Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Insights on the structure of native CNF, an endogenous phospholipase A2 inhibitor from Crotalus durissus terrificus, the South American rattlesnake
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.
References (49)
- et al.
Crotoxin: novel activities for a classic beta-neurotoxin
Toxicon
(2010) - et al.
Natural phospholipase A2 myotoxin inhibitor proteins from snakes, mammals and plants
Toxicon
(2003) - et al.
A phospholipase A2 inhibitor from the plasma of the South American rattlesnake (Crotalus durissus terrificus), protein structure, genomic structure and mechanism of action
J. Biol. Chem.
(1994) - et al.
Structural elements of Trimeresurus flavoviridis serum inhibitors for recognition of its venom phospholipase A2 isozymes
FEBS Lett.
(1998) - et al.
Functional characteristics of a phospholipase A(2) inhibitor from Notechis ater serum
J. Biol. Chem.
(2000) - et al.
Purification and properties of an antivenom factor from the plasma of the South American rattlesnake (Crotalus durissus terrificus)
Toxicon
(1991) - et al.
Purification of gyroxin from a South American rattlesnake (Crotalus durissus terrificus) venom
Toxicon
(1980) - et al.
Steady-state and time-resolved fluorescence spectroscopy of quinine sulphate dication bound to sodium dodecylsulfate micelles: fluorescent complex formation
J. Lumin.
(2014) - et al.
A sensitive and specific plate test for the quantification of phospholipases
Anal. Biochem.
(1972) - et al.
Isolation and characterization of a phospholipase A2 inhibitor from the blood plasma of the Thailand cobra Naja naja kaouthia
Biochem. Biophys. Res. Commun.
(1994)
Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set
Anal. Biochem.
GROMACS: a message-passing parallel molecular dynamics implementation
Comput. Phys. Commun.
Analytical exclusion chromatography
J. Biochem. Biophys. Methods
Identification and characterization of phospholipase A2 inhibitors from the serum of the Japanese rat snake, Elaphe climacophora
Toxicon
Phospholipase A2 biochemistry
Cardiovasc. Drugs Ther.
Action of crotoxin and contractin from the venom of Crotalus durissus terrificus (South American rattlesnake) on the frog neuromuscular junction
J. Physiol.
Crotoxin, a phospholipase A2 neurotoxin from the South American rattlesnake Crotalus durissus terrificus: purification of several isoforms and comparison of their molecular structure and of their biological activity
Biochemistry
Crotoxin, half-century of investigations on a phospholipase A2 neurotoxin
Acta Physiol. Pharmacol. Latinoam.
Snakes as a source of phospholipase A2 inhibitors with biotechnological potential
Purification and characterization of three distinct types of phospholipase A2 inhibitors from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus
Biochem. J.
Molecular structure and mechanism of action of the crotoxin inhibitor from Crotalus durissus terrificus serum
Eur. J. Biochem.
Identification of the B subtype of gamma-phospholipase A2 inhibitor from Protobothrops flavoviridis serum and molecular evolution of snake serum phospholipase A2 inhibitors
J. Mol. Evol.
Inhibition of crotoxin binding to synaptosomes by a receptor-like protein from Crotalus durissus terrificus (the South American rattlesnake)
Biochim. Biophys. Acta
Calibration and application of an X-ray image intensifier/charge-coupled device detector for monochromatic macromolecular crystallography
J. Synchrotron Radiat.
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2020, International Journal of Biological MacromoleculesCitation Excerpt :This finding contrasts the predicted glycosylation site on asparagine 176 in the motif Asn-Ala-Thr suggested for Crotalus durissus collilineatus and PLIs from other venomous snake (UniProt Q90358) [9]. However, it was also demonstrated that the γPLI from e.g. Crotalus durissus terrificus remains its functionality to inhibit PLA2 binding even after deglysosylation [12]. One characteristic of γPLIs is the presence of cysteine rich domains suggested to be relevant for specific recognition of PLA2.
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2017, International Journal of Biological MacromoleculesCitation Excerpt :The mechanism of action of CNF is to displace CA from the native Ctx complex to bind tightly to CB, hence forming a stable CNF.CB complex, devoid of any PLA2 activity. However, the inhibitory activity of CNF is not restricted to CB, the PLA2 from C. d. terrificus venom, but can be extended to similar group II enzymes from other Viperidae snakes [8–11]. Despite the considerable number of sbPLIs described, their structures and mechanisms of action are still under investigation and not well understood.