Molecular isoforms of cobra venom factor-like proteins in the venom of Austrelaps superbus☆
Introduction
Complement system and its activation are important in the host defense against infectious agents and the inflammatory process. Complement protein C3 plays a pivotal role in the activation of both classical and alternative pathways and is indispensable to this cascade of activation. Cobra venom factor (CVF) is a three-chain glycoprotein in the cobra venom that activates the complement system (Eggertsen et al., 1981; Vogel, 1991; Vogel et al., 1996). It is a structural and functional analog of the complement component C3 (Lachmann and Nicol, 1973; Vogel et al., 1984). When added to human (or mammalian) serum, CVF binds to Factor B of the alternative pathway to form the bimolecular complex CVFB (Hensley et al., 1986). Bound Factor B is cleaved by Factor D (Cooper, 1973) in the plasma into Ba (the activation peptide) and Bb that remains bound to the CVF thereby forming a stable complex CVFBb (Vogel and Muller-Eberhard, 1982). The CVFBb complex cleaves C3 and C5 into C3a and C3b, and C5a and C5b, respectively. Thus, CVF functions both as C3 and C5 convertase (DiScipio et al., 1983; Vogel and Muller-Eberhard, 1982). Complement activation by CVF thus results in the consumption of complement components C5, C6, C7, C8, C9 and Factor B leading to their depletion (Birdsey et al., 1971).
Using screening methods based on the immunological cross-reactivity and complement consumption, it has been shown that CVF is present in the venoms of several cobras of three genera (Naja naja, Naja haje, Naja nigricollis, Naja siamensis, Naja nivea, Naja melanoleuca, Naja atra, Naja kaouthia, Hemachatus hemachatus and Ophiophagus hannah) (Birdsey et al., 1971; Eggertsen et al., 1981; Takahashi and Hayashi, 1982). However, CVF-like proteins were not found in the venoms of other elapids including Bungarus multicinctus, Bungarus fasciatus, Bungarus caeruleus and Dendroaspis angusticeps (Birdsey et al., 1971; Eggertsen et al., 1981; Takahashi and Hayashi, 1982). Similarly, no CVF-like proteins were found in the families of Crotalids, Viperids and Hydrophids (Birdsey et al., 1971; Takahashi and Hayashi, 1982). Thus, CVF or CVF-like proteins have never been found in non-cobra snakes. It is to be noted that so far none of the Australian elapids have been screened for the presence of CVF-like proteins.
We have been characterizing novel proteins from snake venoms (Banerjee et al., 2005; Nirthanan et al., 2002; Pung et al., 2005; Torres et al., 2003; Kuhn et al., 2000; Watanabe et al., 2002). In our quest for novel proteins in the venom of A. superbus, we made a comprehensive search for low molecular weight polypeptides. During this study, we identified a polypeptide that showed significant structural similarity to the C-terminal of the α-chain of CVF. Here, we describe the cDNA cloning and sequence analyses of its precursor proteins. The results show the presence of two CVF-like proteins, which were named as AVF-1 (A. superbus venom factor) and AVF-2 in A. superbus. Partial purification and Western blot analyses in combination with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed the presence of two variants of highly expressed isoform AVF-1 in the venom of A. superbus. These variants activate human Factor B and complement C3; however, unlike CVF they do not activate C5. Thus, AVF may be useful in developing therapeutic agents for complement depletion and the Australian snakes may provide an alternative source of CVF-like proteins.
Section snippets
Materials
Venom glands and lyophilized crude venom of A. superbus and lyophilized crude venom of N. kaouthia were purchased from Venom Supplies Pty Ltd. (Tanunda, South Australia, Australia). Columns for reversed-phase high-performance liquid chromatography (RP-HPLC)—Jupiter™ C18 preparative (250 mm×21 mm) and C18 semi-preparative (250 mm×10 mm) columns were purchased from Phenomenex (Torrance, CA, USA). Superdex 200 Hiload™ (16/60) and Sephasil™ C18 (5 μm SC 2.1/10) narrow bore columns were purchased from
Search for novel polypeptides and purification of AVFαc
Following centrifugal filtration of the crude venom of A. superbus, the low molecular weight fraction (<10,000 Da) was subjected to RP-HPLC (Fig. 1A) and the molecular masses of all the fractions were determined (Table 1). This led to the identification of eight potential novel polypeptides as determined by distinctly different molecular masses as compared to well-established polypeptide toxin families in snake venoms. The fractions indicated by an arrow (Fig. 1A) gave a mass of 8327.53±1.24 Da
Discussion
Anti-complementary factors are known to be present in the venoms of several snakes (Ballow and Cochrane, 1969; Eggertsen et al., 1980; Von Zabern et al., 1981). A number of these factors have been purified and characterized. They differ in structure and exhibit their anti-complement activity by distinctly different mechanisms. Flavoxobin, for example, directly cleaves C3 into C3a and C3b (Yamamoto et al., 2002). CVF is a unique snake venom protein, which activates complement pathway. It forms a
Acknowledgments
We thank Dr. David Fritzinger from University of Hawaii, Hawaii, USA for the generous gift of polyclonal anti-CVF antibodies.
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2017, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :Excellent detailed reviews can be found elsewhere [62,255]. So far, CVF is mainly present in Elapidae snake family and it has been described in four different genera: Hemachatus, Naja, Ophiophagus and Austrelaps [60–62,249]. Nevertheless, complement depletion caused by CVF is most studied using the venoms of N. naja, Naja kaouthia and N. atra, even being found in low amounts in these venoms.
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2012, ToxiconCitation Excerpt :Because Asn131 and Asn136 are so close, it might be that only one of the two potential glycosylation sites will be glycosylated (Vogel et al., 1996). Besides, it is predicted that pre-pro-AVF-2 has three potential glycosylation sites, one is in α chain and two are in β chain (Rehana and Manjunatha Kini, 2007). No potential N-linked glycosylation site has been found in pre-pro-AVF-1 (Rehana and Manjunatha Kini, 2007).
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This research work was supported by a research grant from the Biomedical Research Council, Singapore.