One-Step Affinity Purification of Leucine-Rich α2-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A2-Inhibitory Activities as β-Type Phospholipase A2 Inhibitors

Snakes contain three types of phospholipase A(2) (PLA(2))-inhibitory proteins in their blood, PLI alpha, beta, and gamma, which protect them from their own venom, PLA(2). PLI beta is the snake ortholog of leucine-rich alpha(2) glycoprotein (LRG). Since autologous cytochrome c (Cyt c) serves as an endogenous ligand for LRG, in this study, we purified snake LRGs from various snake serum samples using Cyt c affinity chromatography. All purified snake LRGs were found to be dimers linked by disulfide bonds. Laticauda semifasciata and Naja kaouthia LRGs showed no inhibitory activity against L. semifasciata PLA(2) and weak inhibitory activity against Gloydius brevicauda basic PLA(2). Elaphe climacophora PLI beta had weaker inhibitory activity against G. brevicauda basic PLA(2) than G. brevicauda and Elaphe quadrivirgata PLIs, which are abundant in blood and known to neutralize G. brevicauda basic PLA(2). Protobothrops flavoviridis LRG showed no inhibitory activity against basic venom PLA(2), PL-X, or G. brevicauda basic PLA(2). Binding analysis of P. flavoviridis LRG using surface plasmon resonance showed very strong binding to snake Cyt c, followed by that to horse Cyt c, weak binding to yeast Cyt c, and no binding to P. flavoviridis PL-X or BPI/II. We also deduced the amino acid sequences of L. semifasciata and P. flavoviridis LRG by means of cDNA sequencing and compared them with those of other known sequences of PLIs and LRGs. This study concluded that snake LRG can potentially inhibit basic PLA(2), but, whether it actually functions as a PLA(2)-inhibitory protein, PLI beta, depends on the snake.