Differences in primary structure among five phospholipases A₂ from Heloderma suspectum

Abstract

Five increasingly anionic phospholipases A, (Pal -Pa5) exist in the venom of the lizard Heloderma suspectum. We recently elucidated the sequence of Pa,, the most abundant and most active variant, towards emulsified phosphatidylcholines. Here we present the primary structures of Pa,, Pa3 (subvariants a and b) and Pa,, based on Edman degradation of tryptic, endoproteinase Arg-C and chymotryptic fragments of the reduced and Scarboxymethylated proteins. Pa, -Pa,, considered collectively, belong to an original class of secretory phospholipases A, with 141 -143 residues, a short hydrophobic N-terminus, 10 half-cystine residues and an extended C-terminus. The only known phospholipase A, with characteristics close enough to be a member of the same class is that present in the venom from the insect Apis mellifera.More specifically, the sequences of Pa3 and Pa5 are almost identical, and those of Paz and Pa4 are also quite similar. Both groups diverge enough to indicate the translation of two mRNA species in the venom gland. The primary structure of Pa, reveals the existence of subvariants a and b, the sequence of which is identical to that previously defined for Pa5, except that the C-terminal tripeptide GEG in Pa5 is replaced by the dipeptide GE in Pa3, and the tetrapeptide GEGR in Pa,,,. Pa,, when compared to Pa5, shows 21 substitutions with a cluster of five modified amino acids in positions 40 -44, immediately after the catalytic segment amino acids 30 -39, and added changes scattered before the C-terminus. Paz differs from Pa, only by the absence of the Gly142 C-terminal residue. The 15% difference in primary structure observed between the Pa3 -Pa5 and Paz -Pa, subgroups might be largely responsible for their distinct biological properties.In 1984, Vandermeers et al. [l, 21 purified from the venom of the North American lizard Heloderma suspectum (Gila monster) a calcium-dependent phospholipase A,, later called Pa, [3], that induces high amylase release from dispersed rat pancreatic acini. In 1989, four more isoenzymes (from the same venom) were purified to homogeneity [3]. The specific activity of the increasingly anionic Pal -Pa, enzymes, tested by their capacity to hydrolyze phosphatidylcholines in the presence of Triton X-100, at the optimum pH of 9.0, decreases as follows: Pa3 > Pa, > Pa4 > Pal > Pa,. All variants stimulate amylase release from dispersed rat pancreatic acini, at pH 7.4, with the following decreasing potency: Pa, > Pal z Pa, > Pa3 =Pa5, in an inverse relationship to the k,,, of enzymatic activity. This is why Pa,, which efficiently induces amylase release, was called pancreatic secretory factor when initially purified in 1984 [l, 21. We have already established the full sequence of Pa5, the most abundant variant [3]. Its 142-amino-acid sequence conCorrespondence to J. Christophe,

Novel Human Secreted Phospholipase A2 with Homology to the Group III Bee Venom Enzyme

“…Interestingly, the hGIII domain is more similar to venom group III sPLA 2 s identified from vertebrates. Indeed, higher levels of identity are found with the isoforms PA-2 and PA-5 (43 and 46%, respectively) purified from the lizard Gila monster (27), whereas lower levels are observed with venom group III sPLA 2 s from honey bee, bumble bee, and the scorpion Pandinus imperator (Fig. 2).…”

“…This sPLA 2 has been cloned (20), and determination of its three-dimensional structure (11) has revealed important differences with group I and II sPLA 2 s, although the catalytic site is similar to that of vertebrate sPLA 2 s (13). Interestingly, sPLA 2 s similar to the bee venom enzyme were discovered in lizard venom (26,27), indicating that group III sPLA 2 s also exist in vertebrates and thus may occur in mammals as well.…”

Localization of Structural Elements of Bee Venom Phospholipase A2 Involved in N-type Receptor Binding and Neurotoxicity

“…A second set of sequence alignments was made with group III sPLA 2 s, since bvPLA 2 is a typical member of this group and since it also binds with very high affinity and specificity to N-type receptors. Other members of group III sPLA 2 s have been purified from the venom of the Gila monster lizard H. suspectum (45,46) and of the Mexican beaded lizard Heloderma horridum horridum (47) as well as from the mediterranean medusa Rhopilema nomadica (48). Of interest for our study are the two sPLA 2 s, namely Pa2 and Pa5, which are major components of Gila monster (H. suspectum) venom and which have been entirely sequenced (46).…”

“…Other members of group III sPLA 2 s have been purified from the venom of the Gila monster lizard H. suspectum (45,46) and of the Mexican beaded lizard Heloderma horridum horridum (47) as well as from the mediterranean medusa Rhopilema nomadica (48). Of interest for our study are the two sPLA 2 s, namely Pa2 and Pa5, which are major components of Gila monster (H. suspectum) venom and which have been entirely sequenced (46). These two sPLA 2 s display an overall identity of 38% with the bvPLA 2 , the level of identity increasing to 58% when the Ca 2ϩ -binding loop and the active site region were compared.…”

Snake‐venom phospholipase A2 neurotoxins

“…Secreted PLA2s are abundant in mammalian pancreas, snake venoms and many other tissues [l]. According to their sequences, they have been grouped into three classes [2,3]: group I contains PLA2s from mammalian pancreas and from Hydrophiidae or Elapidae venoms ; group 11 contains mammalian non-pancreatic PLA2s and PLA2s from Viperidae (Crotalinae and Viperinae) venoms; group 111, PLAzs from Apis mellifera and Heloderma venoms.…”