Structural elements of Trimeresurus flavoviridis serum inhibitors for recognition of its venom phospholipase A₂ isozymes

Abstract

Five inhibitors (PLI-I^V) against Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) venom phospholipase A P (PLA P ) isozymes have been isolated from its serum. PLI-I, which is composed of two repeated three-finger motifs, and PLI-IV and PLI-V, which contain a sequence similar to the carbohydrate recognition domain (CRD) of C-type lectins, were expressed in the forms fused with glutathione S-transferase (GST). The resulting GST-PLIs showed ability to bind to three Tf venom PLA P isozymes. The binding study with the truncated forms indicated that one of two three-finger motifs of PLI-I was able to bind to PLA P isozymes. The N-terminal 37-amino acid fragment and the CRD-like domain of PLI-IV and PLI-V were bound to PLA P isozymes. On the other hand, their C-terminal 12-amino acid segment also associated with PLA P isozymes. When either of two units of a hydrophobic tripeptide in this sequence was replaced by trialanine, the binding was completely abolished, indicating that the C-terminal hydrophobic cores of PLI-IV and PLI-V were critically responsible for the binding to venom PLA P isozymes.z 1998 Federation of European Biochemical Societies.

Mapping the Region of the α-Type Phospholipase A2 Inhibitor Responsible for Its Inhibitory Activity

“…Therefore, residues 13-36 of GbPLI␣ are important both in binding of acidic PLA 2 and in trimerization of PLI␣ subunits. It should be noted that the N-terminal 37-amino acid fragment of P. flavoviridis PLI␣ was reported to bind directly to its venom PLA 2 isoenzymes (22). As was depicted in Fig.…”

“…However, the loss of PLA 2 inhibitory activity in EqPLI␣-LP could not be rationalized by the replacement of residues 136 -147, because EqPLI␣-LP was found to exhibit the inhibitory activity solely by replacing residues 13-36 with those of GbPLI␣. Moreover, they reported that the N-terminal fragment (residues 1-37), and the CTLD fragment (residues 38 -147) of P. flavoviridis PLI␣ also bound to the PLA 2 isozymes, although their affinities and stoichiometry were not quantified (22). Because Eq37Gb and GbCTLD lost the binding and inhibitory activities toward PLA 2 , the role of CTLD in the PLA 2 binding appears to be inconsistent with their results for the P. flavoviridis CTLD fragment.…”

“…Earlier, Nobuhisa et al (22) investigated the binding of various fragments of P. flavoviridis PLI␣ to the venom PLA 2 isozymes and reported that hydrophobic residues 136 -147 of P. flavoviridis PLI␣ were critical for binding to the PLA 2 isozymes. In the present study, Tyr-144 of GbPLI␣ was found to contribute to the PLA 2 inhibition to some extent.…”

Identification and Characterization of a Serum Protein Homologous to α‐type Phospholipase A2 Inhibitor (PLIα) from a Nonvenomous Snake, Elaphe quadrivirgata

“…The reason why PLIa-LP does not function as a PLA 2 inhibitor may be that the critical residues for the PLA 2 binding might be replaced by these unique residues in the PLIa-LP sequence. Nobuhisa et al suggested that the C-terminal 12 residues of T. flavoviridis PLIa-A and PLIa-B were essential for the interaction of these inhibitiors with T. flavoviridis acidic PLA 2 or its Lys-49 myotoxic isozymes, BP-I and BP-II (18). In the C-terminal 11 residues conserved completely among all of the PLIas, the deletion of C-terminal leucine and the replacement of tyrosine by serine at the position 146 in PLIa-LP might also be involved in the failure to inhibit PLA 2 .…”

Análise do transcriptoma do fígado da serpente Bothrops jararaca utilizando expressed sequences tags (ESTs).

“…Este inibidor apresenta inibição específica para PLA 2 s ácidas do grupo II da própria serpente. Os PLI-α podem inibir a atividade miotóxica induzida pelas PLA 2 s e foram isoladas de diversas serpentes (INOUE et al, 1991(INOUE et al, , 1997LISANO et al, 1997;NOBUHISA et al, 1998;OHKURA et al, 1993). Os PLI-β são compostos por três subunidades idênticas com 50 kDa, com 33% de identidade com um domínio rico em leucina encontrado em uma proteína denominada α 2 -glicoproteína de humanos.…”