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

Abstract

␣-Type phospholipase A 2 inhibitory protein (PLI␣) from the serum of the venomous snake Gloydius brevicaudus, GbPLI␣, is one of the protective endogenous proteins that neutralizes its own venom phospholipase A 2 (PLA 2 ), and it is a homotrimer of subunits having a C-type lectin-like domain. The nonvenomous snake Elaphe quadrivirgata has a homologous serum protein, EqPLI␣-LP, that does not show any inhibitory activity against various snake venom PLA 2 s (Okumura, K., Inoue, S., Ikeda, K., and Hayashi, K. (2003) IUBMB Life 55, 539 -545). By constructing GbPLI␣-Eq-PLI␣-LP chimeric proteins, we have mapped the residues important in conferring GbPLI␣ inhibitory activity on region 13-36 in the primary structure of GbPLI␣. Noninhibitory EqPLI␣-LP showed comparable inhibitory activity only when this region was replaced with that of GbPLI␣. Further, mutational analysis of the candidate residues revealed that the individual GbPLI␣ to EqPLI␣-LP residue substitutions N26K, K28E, D29N, and Y144S each produced a mutant GbPLI␣ protein with reduced inhibitory activity, with the single N26K substitution having the most significant effect. Residues 13-36 were suspected to be located in the helical neck region of the GbPLI␣ trimer. Therefore, the region of GbPLI␣ responsible for PLA 2 inhibition was distinct from the carbohydrate-binding site of the homologous C-type lectin.Phospholipases A 2 (PLA 2 s, EC 3.1.1.4) 2 catalyze the hydrolysis of the acyl-ester bond at the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. Secretory PLA 2 s are a growing family of low molecular weight, highly disulfide-linked, Ca 2ϩ -requiring secretory enzymes with a His-Asp catalytic dyad and are classified into six main groups (I, II, III, V, X, and XII) according to their primary structures (1). Snake venom is one of the most abundant sources of secretory PLA 2 s, which exhibit a wide variety of pharmacological effects including neurotoxicity and myotoxicity (2). Elapidae venom contains group I PLA 2 s, and Viperidae venom contains group II ones (3). Venomous snakes have three distinct types of PLA 2 inhibitory proteins (PLI␣, PLI␤, and PLI␥) in their blood to protect themselves from the leakage of their own venom PLA 2 s into the circulatory system (4 -6). PLI␣ has only been identified in the blood of Viperidae snakes, such as Protobothrops flavoviridis (renamed from Trimeresurus flavoviridis according to the present taxonomy) (7), Gloydius brevicaudus (renamed from Agkistrodon blomhoffii siniticus) (8), Bothrops asper (9), and Cerrophidion godmani (10). It is a 75-kDa trimeric glycoprotein of 20-kDa subunits having a C-type lectin-like domain (CTLD), which is homologous to that of collectins, such as serum mannose-binding protein (MBP) and lung surfactant-apoproteins (11). G. brevicaudus, B. asper, and C. godmani PLI␣s are composed of three identical subunits, whereas P. flavoviridis PLI␣ is a trimer of two homologous subunits. P. flavoviridis and G. brevicaudus PLI␣s inhibit specifically the group II acidic PLA 2...

Alpha-type phospholipase A2 inhibitors from snake blood

“…In this region, two hydrophobic tripeptides and Tyr144 residue appear to be involved in the interaction PLI/PLA 2 [37, 69, 79]. …”

“…Thereafter, Okumura et al [69] studied the relationship of the structure/function of the αPLI previously purified from the snake Agkistrodon blomhoffii siniticus , named GbPLIα, and the αPLI-like protein EqPLIα-LP, purified from the nonvenomous snake Elaphe quadrivirgata, and which does not show inhibitory activity against PLA 2 s [42, 68]. In that work, by constructing chimeric proteins, they mapped important residues to the inhibitory activity of the αPLIs; for example, the region 13-36 of the neck C-terminal portion of the trimer.…”

“…Interestingly, the region found as the responsible for PLA 2 inhibition was distinct from the carbohydrate-binding site. Furthermore, other residues were pointed as candidate, including Asn26, Lys28, Asp29, and Tyr144 [69]. …”

Endogenous phospholipase A2 inhibitors in snakes: a brief overview

“…The sbαPLI from G. brevicaudus (formerly Agkistrodon blomhoffii siniticus ) is a homotrimer, in which the α-helical coiled-coil neck subunit forms a central pore that constitutes the binding site for the target PLA 2 s [28–30]. The C-type lectin-like domain was discarded as responsible for PLA 2 binding [30].…”

“…Chimeric constructions of GbαPLI and the non-functional sbαPLI homolog from E. quadrivirgata allowed the mapping of important amino acids for PLA 2 inhibition in the 13–36 segment, which are expected to be located in the helical neck of the GbαPLI trimer based on the three-dimensional structural model constructed by homology modeling [29, 30]. The trimerization occurs only among subunits having the same α-helical motif in the regions 13–36 and the oligomer is structurally stabilized by intermolecular electrostatic interactions.…”