Marine Toxins - ACS Publications - American Chemical Society

Chapter 23

Cytolytic Peptides of Sea Anemones Alan W. Bernheimer

Downloaded by RUTGERS UNIV on December 28, 2017 | http://pubs.acs.org Publication Date: January 29, 1990 | doi: 10.1021/bk-1990-0418.ch023

Department of Microbiology, New York University School of Medicine, New York, NY 10016 The characteristics and possible mechanisms of action of cytolytic peptides isolated from sea anemones during the past 20 years are described. These agents fall into three categories: (1) sphingomyelin-inhibitable basic peptides of molecular weights between 15,000 and 21,000, present in at least 16 species; (2) metridiolysin, a cholesterol-inhibitable, thiol-activated peptide of molecular weight ca. 80,000, known so far to be present only in Metridium senile; (3) Aiptasiolysin, a hemolytic system involving the cooperative action of phospholipase A and two other proteins, known so far to be present only in Aiptasia pallida. At a conservative estimate about 65 cytolytic peptides have been isolated from natural sources and characterized (2). They are found (a) as products of a variety of bacterial species, frequently although not always pathogens, and most commonly extracellularly, but sometimes intracellularly, (b) as constituents of the basidiocarps of various kinds of higher fungi, (c) as components of snake venoms, (d) in the venoms of hymenopteran insects, (e) in a variety of marine invertebrates, particularly cnidarians, and (f) in at least one vertebrate, the flatfish Pardachirus marmoratus. Among the most thoroughly studied cytolytic peptides are melittin from the honey bee, staphylococcal alpha-toxin, and the thiol-activated cytolysins of streptococci and other bacteria. The present survey is concerned with cytolytic peptides occurring only in sea anemones. However, the existence of cytolytic peptides or proteins of importance to public health, in the Atlantic Portugese Man-of-war (Physalia physalix) and in the North Australian coastal cubomedusan, Chironex fleckeri, is worthy of mention in passing. Much attention has been devoted to anemone peptides having molecular weights in the range of 2700 to 7000, especially because of their inotropic effect on heart muscle and because of their potential usefulness in the design of agents for the treatment of cardiopathies. In our experience these peptides are not cytolytic, but at least one has been reported to be (2). It is evident that these peptides constitute a class of agents distinct from those considered in this paper. Among several thousand species of sea anemones the toxins of only a few dozen have been investigated. Knowledge of the sea anemone peptides permits them to be categorized as (a) sphingomyelin-inhibitable cytolysins, (b) metridiolysin, and (c) Aiptasia lysin.

Sphingomyelin-inhibitable Group Cytolytic peptides derived from 16 species of anemones currently comprise this group (Table I). It is likely that as additional species are examined in the future

0097-6156/90/0418-0304506.00/0 o 1990 American Chemical Society

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


23.

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Cytolytic Peptides of Sea Anemones

Table I. Sphingomyelin-inhibitable Peptides

Proposed Name

Source

Detection and/or Characterization Reference No.

Gigantolysin Kentolysin Equinolysin Heliantholysin Lofotensolysin Epiactolysin A Epiactolysin B Xantholysin Carilysin Koseirolysin Gyrolysin Variolysin Flagolysin Michaelsenolysin Parasolysin Japonicolysin Felinolysin

Condylactis gigantea Stoichactis kenti Actinia equina Stoichactis helianthus Tealia lofotensis Epiactis prolifera Epiactis prolifera Anthopleura xanthogrammica Actinia cari Radianthus koseirensis Gyrostoma helianthus Pseudactinia varia Pseudactinia flagellifera Anthopleura michaelseni Parasicyonis actinostoloides Anthopleura japonica Tealia felina

(3,4) (5,6) (7,8) (9-11) (12) (12,13) (12,13) (12) (14) (15) (15) (16) (16) (16) (17) (17) (18)

S O U R C E : Adapted and reproduced with permission from Ref. 26. Copyright 1986 Pergamon Journals, L t d .


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MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY


other toxins w i l l b e added. Individual members o f this group have been characterized t o various degrees. T h e first t o b e described was t h e lysin o f Condylactis gigantea (3) a n d t h e most thoroughly studied is heliantholysin from Stoichactis (Stichodactyla) helianthus. A l l are single, basic polypeptide chains having molecular weights between 15,000 and 21,000. They are broadly similar i n amino acid c o m p o sition but exhibit individual differences. Because o f convenience and rapidity, capacity t o lyse mammalian erythrocytes i n vitro is c o m m o n l y used for assaying activity, but it is clear that these peptides are also toxic for other cells, such as cultured fibroblasts and b l o o d platelets. S o m e o r a l l are lethal for w h o l e animals such as mice, rats, and crustaceans. T h e i r biological effects, o r more accurately those that have been examined, are specifically inhibited by l o w concentrations o f sphingomyel i n (Table II). H e l i a n t h o l y s i n . T h e major form o f heliantholysin is a basic polypeptide chain (pi i n the region o f 9.8) having a molecular weight o f 16,600. Its amino acid sequence has been determined (11). It is powerfully hemolytic for washed erythrocytes derived from a variety o f animals, those o f the cat being t h e most sensitive, and those o f the guinea p i g the most resistant (10). A s is true o f most hemolytic systems, t h e biochemical basis for the very large differences i n sensitivity o f erythrocytes from different animal species is u n k n o w n . T h e capacity o f sphingomyelin to inhibit the hemolytic activity o f heliantholysin suggests that this l i p i d may b e the receptor for t h e toxin. This idea is supported by the fact that prior treatment o f erythrocytes w i t h sphingomyelinase renders them resistant t o lysis. Washed erythrocyte membranes also inhibit the toxin from lysing red cells, and prior treatment o f membrane suspensions w i t h enzymes k n o w n to destroy sphingomyelin abolishes the i n h i b i t i o n (10). Lysis appears t o depend u p o n the formation o f transmembrane channels that are formed by aggregation o f toxin molecules i n t h e bilayer (19-20). There are observations suggesting that lysis may also occur by way o f a detergent-like action (21). K e n t o l y s i n C o m p a r e d to H e l i a n t h o l y s i n . Stoichactis helianthus occurs i n t h e Caribbean region whereas another species, Stoichactis kenti is distributed i n t h e IndoPacific area. T h e latter produces a toxin, kentolysin, that is similar t o , b u t n o t identical w i t h heliantholysin (6). T h e amino acid compositions o f the two polypeptides show a distinct resemblance but appear t o differ significantly i n number o f residues o f lysine, methionine, tyrosine and histidine. I g G from a rabbit i m m u n i z e d against heliantholysin neutralizes b o t h heliantholysin and kentolysin, but neutralizat i o n o f the homologous toxin is more efficient (Table III). It can b e seen that i n the concentrations used, t h e I g G failed t o neutralize t h e related lytic peptides o f Condylactis gigantea and Epiactis prolifera. M u l t i p l e Toxins from a Single Anemone Species Stoichactis (Stichodactyla) helianthus. It has recently been shown by C M - c e l l u l o s e chromatography that heliantholysin consists o f four isotoxins having different Nterminal amino acid sequences ( K e m and D u n n , i n press). Designated I t o I V i n order o f increasing isoelectric p H , toxins I and II had o n e additional amino acid at the amino terminus than d i d toxin III. T o x i n I V had a seven residue extension at the amino terminus relative t o toxin III. T o x i n ni and toxin I I contributed 83% and 14% o f the total hemolytic activity, respectively, and toxins I and I V together about 3%. Epiactis prolifera. Extracts o f this relatively c o m m o n species contain t w o toxins, epiactolysins A and B (13). They have the same molecular weight, namely about 19,500, b u t differ i n pi values as determined by isoelectric focusing. B o t h lack


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Table II. Inhibition of Heliantholysin by Sphingomyelin Amount required to inhibit 2/3 the test amount* of toxin (pg)


Inhibitor

>500 >500 >500 >500 >500 >500 1.5 2.0 70 35 >500 >500 >500 >500

Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Phosphatidylinositol Phosphatidylglycerol Diphosphatidylglycerol Sphingomyelin (prep. A ) Sphingomyelin (prep. B ) Ceramide (prep. A ) C e r a m i d e (prep. B ) Sphingosine Cholesterol Cerebrosides Partially purified brain gangliosides Chloroform—methanol-extracted brain lipids H u m a n serum a - G l o b u l i n fraction o f h u m a n serum ^ - L i p o p r o t e i n fraction o f h u m a n serum H u m a n serum albumin Concavanalin A b

2.5 0.008 5 10 >500 >500 c

SOURCE: R e p r o d u c e d w i t h permission from R e f . 10. Copyright 1976 N a t i o n a l Academy o f Science. Three hemolytic units. T w o additional specimens from commercial sources gave the same result. Measured i n pL. a

b

c

Table III. Neutralizing Capacity of Anti-helianthin IgG for Helianthin, Kentin and Lysins from Other Sea Anemones

Source of cytolysin S. helianthus S. kenti C. gigantea E. prolifera E. prolifera

(epiactin A) (epiactin B )

Volume of IgG solution required to neutralize 2 hemolytic units of test lysin (mL)

Reciprocal of initial dilution of IgG solution required to neutralize 2 hemolytic units of test lysin

0.006 0.029 >0.1 >0.1 >0.1

167 34

Marine Toxins - ACS Publications - American Chemical Society

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