r e g i o n postulated to be o c c u p i e d by water and near L y s - 8 and Lys-16 o f adjacent helices. Initially, the c o n f o r m a t i o n s o f Ser-11 and Ser-12 were adjusted m a n u a l l y using c o m p u t e r graphics so that N - h e l i c e s c o u l d have apparently favorable interactions w i t h the m e m b r a n e l i p i d and w i t h o t h e r p r o t e i n segments. C o n n o l l y (14) surfaces were added to the structures and m o n o m e r s were m a n u ally d o c k e d to form c h a n n e l structures using the M o g l i p r o g r a m o n an E v a n s and S u t h e r l a n d c o m p u t e r graphics m o n i t o r . C-helices were packed so that C o n n o l l y surfaces f o r m e d a tight barrier between the inside and outside o f the c h a n n e l . M o s t h y d r o p h i l i c groups n o t i n v o l v e d i n i n t e r n a l hydrogen bonds w e r e p o s i t i o n e d to b e exposed to water and most h y d r o p h o b i c groups were p o s i t i o n e d to be i n contact w i t h t h e h y d r o c a r b o n p o r t i o n o f the l i p i d membrane. E n e r g i e s for i n d i v i d u a l dimers created this way were refined w i t h the C H A R M M p r o g r a m (15) u s i n g a d o p t e d basic N e w t o n - R a p h s o n m e t h o d . A c o n v e r s i o n c r i t e r i o n o f .01 A was used for t h e rms gradient d u r i n g a cycle o f m i n i m i z a t i o n . These energy refined dimers w e r e t h e n used to reconstruct aggregates and a final energy refinement was perf o r m e d o n t h e c o m p l e t e aggregate. E a c h m o n o m e r was i n an i d e n t i c a l e n v i r o n m e n t and h a d t h e same c o n f o r m a t i o n . E x p e r i m e n t a l data indicates that i n aqueous s o l u t i o n , pardaxin appears to exist p r i m a r i l y as a tetramer (5) and shows a concentration-dependent o l i g o m e r i z a t i o n (5). O n this basis, a tetramer ( F i g u r e 2) and "raft" models were developed using dimers that have the same p a c k i n g for the C-helices as the c h a n n e l models. T h e major differences a m o n g the m o n o m e r c o n f o r m a t i o n s o f these models i n v o l v e t h e C - t e r m i n u s and residues Ser-11 and Ser-12 that c o n t r o l the relative p o s i t i o n s o f the N - a n d C-helices to each other. In the tetramer the dimers were packed next to each o t h e r so that the row o f large h y d r o p h o b i c side chains o n the C-helices o f each d i m e r p a c k e d next to those o f the adjacent d i m e r ( F i g u r e 2 A , B ) . T h i s p r o d u c e d a structure s i m i l a r to that o f m e l i t t i n tetramers (16). T h e tetramer structure h a d 2-fold symmetry about each o r t h o g o n a l axis ( F i g u r e 2 A , B ) . H e l i c a l interact i o n s between C - h e l i x dimers had "4-4 ridges-into-grooves" type p a c k i n g . T h e N helices w e r e p o s i t i o n e d so they fit i n t o a h y d r o p h o b i c g r o o v e f o r m e d by t h e C helices. T h e N - t e r m i n i o f two N - h e l i c e s a p p r o a c h e d each o t h e r o n each side o f t h e tetramer. A l l h y d r o p h i l i c side chains were exposed o n t h e surface and most h y d r o p h o b i c side chains were b u r i e d . T h i s m o d e l is i n accordance w i t h the u n i q u e p r o p e r t y o f pardaxin to reduce water surface tension (17) at c o n c e n t r a t i o n s m o r e t h a n 10" M , the critical micellar c o n c e n t r a t i o n o f this p e p t i d e i n aqueous s o l u t i o n s ( L e l k e s a n d L a z a r o v i c i , u n p u b l i s h e d data). 6


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Mechanism of Pardaxin-Produced Repellency Location of the Pardaxin Target in Fish.

I n free-swimming sharks, m i x i n g bait w i t h the g l a n d secretion stops the shark from c o m p l e t i n g its b i t e a n d causes it to leave w i t h an a b n o r m a l l y o p e n jaw (18). I n sharks, the m a i n site for c h e m i c a l sensing is l o c a t e d w i t h i n the lateral-line organ o f the head. T o d e t e r m i n e w h e t h e r the response t o pardaxin was mediated t h r o u g h the lateral l i n e system o r v i a the pharyngeal cavity a n d the gills, an apparatus was constructed w h i c h prevented a m i x i n g o f t h e o u t f l o w from shark gills w i t h water b a t h i n g its surface s k i n (19). Pardaxin administered to the m e d i u m bathing the s k i n surface o f the shark's head d i d not elicit the b e h a v i o r a l responses set up by a repellent. A d d i t i o n o f pardaxin to the m e d i u m b a t h i n g the shark's pharynx and gills caused the shark to struggle v i o l e n t l y i m m e d i a t e l y (19). It was rather unexpected to find that the shark's m a i n sensory system, l o c a t e d at the head surface, d i d not respond to pardaxin (19). Furthermore, pardaxin e l i c i t e d its repellent effect ( 1 0 " - 1 0 " M ) o n l y w h e n a p p l i e d i n sea water, a n d s h o w e d n o effect w h e n injected i n t o the c i r c u l a t i o n (20). P r e v i o u s studies have s h o w n that the N a - K A T P a s e o f the gills o f teleost fish is affected by pardaxin (10" —10" M ) and h i s t o p a t h o l o g i c a l effects have been described (21). 6

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Pardaxin Impairment of Ion-Transport of the Opercular Epithelium. P a r d a x i n affects gills i n b o t h teleosts (21) and elasmobranchs (20), and its toxicity is higher i n fish preadapted to a m e d i u m o f h i g h salinity (21). Therefore, its m o d e o f a c t i o n u p o n i o n i c transport by the o p e r c u l a r e p i t h e l i u m o f the k i l l i f i s h Fundulus heteroclitus, w h o s e i o n - o s m o r e g u l a t o r y properties closely resemble those o f teleost gills (22), was e x a m i n e d . It is w e l l established that fish adapted to sea water actively secrete N a C l by means o f specific cells p o s i t i o n e d i n the gills and o p e r c u l a r e p i t h e l i a l s k i n (22). A d m i n i s t r a t i o n o f pardaxin to the m u c o s a l (seawater) side o f the isolated shortc i r c u i t e d o p e r c u l a r e p i t h e l i u m caused a transient s t i m u l a t i o n o f the active transport o f i o n s (1^), f o l l o w e d by an i n h i b i t i o n ( F i g u r e 3). T h e s t i m u l a t i o n was abolished by o u a b a i n and/or r e m o v a l o f N a from the R i n g e r (23). P a r d a x i n d i d n o t affect the 1^ w h e n a p p l i e d to the serosal (blood) side. O n the m u c o s a l side, pardaxin p r o d u c e d a net transient N a current from the m u c o s a l to the serosal side o f 2.2 /xequiv c m " h " . T h e s o d i u m flux was 1000-fold higher than the passive p e r m e a b i l ity measured by the u n i d i r e c t i o n a l i n u l i n fluxes (23). It was c o n c l u d e d that the increased N a influx underlay the s t i m u l a t i o n , and this was suggested to be the m e c h a n i s m responsible for the toxicity o f pardaxin. +

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Pardaxin Evoked Increase of Intracellular C a

2+

in Chromaffin Cells. T h e previously described p o r e f o r m i n g properties i n artificial membranes (5) warranted e x a m i n a t i o n o f the effect o f pardaxin o n intracellular free C a concentrations, using c h r o m a f f i n cells as a m o d e l and the fluorescent dye F u r a - 2 to measure i n t r a c e l l u l a r [ C a ] . T h e basal level o f [ C a ] for these cells is 50-90 n M (28). Stimulation o f the cells w i t h potassium triggers a rapid, transient increase o f [ C a ] (Figure 4). A s expected, i o n o m y c i n , a c a l c i u m i o n o p h o r e , causes a sustained rise i n the free i n t r a c e l l u l a r c o n c e n t r a t i o n to approximately 450 n M ( F i g u r e 4). I n this system, pardaxin i n d u c e d an increase i n intracellular [ C a ] o n l y i n the presence o f extracellular C a ( F i g u r e 4). These results indicate that pardaxin mediated a C a influx but d i d n o t release C a from intracellular stores. T h i s influx is most p r o b ably m e d i a t e d directly by pardaxin channels and possibly also i n d i r e c t l y by activation o f the C a channels o f the c h r o m a f f i n cells by the d e p o l a r i z a t i o n p r o d u c e d by the pardaxin channels (data not shown). These observations further substantiate o u r hypothesis (10) that transmembrane fluxes o f N a and C a are i n v o l v e d i n the p a t h o l o g i c a l a c t i o n o f pardaxin. 2 +

+

2 +

i n

2 +

i n

2 +

2 +

2 +

2 +

2 +

+

2 +


356


0

20 40 T I M E (min.)

60

0

20

40 T I M E (min.)

60

Figure 3. T h e effect o f pardaxin o n the short circuit current o f the isolated epithelial preparations. (Left) Isolated skin o f the frog R. catesbeiana. Pardaxin (6 x 10" M ) was applied to the mucosal side. A D H (5 x 10" M ) was applied to the serosal side. (Right) Isolated opercular skin o f seawater-adapted killifish F. heteroclitus. Pardaxin (10" M ) (full line) and m e l i t t i n (3 x 10" ) (circles) were added (first arrow) to the mucosal (seawater) side o f the skin. Isoprenaline (2 x 10 M ) was added to the serosal side (second arrow). T h e continuous tracing shows the short-circuit current (1^). 5

7

5


5


Presynaptic Excitatory, Ionophore Polypeptide357

Time (sec) Figure 4. Effect o f pardaxin o n intracellular ionized calcium level i n bovine adrenal chromaffin cells. Isolated chromaffin cells were maintained i n suspension culture and loaded w i t h the fluorescent calcium indicator F u r a 2 as previously described (28). 2 x 1 0 cells/ml were added into a cuvette containing standard buffer without (dotted line) or w i t h (full line) 2 m M calcium. A t the arrow, 10" M pardaxin was added. A rise i n [ C a ] was observed only i n the presence o f calcium. Inset, typical control experiment. D e p o l a r i z a t i o n o f the cells by 30 m M K G resulted i n rapid, partially reversible increase i n [ C a ] . Further, addition o f 0 . 1 / i M ionomycin, a calcium ionophore, increased the intracellular calcium level. 5

7

2 +

i n

2 +

i n



358

P r e s y n a p t i c Effects of P a r d a x i n . T h e e p i t h e l i a l s k i n isolated from t h e k i l l i f i s h operc u l u m contains an extensive supply o f b l o o d vessels and nerves o n b l o o d side (serosal) i n close p r o x i m i t y to the e p i t h e l i a l cells; however t h e possible exposure o f nerve terminals o n the m u c o s a l (seawater) side and the detailed ultrastructural m o r p h o l o g y are u n k n o w n . It is possible that the predator repellent a c t i o n o f pardaxin m i g h t i n v o l v e sensory and m o t o r n e u r o n a l pathways i n a d d i t i o n t o i o n i c transport systems. Therefore, we examined the possible effects o f pardaxin o n n e u r o m u s c u l a r transmission using the frog sartorius nerve muscle p r e p a r a t i o n . I n this system, pardaxin ( 1 0 " - 1 0 " M ) p r o d u c e d presynaptic but n o t postsynaptic effects (29). It increased t h e frequency o f the spontaneous release o f transmitter q u a n t a i n a dosedependent and temperature-influenced way u p to m o r e t h a n 100 times the c o n t r o l values. A t the same time the quantal content o f the e v o k e d end-plate potentials was greatly elevated (29). T h e glycosteroids isolated from the gland secretion were relatively ineffective o n neurotransmitter release; however, at h i g h doses they had postsynaptic effects, as s h o w n by a d i m i n u t i o n o f the a m p l i t u d e o f the e v o k e d end-plate potentials. T h e y d i d not reinforce the effect o f the pardaxins. 8

6

A t h i g h e r doses pardaxin d e p o l a r i z e d the postsynaptic membranes, p r o d u c i n g m u s c l e c o n t r a c t i o n s w h i c h c o u l d not be b l o c k e d by ( + ) - t u b o c u r a r i n e o r tetrodot o x i n , and eventually also physical d i s r u p t i o n o f muscle cells. N o effects o n nerve c o n d u c t i o n were observed (29). It is suggested that the pardaxin effects o n nerve terminals are due to the fact that the absence o f m y e l i n makes t h e terminals m o r e susceptible to the toxin and the smaller surface/volume ratio o f t h e t e r m i n a l means that a few channels can p r o d u c e a large effect o n the i n t r a c e l l u l a r i o n i c contents and so o n the m e m b r a n e p o t e n t i a l . In a d d i t i o n , small changes i n the i n t r a c e l l u l a r i o n i c contents and i n the m e m b r a n e p o t e n t i a l o f the nerve t e r m i n a l c o u l d easily p r o d u c e large changes i n the rate o f release o f q u a n t a o f transmitter. This prof o u n d presynaptic activity o f pardaxin led us to suggest that pardaxin may act o n t h e seawater-facing cells o f the gills and/or the pharynx by two mechanisms: (1) repellency ( 1 0 " - 1 0 " M ) as a result o f d e p o l a r i z a t i o n and a c t i v a t i o n o f s e n s o r i m o t o r n e u r o n a l pathways involved i n the escape behavior, and (2) toxicity (10 10" M ) as a result o f the collapse o f the osmoregulatory homeostasis o f the fish. 8

6

4

I o n o p h o r e A c t i v i t y of P a r d a x i n P o r e F o r m i n g A c t i v i t y i n L i p o s o m e s . W h e n a p o r e f o r m i n g factor inserts i n t o the h y p e r p o l a r i z e d m e m b r a n e o f l i p i d vesicles, the existing i o n gradients can p r o d u c e p o t e n t i a l differences. A p o t e n t i o m e t r i c cyanine dye (9) can be used to elucidate s o m e o f the characteristics o f pardaxin p o r e i n p h o s p h a t i d y l c h o l i n e l i p o s o m e s (9). I n this system, pardaxin acts at higher concentrations ( 1 0 " - 1 0 ' M ) , and i n a m o r e c o m p l e x way to p e r m e a b i l i z e the bilayer, c o m p a r e d to g r a m i c i d i n , a w e l l known ionophore. T h i s pardaxin c h a n n e l does not d i s c r i m i n a t e significantly between cations and anions and it is equally effective i f sucrose o r c h o l i n e sulfate replaces the N a S 0 i n the external buffer (9). F r o m the fraction o f d e p o l a r i z e d vesicles a n d the a m o u n t o f radioactive pardaxin b o u n d , it was estimated that 4-12 pardaxin m o n o m e r s are required to form a pore, sufficient to alter the p o t e n t i a l differences across the m e m b r a n e o f o n e vesicle (9). 1 0

2

9

4

A g g r e g a t i o n of P h o s p h a t i d y l s e r i n e Vesicles by P a r d a x i n . Besides f o r m i n g pores i n l i p i d membranes, pardaxin induces aggregation o f p h o s p h a t i d y l s e r i n e vesicles (26), a p h e n o m e n a w h i c h has been visualized w i t h negative contrast e l e c t r o n m i c r o s c o p y (5). T h e pardaxin-induced aggregation is very r a p i d and c o n t i n u e s for an extended p e r i o d o f t i m e (>30 m i n ) . F u r t h e r m o r e , pardaxin-induced aggregation is strongly m o d u l a t e d by the transmembrane and/or surface p o t e n t i a l (26). I n spite o f the extensive aggregation, n o exchange o f lipids between vesicles o r fusion was observed



Presynaptic Excitatory, Ionophore Polypeptide 3 5 9

(26). W e have p r o p o s e d that vesicle aggregation is p r o b a b l y related t o t h e d i s p o s i t i o n o f pardaxin b o u n d i n t h e phosphatidylserine vesicle l i p i d bilayer (26). T h i s c o n c l u s i o n is s u p p o r t e d by t h e observation that p h o s p h a t i d y c h o l i n e vesicles are n o t i n d u c e d t o aggregate a n d that t h e pardaxin-induced phosphatidylserine vesicle aggreg a t i o n is affected by charge p o l a r i z a t i o n o f t h e vesicle (26). T h i s suggestion seems t o b e consistent also w i t h t h e voltage dependence o f fast "pore" activity o f pardaxin, t h e channels w h i c h are o p e n o n l y at positive m e m b r a n e potentials.

Single Channel Recordings and Selectivity of Pardaxin Channels on Planar Bilayers of Phosphatidylethanolamine at the Tip of Patch Pipets.

T o learn about t h e i o n

selectivities o f t h e pardaxin channels, their behavior i n l i p i d bilayers was examined. Bilayers o f p h o s p h a t i d y l e t h a n o l a m i n e were f o r m e d at t h e tips o f m i c r o p i p e t t e s ( F i g u r e 5a) by t h e d o u b l e - d i p m e t h o d (27). A f t e r t h e bilayer was f o u n d t o b e stable, pardaxin was added t o t h e s o l u t i o n o n o n e side, whose p o t e n t i a l was set at + 2 0 mV. T h e currents across t h e bilayer i n t h e voltage c l a m p m o d e were m o n i t o r e d . A t l o w c o n c e n t r a t i o n s o f pardaxin ( 1 0 " - 1 0 " M ) single c h a n n e l events ( F i g u r e 5b) usually appeared w i t h i n 10-20 m i n . A t higher t o x i n concentrations t h e latencies w e r e shorter. A p o t e n t i a l gradient was required for t h e t o x i n m o l e c u l e s t o insert and/or express c h a n n e l activity i n t o t h e membrane; l i t t l e o r n o activity was f o u n d i n t h e absence o f a p o t e n t i a l gradient. 9

8

T o d e t e r m i n e t h e i o n i c selectivity o f t h e pardaxin channels, various i o n substit u t i o n s were p e r f o r m e d a n d t h e b i o n i c reversal p o t e n t i a l , i.e., t h e p o t e n t i a l at w h i c h t h e current across a bilayer w i t h many o p e n pardaxin channels changed sign, was d e t e r m i n e d . T h e relative permeabilities o f t h e ions c o u l d then b e d e t e r m i n e d from t h e general e q u a t i o n :

E

rev -

R

T

(

P

C

C l

[ ll

+

P

A

A,[ ,])

n

F

P

( C I 2l C

+

2

C p C , A p a n d A2 are t h e concentrations o f cations a n d anions o n sides o n e a n d two. I n practice, it was f o u n d that Tris was an i m p e r m e a n t c a t i o n a n d H E P E S an i m p e r m e a n t a n i o n , a n d so salts o f these c o m p o u n d s were used t o simplify t h e e x p e r i m e n t a l c o n d i t i o n s . F o r cations t h e selectivity sequence was 2

+

+

+

+

Tl >Cs >Rb >K ,

NH

+ 4

+

>Li >Na

+

+

w h i c h , except f o r L i , is t h e same as that o f t h e relative hydrated sizes. F o r anions t h e sequence was I" > N O " > B r " > C P > C I O / > S C N " > H C O O - , 3

4

'

w h i c h is q u i t e different from that o f the relative hydrated sizes. Therefore, t h e mechanisms g o v e r n i n g the permeabilities o f cations a n d anions appear t o b e different.

Modeling Pardaxin Channel.

T h e remarkable switching o f c o n f o r m a t i o n i n t h e presence o f detergents o r p h o s p h o l i p i d vesicles (5) suggests that pardaxin is a very flexible m o l e c u l e . T h i s property helps t o e x p l a i n t h e apparent ability o f pardaxin t o insert i n t o p h o s p h o l i p i d bilayers. I n a d d i t i o n , it is consistent w i t h t h e suggestion that t h e d e o x y c h o l a t e - l i k e aminoglycosteroids (5,7) present i n t h e natural secretion f r o m w h i c h pardaxin is purified (5) serve to stabilize its dissociated c o n f o r m a t i o n . T h e q u e s t i o n o f t h e mechanism by w h i c h pardaxin assembles w i t h i n membranes is i m p o r t a n t f o r understanding p o r e f o r m a t i o n a n d its cytolytic activity (5). W e have developed a series o f models i n w h i c h 8-12 m o n o m e r s were p a c k e d i n an a n t i p a r a l l e l m a n n e r t o form t h e c h a n n e l . T h e kinetics o f p o r e f o r m a t i o n i n l i p o s o m e s (9) a n d t h e dependence o f planar bilayer c o n d u c t a n c e o n t o x i n


360


Open

JIIUUJ^^

J

1 pA

Closed

10 sec Figure 5. Scheme of the bilayer formation at the tip of the patch pipets (a) and single channel recordings of pardaxin pores (b). Step 1 - Monolayer of phosphatidylethanolamine is prepared at the air water interface with a glass rod; Step 2 - A patch pipet is removed from the solution, the polar head groups of the monolayer lipids are adsorbed to the interface while the fatty acid hydrophobic tails are exposed to the air; Step 3 - The pipet is reinserted into the liquid, resulting in apposition of the hydrocarbon tails of the attached monolayer to those of the original monolayer, forming a bilayer (27); Step 4 - Addition of the synthetic pardaxin (10 M) to the bath results after 10-20 min in tetramer insertion into the bilayer and pore formations as measured by the current fluctuations depicted in B at a positive potential of 100 mV. The single channel conductance can be estimated from the amplitude of the current steps divided by the applied voltage and was in the range of 10 pS. 9



Presynaptic Excitatory, Ionophore Polypeptide 3 6 1

c o n c e n t r a t i o n yield linear relationships w i t h slopes a r o u n d 8-12. The common i n t e r p r e t a t i o n o f such results is that an aggregate o f 8-12 m o l e c u l e s forms the i o n c o n d u c t i n g c h a n n e l . A n t i p a r a l l e l models appear to be m o r e p r o b a b l e because they allow: 1. 2. 3.

4.

t h e i n t e r a c t i o n o f negative carboxyls o f the C - t e r m i n u s glutamate w i t h p o s i t i v e charges o f Lys-16 and Lys-8; t h e favorable i n t e r a c t i o n o f the b a c k b o n e d dipoles o f adjacent C-helices; t h e o n l y negatively charged p o r t i o n o f the m o l e c u l e , t h e C - t e r m i n u s , t o l i e near to the radial center o f the p o r e w h e r e it c o u l d m a k e the c h a n n e l selective t o cations; and large h y d r o p h o b i c side chains o n the C - h e l i x to pack tightly next to each other.

M o d e l s i n w h i c h the m o n o m e r s are p a r a l l e l , w h i c h w e have previously c o n sidered (10)> appear u n l i k e l y because: (1) a l l models w i t h p a r a l l e l a helices have h i g h l y positively charged regions that s h o u l d strongly reduce c a t i o n permeability; and (2) w e c o u l d find n o parallel models i n w h i c h side chains pack next to each other. T w o ways were f o u n d to pack C-helices i n an a n t i p a r a l l e l m a n n e r to f o r m dimers i n w h i c h all the large alkyl side chains and the p h e n l y a l a n i n e side c h a i n f o r m a h y d r o p h o b i c c o l u m n o n o n e side o f the d i m e r and several serines form hydrogen bonds w i t h each o t h e r o n the o p p o s i t e side o f t h e dimers. I n a d d i t i o n , t h e p o i n t o f closest contact between a - h e l i x backbones is G l y - 2 3 , t h e positively charged Lys-16 extends over the C - t e r m i n u s o f the adjacent a helix w h e r e it interacts favorably w i t h the negative end o f the helix d i p o l e a n d carboxyls o f the t e r m i n u s G l u - 3 3 , and the helices cross each o t h e r at an angle o f - 1 5 * predicted by 3 - 4 ridges i n t o grooves' helix p a c k i n g theory (24). These dimers for the C-helices were used t o construct models o f pardaxin i n s o l u t i o n as a tetramer ( F i g u r e 2, t o p p h o t o g r a p h s ) , o n the m e m b r a n e surface as a "raft-like" structure (data n o t shown), and i n t h e transmembrane o r i e n t a t i o n as a c h a n n e l ( F i g u r e 2, b o t t o m photographs). T h e s e dimers were used to construct several c h a n n e l m o d e l s ( F i g u r e 2, b o t t o m right) that have n a r r o w regions formed by the C - t e r m i n i near t h e i r entrances ( F i g u r e 2, b o t t o m center), a large noncharged p o r e ( F i g u r e 2, b o t t o m center) f o r m e d by t h e C-helices t h r o u g h the m i d d l e o f the membrane, and a positively charged hydrop h o b i c ring a r o u n d the c h a n n e l formed by the N - h e l i c e s ( F i g u r e 2, b o t t o m right). F i g u r e 2 ( b o t t o m center) shows the entrance o f the c h a n n e l f o r m e d by residues 1-16 and 31-33 i n o n e o f these models. T h e s e segments c o n t a i n a l l t h e charged groups. T h e N - h e l i c e s form a h y d r o p h o b i c and positively charged o u t e r ring that surrounds the negatively charged C - t e r m i n i . T h e narrowest p o r t i o n o f the c h a n n e l is f o r m e d by a r i n g o f six a m i d e groups from the G l n - 3 2 side chains. These f o r m a n e t w o r k o f hydrogen bonds w i t h each o t h e r and w i t h the carboxy t e r m i n i . L y s - 8 a n d Lys-16 f o r m salt bridges to the two carboxyl groups o f G l u - 3 3 . A sphere o f diameter 4.8 A c o u l d just pass t h r o u g h this ring. T h i s is large e n o u g h to pass the k n o w n permeant cations but small e n o u g h to exclude Tris, w h i c h is i m p e r m e a n t . T h e central r e g i o n o f this c h a n n e l m o d e l formed by residues 17-30 is s h o w n i n F i g u r e 2 ( b o t t o m right). O n l y serine side chains l i n e the hexagonally shaped pore. T h e p o r e is about 20 A wide. T h e exterior o f the structure w h i c h w o u l d be exposed t o l i p i d contains o n l y alkyl side chains. T h e c h a n n e l is large e n o u g h to c o n t a i n an hexagonally shaped Ice I type water structure (25) i n w h i c h each crosssectional "layer" o f water molecules contains 42 water m o l e c u l e s . T h e hydroxyl groups o f the c h a n n e l l i n i n g may interact w i t h this o r a s i m i l a r k i n d o f water struct u r e that has 3-fold o r 6-fold symmetry.


362


T h i s pardaxin m o d e l is n o t u n i q u e . W e have d e v e l o p e d several s i m i l a r models that are equally g o o d energetically a n d equally consistent w i t h present experimental results. It is difficult t o select a m o n g these models because t h e helices c a n b e p a c k e d a n u m b e r o f ways a n d t h e C - t e r m i n u s appears very flexible. O u r energy c a l c u l a t i o n s are far from definitive because they d o n o t i n c l u d e l i p i d , water, ions, m e m b r a n e voltage, o r e n t r o p y a n d because every c o n f o r m a t i o n a l possibility has n o t been e x p l o r e d . T h e m o d e l presented here is intended t o illustrate t h e general foldi n g pattern o f a family o f pardaxin models i n w h i c h t h e m o n o m e r s are a n t i p a r a l l e l a n d t o d e m o n s t r a t e that these models are feasible. T h e most l i k e l y way for pardaxin molecules t o insert across t h e m e m b r a n e i n an a n t i p a r a l l e l m a n n e r is for t h e m t o f o r m antiparallel aggregates o n t h e m e m b r a n e surface that then insert across t h e membrane. W e developed a "raft"model (data n o t s h o w n ) that is s i m i l a r to t h e c h a n n e l m o d e l except that adjacent dimers are related t o each o t h e r by a linear translation instead o f a 6 0 ° r o t a t i o n about a c h a n n e l axis. A l l o f the large h y d r o p h o b i c side chains o f t h e C-helices are o n o n e side o f t h e "raft" a n d a l l h y d r o p h i l i c side chains are o n t h e o t h e r side. W e p o s t u late that these "rafts" displace t h e l i p i d molecules o n o n e side o f t h e bilayer. W h e n t w o o r m o r e "rafts" meet they c a n insert across t h e m e m b r a n e t o f o r m a c h a n n e l i n a way that never exposes t h e h y d r o p h i l i c side chains t o t h e l i p i d alkyl chains. T h e c o n f o r m a t i o n a l change from the "raft" t o t h e c h a n n e l structure p r i m a r i l y involves a p i v o t i n g m o t i o n about the "ridge" o f side chains f o r m e d by T h r 17, A l a - 2 1 , A l a - 2 5 , a n d Ser-29. These small side chains present few steric barriers for t h e postulated c o n f o r m a t i o n a l change.

Conclusions P a r d a x i n is the p r i n c i p a l p o l y p e p t i d e toxic c o m p o n e n t o f t h e secretion o f t h e flatfish Pardachirus marmoratus (5,6). It is secreted i n t o t h e water by a series o f d o u ble, c l i n d r i c a l acinar glands. A l t h o u g h it is water soluble, at concentrations o f 10" —10" M , this p o l y p e p t i d e spontaneously inserts i n t o artificial o r b i o l o g i c a l membranes t o f o r m voltage-gated pores w h i c h are permeable t o cations a n d anions. A t concentrations higher than 1 0 M pardaxin acts as a lytic agent. B o t h properties p r o b a b l y underly t h e toxicity a n d repellency t o m a r i n e organisms. T h e p r i m a r y sequence o f pardaxin indicates a strong h y d r o p h o b i c segment at t h e a m i n o - t e r m i n a l , f o l l o w e d by an a - h e l i c a l a m p h i p a t h i c region a n d a h y d r o p h y l i c carboxy t e r m i n a l . 11

7

- 7

I n analogy t o a series o f p o l y p e p t i d e c h a n n e l f o r m i n g quasi i o n o p h o r e s , m o d e l o f pardaxin tetramer i n water a n d i n the m e m b r a n e is presented:

a

1.

I n aqueous buffer, pardaxin is c o m p r i s e d o f four a n t i p a r a l l e l m o n o m e r s tightly p a c k e d w i t h 2-fold symmetry o f t h e "4-4 ridges i n t o grooves" type; t h e hydrop h o b i c a m i n o - t e r m i n a l segments o f pardaxin m o n o m e r s are shielded f r o m t h e aqueous surface i n t h e tetramer w h i c h most probably exposes t h e p o l a r side c h a i n t o water.

2.

I n t e r a c t i o n w i t h a l i p i d bilayer driven by a p o t e n t i a l difference a n d by p o l a r and/or h y d r o p h o b i c forces between the a m i n o acid side chains o f t h e pardaxin tetramers a n d t h e p o l a r m e m b r a n e l i p i d head group triggers i n s e r t i o n from a "raft" l i k e structure. I n t h e bilayer o r u p o n i n t e r a c t i o n w i t h detergent micelles, a structural reorg a n i z a t i o n o f pardaxin aggregates takes place, i n w h i c h t h e p o l a r side chains interact w i t h themselves a n d t h e h y d r o p h o b i c residues are externally o r i e n t e d i n t h e pardaxin aggregate, therefore a l l o w i n g interactions w i t h t h e l i p i d backb o n e hydrocarbons. I n this pardaxin o l i g o m e r c h a n n e l , the a helices cross at an angle o f 2 5 ° a n d the m o d e l predicts a h y d r o p h i l i c i n t e r i o r a l l o w i n g for passage o f ions.

3.

4.


27. 5.

6.

LAZAROVICI ET AL.


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The voltage induces the opening of pardaxin pores, or opens them indirectly, by affecting pardaxin oligomers, supposed to be stabilized by dipole-dipole interactions of monomer helices. Most probably the presence of pardaxin pores alters the structure of the bilayer resulting in aggregation of phosphatidylserine vesicles mediated by contact but not by partial merging of their membranes.

Although for the moment this model is only partially supported by experimental data it offers the opportunity to design new experiments which will help to understand the mechanisms of pardaxin insertion and pore formation in lipid bilayers and biological membranes which at a molecular level are the events leading to shark repellency and toxicity of this marine toxin.

Acknowledgment This work was supported in part by Research Grant N-00014-82-C-0435 from the Department of Defense, Office of Naval Research, United States Navy and Koret Foundation, San Francisco.

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RECEIVED August 18, 1989


Origin, Chemistry, and Mechanisms of Action of a Repellent

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