Proteomic Analysis of Venomous Fang Matrix Proteins of Protobothrops flavoviridis (Habu) Snake

Venomous animals have specialized venom delivery apparatus such as nematocysts, stings, and fangs in addition to the poisonous organs consisting venom gland or sac, which produce and stock the venom. Snake is one of the major venomous animals, of which fangs are connected to the venom gland to inject the venom into prey. Snake’s venomous fangs showed the unique characteristics including mechanical strength and chemical stability. Especially, Protobothrops flavoviridis (habu) snake fangs showed the resistance against its venom digestive proteases, whereas the bones and teeth of mouse were completely digested in the gastrointes-tinal tract, although habu fangs were also drawn into the body with the prey. These observations suggest that structural differences exist between venomous fangs and mammalian bones and teeth. In this study, to reveal the molecular properties of venomous snake fangs, the matrix proteins of P. flavoviridis (habu) snake venom fang were analyzed by using proteomics experiments using 2D-PAGE and TOF MS/MS analyses. As a result, several biomineralization-related proteins such as vimentin, tectorin, adaptin, and collagen were identified in the venomous fang matrix proteins. Interestingly, the inhibitory proteins against venomous proteins such as metalloproteinase and PLA2 were also identified in fang’s matrix proteins.


Introduction
Venomous animals such as sea anemone, jellyfish, lizards, scorpion, fish, arachnids, bees, and snakes produce chemical weapon, toxic proteins, and peptides cocktail to kill and capture pray. They deliver the toxins as venom into prey through the sophisticated venom delivery systems consisting of an exocrine gland, a lumen, venom duct, and also injector such as nematocyst, sting, fangs, harpoon-like sting, and spine. These venomous apparatuses are thought to have evolved from the general biological organs, namely, an ovipositor, a tooth, radula, and dorsal fin, respectively. Snake is one of the major venomous animals, of which fangs are connected to the venom gland to inject the venom into prey. Venomous snakes can be classified into two groups according to the fang systems, front fanged (elapid and vipers) and rear fanged (grass snakes), and frontal fangs are further divided into two types, grooves and tubes (Kardong 1979;Savitzky 1980;Jackson 2002;Kuch et al. 2006). Vonk et al. (2008) reported the evolutionary origin and development of snake fangs, showing that front fangs develop from the posterior end of the upper jaw and are strikingly similar in morphogenesis to rear fangs. In the anterior part of the maxilla of front-fanged snakes, gene expression of sonic hedgehog, which is responsible among other things for the formation of the teeth, is suppressed. Despite such extensive studies and the recent genome sequence analyses for two venomous snakes, the king cobra (Ophiophagus hannah) (Vonk et al. 2013) and the five-pacer viper (Deinagkistrodon acutus) (Yin et al. 2016), the matrix proteins of venomous fangs, their evolutionary origins, and the biomineralization mechanisms of venomous fangs are still poorly understood.
Protobothrops flavoviridis (habu) snake that inhabits Ryukyu (Okinawa, Tokunoshima, and Amami) Islands are dangerous snakes having various toxic peptides and proteins (multiple protein families) as venom. Their venomous fangs are frequently lost and drawn into their own body with the prey after injection of the venom. Interestingly, venomous fangs are excreted with no change and no digestion, whereas the bones and teeth of the mouse (prey) are completely digested. These observations suggest that structural differences between venomous fangs and mammalian bones and teeth exist. In addition, it is conceivable that the adaptive evolution of the venomous organ and venomous fang bestowed them to have resistance to digestive juices. Thus, the snake fangs show the unique characteristics including mechanical strength and chemical stability.
In this study, to reveal the characteristics of habu snake fangs such as chemical stability, and their molecular evolution, proteomic analyses of fang matrix proteins were conducted by using 2D-PAGE and MALDI-TOF MS/MS.

Materials
The crude venomous fangs of Protobothrops flavoviridis (habu) snakes captured in Amami Island, Kagoshima Prefecture, Japan, were collected by dissection of the head from sacrifice. Subsequently, fangs and tissues were separately rinsed with phosphate-buffered saline and stored at −80 °C until use. Immobiline DryStrip for two-dimensional electrophoresis and the IPG buffer (pH 3-11) were obtained from GE Healthcare UK Ltd. (Buckinghamshire, England). Silver Stain MS kit was purchased from Wako Pure Chemical Industry, Ltd. (Osaka, Japan). Achromobacter protease I and Staphylococcus aureus V8 protease were obtained from Wako Pure Chemicals (Osaka, Japan) and Sigma-Aldrich Co. (St. Louis, MO, USA), respectively. ZipTip C18 was purchased from Millipore (Massachusetts, USA). All other reagents were of the best commercially available grade from Wako Pure Chemicals (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan).

Isolation and Characterization of the Matrix Proteins from the Venomous Fang
Venomous fangs of habu snakes were decalcified with 50% formic acid at room temperature for 2 days. Then, the decalcified matrix proteins were dissolved in 6 M guanidinium hydrochloride in 50 mM Tris-HCl buffer (pH 8.8) containing 200 mM NaCl at 60 °C. After TCA-acetone precipitation, the pellet was dissolved in 8 M urea in 100 mM Tris-HCl buffer (pH 8.2) at 60 °C. For two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), fang matrix proteins were directly dissolved in 400 μl of rehydration buffer (8 M urea, 4% CHAPS, 2% immobilized pH gradient (IPG) buffer (pH 3-11NL), DeStreak reagent (15 mg/ml), and 0.002% bromophenol blue) and were loaded onto IPG strips. After rehydration for 12 h, isoelectric focusing (IEF) was performed at 20 °C using the running conditions of the following focusing program: 500 V for 1 h, a gradient to 1000 V for 1 h, a gradient to 8000 V for 3 h, and 8000 V for 1.5 h (3225 V, 50 μA, 19,742 Vhs). After running IEF, IPG strips were equilibrated in a reducing equilibration buffer for 15 min and subsequently alkylated with iodoacetamide. Then, IPG strips were transferred onto 15% polyacrylamide gel (18 × 16 cm) and embedded with 0.5% agarose and electrophoresed. Gels were stained using Silver Stain MS kit or Coomassie Brilliant Blue.

Proteome Analysis
The spots on 2D gel were cut into pieces and washed with Milli-Q water. After the gels were dehydrated by acetonitrile with gentle agitation and completely dried in vacuo, gel samples were reduced by 10 mM DTT for 1 h at 56 °C. After cooling and washing by 25 mM ammonium bicarbonate buffer for 10 min, the gel samples were treated with 55 mM iodoacetamide in 25 mM ammonium bicarbonate solution in the dark. After removal of the solvent to be completely dried, gel particles were digested by Achromobacter protease I (Lys-C) or V8 protease at 37 °C for one night.
After concentrating the digest in speed vacuum, samples were desalted on ZipTip C18 (Millipore). Samples were separated by using a DiNa Nano LC system equipped with a DiNa MALDI spotting device (KYA Technologies Co., Tokyo, Japan) and applied to MALDI-TOF MS and tandem MS/MS analysis using TOF/TOF™ 5800 Analyzer (AB SCIEX). Enzyme-digested matrix proteins without 2D-PAGE were also analyzed by nanoLC-MALDI-TOF MS/MS. Molecular masses were calibrated using the Sequazyme Peptide Mass Standards Kit (Applied Biosystems). Protein identification was performed by searching of each MS/MS spectrum against the protein sequence databases derived from the RNA-seq data of P. flavoviridis snake fang-forming tissues by using ProteinPilot software (version 3.0; AB Sciex) with the Paragon method.

Isolation and Characterization of the Matrix Proteins from P. flavoviridis Venomous Fangs
First, the decalcification conditions of venomous fangs were investigated by using hydrochloric acid and formic acid, respectively. The complete decalcification of the venomous fang without protein degradation was achieved by 50% formic acid at 30 °C for 2 days, resulting typical yield of 5.6 mg from 1.0 g of P. flavoviridis fangs, while the decalcification of fang by 10% HCl treatment caused the degradation of proteins ( Fig. 5.1a). Then, the matrix proteins were subjected to the proteome analysis, in-gel enzymatic digestions for mass spectrometry characterization with 2D-PAGE and the shotgun proteomics of enzymatic digestions of total matrix proteins by nanoLC-MS/MS, respectively, after dissolved in 6 M guanidinium at 60 °C and concentrated by TCA-acetone precipitation.

Proteome Analysis of the Fang Matrix Proteins
To identify the array of proteins in P. flavoviridis venomous fangs, the extracted matrix proteins were subjected to the 2D-PAGE (pH 3-11), resulting in identification in acidic region of around 20 appreciable major spots, of which pI values ranging from 4 to 6 ( Fig. 5.2). These fang matrix proteins were roughly divided into five groups based on the molecular mass numbers: 55 kDa (sample #1), 40 kDa (#2), 35 kDa (#3), 30 kDa (#4), and 25 kDa (#5) proteins. Preliminary proteomic analyses of these protein spots allowed the identification of major components of fang matrix proteins including type I collagens alpha-1 and alpha-2 and UV excision repair protein RAD23-like protein (Table 5.1). Interestingly, antihemorrhagic factor HSF, which is a proteinaceous serum inhibitor against own venom metalloproteinases, was also detected as a matrix protein. However, these proteomic data from 2D-PAGE could not provide satisfactory results.
To improve the proteomic data of P. flavoviridis fang matrix proteins, a direct shotgun proteomic analysis was conducted. As a result of 4 independent experiments of shotgun proteomics, 36 proteins were identified as fang matrix proteins (Table 5.2). In addition to the type I collagen alpha1 (isoform X1 and X2) and alpha2 chains, the collagens type VI alpha2 and alpha3 chains and type XI alpha1 and alpha2 chains were identified. Because type I collagen has been reported to be related to the formation of dentin and enamel, contributing to the nanoscale architecture in the teeth (Wallace et al. 2010), type I collagen seems to be an important  Titin-like component of venomous snake fang. Furthermore, the type VI collagen, which forms microfibrils and is primarily associated with the extracellular matrix of skeletal muscle and bone marrow, and type XI collagen, which is found in the cartilage of the nose and external ears in human, were also identified as matrix proteins in venomous fang, suggesting the unique distribution of type VI and type XI collagens as part of the fang matrix. On the other hand, noncollagenous dentin matrix proteins including proteoglycans (PGs), glycoproteins, serum proteins, enzymes, and growth factors are deemed to play structural, metabolic, and functional roles as key components in the mineralization process of dentin (Orsini et al. 2009). Shotgun proteomic analysis showed the fang noncollagenous dentin matrix proteins include proteoglycan such as decorin (1_2, 3_4, 4_13 in Table 5.2) and biglycan (1_8, 4_4), glycoproteins such as osteonectin (secreted protein acidic and rich in cysteine: SPARC) (1_5, 4_8), the SIBLING proteins such as dentin matrix acidic phosphoprotein 1 (4_12), and serum proteins such as albumin (1_4, 3_5, 4_1), phospholipase A 2 inhibitor (1_14) and antihemorrhagic factors, HSF (1_9, 2_2, 3_2, 4_2), and HSFlike protein (1_7, 3_6). The coexistence of these serum inhibitors as fang matrix proteins explains why venomous fang is stable against own venom enzymes compared with mouse-derived teeth and bones. Compared with the homologous proteins in mouse, several fang matrix proteins such as dentin matrix acidic phosphoprotein 1 (36%), titin-like protein (31%), transferrin-like protein (26%), and serum inhibitors including albumin (32%) and PLA2 inhibitor (29%) showed lower sequence similarities, suggesting that these differences in matrix proteins might be related to the functional differences and distinctive properties between venomous fang and mouse's teeth.
In this study, we identified 36 matrix proteins from P. flavoviridis snake fangs by proteomics analyses. They include proteinaceous inhibitor against own venom enzymes in addition to several types of collagens (types I, VI, and XI) and noncollagenous dentin matrix proteins. More recently, we have decoded the whole genome sequence of P. flavoviridis snakes (Shibata et al. 2018, in press). Further investigations are needed to elucidate the biomineralization mechanisms of venomous fang and their biological functions.
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