J Mol Evol (1990) 31:228-238

Journal of Molecular Evolution @ Springer-VerlagNew York Inc. 1990

Evolutionary Relationships and Implications for the Regulation of Phospholipase A2 from Snake Venom to Human Secreted Forms Florence F. Davidson and Edward A. Dennis Department of Chemistry and the Center for MolecularGenetics, Universityof California at San Diego, La Jolla, California 92093, USA

Summary.

The amino acid sequences of 40 secreted phospholipase A2's (PLA2) were aligned and a phylogenetic tree derived that has three main branches corresponding to elapid (group I), viperid (group II), and insect ve nom types of PLA2. The human pancreatic and recently determined nonpancreatic sequences in the comparison align with the elapid and viperid categories, respectively, indicating that at least two PLA2 genes existed in the vertebrate line before the divergence of reptiles and mammals about 200-300 million years ago. This allows resolution for the first time of major genetic events in the evolution o f current PLA2's and the relationship o f human PLAE'S to those o f snake venom, many o f which are potent toxins. Implications for possible mechanisms of regulation of m a m m a lian intra- and extracellular PLAa's are discussed, as well as issues relating to the search for the controlling enzymes in arachidonic acid release, prostaglandin generation, and signal transduction.

Key words: Phospholipase

A 2 --

Snakes -- Pan-

creas -- Bees

Introduction Phospholipase A2 (PLA2) is an enzyme whose catalytic function is to cleave phospholipids at the sn-2 position o f the glycerol backbone (Dennis 1983). The products, free fatty acid and lysophospholipid, exhibit a number of biological actions, depending in large part on the location in which they are generated. In the gastro-intestinal tract, PLA2 that has Offprint requests to: E.A. Dennis

been secreted by the pancreas breaks down phospholipids for dietary adsorption (Verheij et al. 1981). However, in other mammalian tissues, arachidonic acid released by PLA 2 serves as the c o m m o n precursor to the leukotrienes and prostaglandins involved in inflammatory diseases (Dennis 1987). In addition, lysophospholipids are potent biological detergents that have been proposed to function in a variety of physiological events, including cell fusion (Stafford and Dennis 1988). PLA2's are undoubtedly involved in modulation of membrane composition and signal transduction (Dennis et al. 1989) and their regulation is key to many aspects o f homeostasis. Homeostasis o f cellular membranes was probably one of the earliest functions of PLA2. Now, however, secreted PLA2's have acquired an impressive array of pharmacological and higher-order digestive actions. All o f the PLA2"s that have been structurally characterized so far are extracellular forms, the vast majority isolated from the venoms o f snakes. Snake venom glands are thought to have evolved from digestive glands, because of their location and morphology and their secretion of a great variety of lyric enzymes. Indeed, there is a high degree o f similarity between mammalian pancreatic PLA2 and many venom PLA2's. However, it will be shown here that at least two distinct PLA2 genes already existed at the time that the ancestral lines o f reptiles and mammals diverged, before snakes arose. The two major series ofpoisonous snakes, the Proteroglypha (fixed front-fanged) and Solenoglypha (movable frontfanged), each conscripted a different one of these two PLA2 genes for use in the production of their venoms, and only one of them corresponds to the currently expressed mammalian pancreatic-type PLA2.

229 PLA2 s e q u e n c e s h a v e p r e v i o u s l y b e e n classified as belonging to g r o u p s I or II o n the basis o f Cys Positions ( H e i n r i k s o n et al. 1977). S u b s e q u e n t sequence c o m p a r i s o n s h a v e resulted in t w o types o f e v o l u t i o n a r y trees ( D u f t o n a n d H i d e r 1983) a n d stmilarities a m o n g the t w o v e n o m g r o u p s h a v e b e e n determined. H o w e v e r , at the t i m e t h a t t h o s e trees Were m a d e , o n l y a few s e q u e n c e s were f r o m g r o u p II. M a n y m o r e s e q u e n c e s f r o m g r o u p II h a v e n o w b e c o m e available, i n c l u d i n g o n e f r o m a h u m a n SOurce. W i t h t h e aid o f these, a n d access to n e w e r c o m p u t e r a p p r o a c h e s (Feng a n d D o o l i t t l e 1987), we have g e n e r a t e d a s e q u e n c e a l i g n m e n t o f 37 n o n r e dUndant P L A 2 sequences, plus 3 i s o e n z y m e s . N o table sequences i n c l u d e the h o n e y b e e a n d Naja naja naja v e n o m P L A 2 ' s , the n e w h u m a n g r o u p II sequence, a n d six P L A 2 h o m o l o g s t h a t are i n a c t i v e b y themselves, b u t f o u r o f w h i c h serve as c h a p e r o n e s , or inhibitors o f p o t e n t l y toxic, a c t i v e PLA2's. F r o m the a l i g n m e n t , an e v o l u t i o n a r y tree was d e r i v e d . The tree is the first to correlate with the m a j o r diVisions o f v e r t e b r a t e p h y l o g e n y , t h e r e b y a l l o w i n g new insights i n t o P L A 2 r e l a t i o n s h i p s t h a t were n o t Possible using o l d e r d a t a bases. T h e s e i n c l u d e a clearer u n d e r s t a n d i n g o f the c o n t r i b u t i o n o f P L A 2 gene d u p l i c a t i o n s to the e v o l u t i o n o f c u r r e n t PLA2's. By integration o f the e x p e r i m e n t a l results herein With w h a t is n o w k n o w n a b o u t P L A 2 f u n c t i o n s , p o s Sible selective pressures i n v o l v e d in PLA2 e v o l u t i o n have b e e n c o n s i d e r e d . T h e results suggest t h a t true intracellular r e g u l a t o r y PLA2"s will n o t be f o u n d Solely using p r o b e s b a s e d o n s e q u e n c e s o f secreted PLA2's, a n d o t h e r m e t h o d s will be n e e d e d to isolate these f o r m s .

Experimental Procedures The PLA2 sequences were obtained from the National Biomedical Research Foundation--Protein Identification Resource Protein Sequence Database, release 15.0, with the exception of the follOWing,obtained from the sources indicated: N. n. naja (Davidson and Dennis 1990a), human pancreatic (Seilhamer et al. 1986), Naja naja oxiana (Dufton and Hider 1983), and Aspidelaps SCutatus CMII Ooubert 1987) PLA2's, vipoxin PLA~, and its inhibitor (Mancheva et al. 1987), Agkistrodon halys blomhoffi basic PLA~ (Forst et al. 1986a), Agkistrodon piscivoruspiscivorus D49 and K49 (Maraganore and Heinrikson 1986), Laticauda semifasciata LsPLA_, I (Takasaki et al. 1988), and human nonpancreatic PLA2 (Kramer et al. 1989). The N-terminal octapeptide e~tensions of taipoxin "r and the pancreatic proenzymes were not USed in generating the alignment. The alignment was generated using the progressive alignment method (Feng and Doolittle 1987). The similarity weighting scale Was a modified version of DayholTs log odds matrix that has been reported previously (Feng et al. 1985). Progressive alignment uses the Needleman-Wunsch pairwise alignment algorithm iteratively in order to achieve multiple alignments. From the multiple alignment, a difference matrix was derived for construction of the evolutionary tree by Fitch and Margoliash's method (Fitch and Margoliash 1967) as implemented by D.-F. Feng.

Progressive alignment differs from other alignment methods primarily by starting with what is determined to be the closest sequence pair and aligning all else relative to that pair. Alignments are made following the order of closest sequences first, and the rule that once a gap is generated it is retained by the insertion of neutral characters. The method therefore places greater confidence in the comparison of recently diverged sequences over the more distantly related members of the set. It has the advantage of being sufficiently streamlined to allow complete generation of the multiple alignment by computer, without having to resort to subjective adjustments. In the present alignment, all comparisons except one were generated without additional weighting factors. A weighting factor of 3 had to be used, starting with the 35th sequence, in order to allow a sufficient N-terminal gap to be generated at the beginning of the 40th, bee venom, sequence so that alignment of its active site with those of the other sequences could be achieved (see Results). For this reason, the placement of the first two bee Ile with conserved Ile of the other sequences may not be significant. The weighting factor did not change the alignments or branching order of the tree for the other 39 sequences, as confirmed by the fact that a totally nonweighted comparison in the absence of the honeybee sequence gave virtually identical results. All the same residues in the region after C59 are conserved as when the bee venom sequence is omitted.

Results A m i n o Acid Sequence Alignments F o r t y a m i n o acid sequences, s h o w n in Fig. 1, were selected f r o m an available d a t a base o f 57 P L A 2 ' s a n d h o m o l o g s . Because o f c o n s i d e r a t i o n s o f c o m p u t e r time, it was desirable to r e d u c e the n u m b e r o f c o m p a r e d s e q u e n c e s as far as possible while still reflecting the existing range o f types. T h u s , with o n e exception, n o m o r e t h a n o n e P L A 2 s e q u e n c e w a s i n c l u d e d per species unless there w e r e d e m o n s t r a t e d f u n c t i o n a l differences b e t w e e n t h e m ( D a v i d s o n a n d D e n n i s 1990b). I n the case o f L. semifasciata, t w o n e u r o t o x i c P L A z ' s were i n c l u d e d p r i m a r i l y b e c a u s e o n e o f t h e m is a n u n u s u a l acidic n e u r o t o x i n . Also, in three instances, v e r y closely related P L A 2 ' s f r o m t w o different species w e r e used, b u t otherwise, enz y m e s t h a t differed b y less t h a n 10 a m i n o acids a n d h a d n o k n o w n f u n c t i o n a l differences were d r o p p e d f r o m the c o m p a r i s o n . T h e three e x c e p t i o n s w e r e m a d e in the cases o f Crotalus atrox a n d Crotalus adamanteus, N. n. naja, a n d Naja naja atra, a n d two pancreatic enzymes, one human and one bovine. T h e s e three d u p l i c a t i o n s a n d the L. semifasciata fall i n t o different m a j o r s u b g r o u p s o f P L A 2 ' s a n d t h u s s h o w the t y p e s o f distances to be e x p e c t e d b e t w e e n t h o s e close P L A ~ ' s t h a t w e r e o m i t t e d f r o m the analysis. O f five N. n. spp. s e q u e n c e s available, the t w o c h o s e n were d e t e r m i n e d b y a p r e l i m i n a r y d i s t a n c e analysis to be the furthest apart. T h e results o f the m u l t i p l e a l i g n m e n t are s h o w n in Fig. 1. I n all the a c t i v e extracellular PLAE'S, 18 residues are a b s o l u t e l y c o n s e r v e d , i.e., Ile 9, T y r 25, G l y 27, Cys 31, G l y 32, G l y 34, A s p 50, Cys 52, C y s 53, H i s 56, A s p 57, Cys 59, T y r 60, T y r 81,

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~U g~ 40%) of the extracellular P L A z gene products, whose anCeStor genes seem to have diverged m o r e than 200 million years ago. Notably, the bee v e n o m sequence, which has diverged prior to the emergence of Vertebrates, has far lower overall identity with the Other PLAz's. An intracellular PLAz, whose

ancestor had fewer or no disulfides and m a y be pres u m e d to have diverged even m o r e anciently, m a y have a sequence that is unrecognizable as a PLA2. If, then, PLA2's do exist w h i c h are fully exposed to a reducing e n v i r o n m e n t in the cell interior, their characterization m a y be expected to reveal a totally new p r i m a r y structure. In support o f this, m a c r o phage-like cells contain both a cytosolic PLA2 a n d a m e m b r a n e - a s s o c i a t e d PLA2 with different inhibitor profiles than the secreted PLA2"s discussed herein (Lister et al. 1989). Thus, it will be o f great interest to determine the sequence o f the m a m m a l i a n cytoplasmic PLA2's to see if they are very different f r o m the e x t r a c e l l u l a r - t y p e e n z y m e s d i s c u s s e d herein.

Note added in proof." Subsequent to preparation of this manuscript, an article [Kuchler K, Gmachl M, Sippl MJ, Kreil G (1989) Analysisof the cDNA of Phospholipase A2from honeybee venom glands. The deduced amino acid sequence reveals homology to the corresponding vertebrate enzymes. Eur J Biochem 184:249254] appeared that reported the sequence of the bee venom enzyme based on its c-DNA and found several differences from that originally published by Shipolini et al. [Shipolini RA, Callewaert GL, Cottrell RC, Vernon CA (1971) The Primary Sequence of Phospholipase-A from Bee Venom. FEBS Lett 17:3940]. The new sequence differs in the assignment of six residues and contains an additional six residues which are missing in the original sequence. The change in these twelve residues alters the alignment of the bee venom enzyme after the active-site His Asp pair and results in more conserved residues than are reported here, including Asp 108. The new residues include two additional Cys, making a total of ten rather than the eight originally found, and implying five rather than four disulfide bonds. In this case, three of these disulfide bonds are aligned with the extracellular PLAz's, This clarifies some of the ambiguity noted in Table 1. Thus, the new bee venom sequence suggests a slightly altered alignment for this enzyme which would give it a greater homology to the group I and II PLA2's.This finding strengthens one of the overall conclusions of this manuscript that the bee venom PLA2 is divergently related to the vertebrate PLA2's.

Acknowledgments. We thank Professor Russell Doolittle, Dr. Marcella A. McClure, and Dr. Da-Fei Feng for use of their computer programs and constructive help in their utilization. We thank Dr. Ruth Kramer for generously allowing us to utilize the sequence of the nonpancreatic PLA: in advance of publication. Critical reading of the manuscript by Dr. Laure Reynolds and Ray Deems is appreciated. Support for this work was provided by NIH grant GM20,501 and NSF grant DMB89-17392. F.F.D. was a predoctoral fellowof the National Cancer Institute (training grant CA09523).

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Evolutionary relationships and implications for the regulation of phospholipase A2 from snake venom to human secreted forms.

The amino acid sequences of 40 secreted phospholipase A2's (PLA2) were aligned and a phylogenetic tree derived that has three main branches correspond...
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