Characterization and Evolution of a Gene Encoding a Trimeresums flavoviridis Serum Protein that Inhibits Basic Phospholipase A₂ Isozymes in the Snake's Venom

Abstract

The proteins that bind phospholipase A2 (PLA2) isozymes of Trimeresurus flavoviridis (habu snake, crotalinae) venom were ractionated from sera on four columns, each conjugated with one of four PLA2 isozymes. Five proteins, termed PLA2 inhibitors (PLI) I–V, were obtained as the binding components. The combinations of the binding components differed depending on the PLA2 isozymes. PLI‐IV and PLI‐V correspond to PLI‐A and PLI‐B, respectively, which were known to bind to a major [Asp49]PLA2, PLA2, and contained a segment similar to the carbohydrate‐recognition domain of C‐type lectins. PLI‐I, which is a major component of inhibitory proteins against three basic PLA2 isozymes, PLA‐B (a basic [Asp49]PLA2) and basic proteins I and II (both [Lys49]PLA,s), has been isolated, and its partial amino acid sequence has been determined. A cDNA encoding PLI‐I was isolated from a T. flavoviridis liver cDNA library and sequenced. PLI‐I cDNA encoded 200 amino acid residues, including a signal peptide of 19 amino acid residues. One sugar chain was predicted to occur at position 157. A gene coding for PLI‐I was isolated. It is 9.6‐kb long and consists of five exons and four introns. Comparison of the exonintron structure of the PLI‐I gene with those of genes encoding urokinaseype‐plasminogen‐activator receptor (uPAR), Ly‐6, CD59 and neurotoxins showed that they have characteristic unit encoding approximately 90 amino acid residues, which is divided over two exons. This strongly suggests that the PLI‐I gene belongs to the uPAR, Ly‐6, CD59 and neurotoxin gene family. There are two types of structurally different inhibitors against PLA2 isozymes in T. flavoviridis serum with different evolutionary origins.

Purification and inhibitory profile of phospholipase A2 inhibitors from Australian elapid sera

“…This finding has recently been confirmed for another elapid PLI [25]. For the PLIs that have been sequenced, consensus N-linked glycosylation sites have been identified, or glucosamine has been detected after amino acid analysis of the purified inhibitor [24,27,30,[32][33][34]37]. Furthermore, the masses of the carbohydrates identified, although not examined as throughly as here, indicate that the carbohydrate moieties vary from 3 to 5 kDa in molecular mass [23,30], suggestive of a similar glycosylation pattern for Class A and B PLIs.…”

“…These PLIs have been purified from Naja naja kaouthia (Thailand cobra [23,24]), Laticauda semifasciata (Sea Krait [25]), Crotalus durissus terrificus (South American rattlesnake [26][27][28]), Trimeresurus fla o iridis (Habu [29][30][31][32]), Agkistrodon blomhoffii siniticus (Chinese mamushi [33,34]) and Bothrops asper (fer-delance [35]). Additionally, an inhibitory serum factor from Vipera palaestinae (Palaestinian viper) that shares many of the characteristics of PLIs has been identified [36] ; however, it has not been clearly shown that this protein inhibits PLA # enzymes.…”

Caracterização funcional e estrutural de inibidores de fosfolipases A2 isolados do plasma de serpente Bothrops jararacussu

“…Finalmente, os inibidores γPLI foram encontrados em diversas famílias tais como Viperidae, Colubridae, Elapidae, Boiidae e Hydrophiidae, as quais pertencem diferentes espécies de serpentes como a Laticauda semifasciata , Crotalus durissus terrificus (FORTES-DIAS et al, 1994;PERALES et al, 1995), Agkistrodon blomhoffii siniticus , Trimeresurus flavoviridis (NOBUHISA et al, 1997), Elaphe quadrivirgata e E. climacophora (OKUMURA et al, 1999;SHIRAI et al, 2009), Cerrophidion godmani , Naja naja kaouthia (OHKURA et al, 1994a; neuwiedi (ESTEVÃO-COSTA et al, 2008).…”

“…Os γPLIs possuem em comum repetições intramoleculares de domínios ricos em cisteína (OHKURA et al, 1994a;NOBUHISA et al, 1997 …”

“…Considerando-se que vários PLIs estão presentes no sangue de diferentes espécies de serpentes (NOBUHISA et al, 1997;OHKURA et al, 1997;PERALES, 2003) e inibem PLA 2 s tóxicas específicas dentro da mesma espécie (LIZANO et al, 1997;, é possível sugerir que proteínas endógenas de serpentes podem estar envolvidas na neutralização de atividades enzimáticas, farmacológicas e tóxicas. -dependentes, como por exemplo, a apoproteína surfactante do pulmão, que liga os resíduos do carboidrato que contêm manose (INOUE et al, 1991;OHKURA et al, 1993;LIZANO et al, 1997).…”