Insights on the structure of native CNF, an endogenous phospholipase A2 inhibitor from Crotalus durissus terrificus, the South American rattlesnake

https://doi.org/10.1016/j.bbapap.2014.05.001Get rights and content

Highlights

  • Native CNF occurs as a mixture of oligomers, mainly tetramers.

  • Carbohydrates are not essential for CNF oligomerization or PLA2 inhibition.

  • A secondary component was identified in the CNF preparations.

  • This secondary component is similar to the B subunits of heteromeric inhibitors.

  • The role played by this minor component in the functionality of CNF is unclear.

Abstract

Several snake species possess endogenous phospholipase A2 inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A2 (PLA2) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, β and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA2. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.

Introduction

Snake venoms are amongst the richest sources of phospholipases A2 (PLA2, EC 3.1.1.4), a widespread superfamily of structurally related enzymes that hydrolyze glycerophospholipids in lysophospholipids and free fatty acids. Based on molecular and biochemical criteria, secretory sPLA2s (sPLA2s) have been classified into 15 different groups [1]. Snake venom PLA2s belong to groups I and II, which also encompasses pancreatic and inflammatory sPLA2s.

The sPLA2s in snake venoms may exert their deleterious actions as monomeric or multimeric toxins with at least one catalytically active subunit. A well-known example of a multimeric sPLA2 is the crotoxin (Ctx), a β-neurotoxin [2], which is the major toxic component of the venom of the South American rattlesnake, Crotalus durissus terrificus. Ctx has a heterodimeric structure formed by an acidic, non-enzymatic subunit (CA) and a basic, enzymatically active counterpart (CB) [3] that are strongly bound by non-covalent interactions. CA acts as a chaperone, preventing the non-specific adsorption of CB to membrane structures other than its physiological target, thereby enhancing the pharmacological potency and, consequently, the lethal effect of CB [4]. The structural and functional activities of Ctx have been recently reviewed by [5].

Aiming, primarily, at a physiological protection against the eventual presence of venom gland contents in their circulating blood, several snake species have been provided with PLA2 inhibitors, generally referred to as sbPLIs (snake blood phospholipase A2 inhibitors). During the last two decades, a growing number of sbPLIs has been described for several venomous and non-venomous snake species [6]. These inhibitors have an oligomeric structure of, at least, three subunits and, based on known mammalian protein domains, they have been categorized into three structural classes (α, β and γ). Members of these three classes can be concomitantly present in a single snake species. The αPLIs have a C-type lectin domain, whereas the distinguishing feature in the βPLIs is the occurrence of leucine-rich repeats, known as LRR. The γPLIs, in turn, are composed of two structural units of highly conserved tandem repeats of half cysteines known as three-finger motifs [7]. Based on the current understanding, the last class comprises the highest number of sbPLIs that are, typically, oligomers of glycosylated and non-glycosylated subunits. Based on the identity of their subunits, Lizano et al. [8] proposed a sub-classification into heteromeric (subclass I) or homomeric (subclass II) sbγPLIs

A gamma sbPLI from C. d. terrificus snakes, named CNF (for an acronym of Crotalus neutralizing factor) has been extensively studied by the authors of this paper, leading to important conclusions on its mechanism of action. CNF is able to displace CA from the native Ctx complex and to bind tightly to CB, thus forming a stable CNF.CB complex. CNF.CB is devoid of any PLA2 activity and may be considered as reminiscent of the interaction of Ctx with its target receptor at the pre-synaptic neuromuscular junctions [9], [10]. Native CNF is an oligomer of glycosylated and non-glycosylated single subunits of 24 kDa and 20 kDa [9] and has been assigned to subclass II of sbγPLIs [8]. However, fundamental questions remain to be answered such as the number of subunits in the oligomer, the role of glycosylation in the inhibitor functionality and its homomeric character. These issues are not exclusive to CNF but can be extended to most sbγPLIs. Although oligo/polymeric structures have been assigned to all of these inhibitors, the number of forming subunits, in most cases, remains undetermined. Regarding glycosylation, the ability to bind or to inhibit phospholipases A2 was solely demonstrated for homologous recombinants with no carbohydrates from Python and Protobothrops inhibitors [11], [12]. In addition, the fact that a second subtype of gamma inhibitor was isolated from a cDNA library from Protobothrops flavoviridis snake liver [13] made the homomeric character of the subclass II sbγPLIs questionable. The translated protein is similar to the subunit B of heteromeric sbγPLIs.

In the present study, we focused on the native structure of CNF, the sbγPLI from C. d. terrificus snakes, in an effort to increase our understanding of oligomerization, role of glycosylation and subunit composition.

Section snippets

Purification of CNF and crotoxin

Heparinized blood plasma from C. d. terrificus snakes was obtained from the Serpentarium of Fundação Ezequiel Dias, depending on the availability of specimens. The procedure followed the protocol approved by the Committee for Ethics in Animal Use of the Fundação Ezequiel Dias (CEUA FUNED 022/2012). Native CNF was purified in two steps, i.e., ion exchange in DEAE-Sephacel [14] followed by a hydrophobic interaction chromatography on a Hitrap Phenyl FF (GE HealthCare) [7], starting with plasma

Oligomerization of CNF: insights in its quaternary structure

The number of forming subunits remains undetermined for most polymeric sbγPLIs with the exception of the inhibitors from Laticauda semifasciata [24] and Naja naja kaouthia [23] elapid snakes, which have been identified as trimers (Table 1). In order to determine the number of subunits in native CNF, we combined several biochemical and biophysical approaches, namely, chemical cross-linking, gel filtration chromatography, fluorescence spectroscopy, dynamic light and small angle X-ray scatterings

Discussion

The determination of the quaternary structure of native sbγPLIs is a crucial step for a better understanding of the functionality of those complex molecules with high biotechnological potential. Although the molecular masses of the individual subunits have been estimated for the great majority of sbγPLIs by classical gel filtration (Table 1), the number of forming subunits in the native molecules remained to be determined.

In a first attempt to better understand its quaternary structure,

Acknowledgements

We acknowledge Miss Ana do Carmo Valentim for excellent technical help and Dr. Amando Siuiti Ito for fruitful discussions concerning fluorescence spectroscopy. We are thankful to the Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG CBB 1271/11), Fundação de Amparo à Pesquisa de São Paulo (FAPESP), INCTTox (CNPq/FAPESP) and CAPES (Toxinology 063/2011) for financial support.

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