Cone snail venoms are considered an untapped reservoir of extremely diverse peptides, named conopeptides, displaying a wide array of pharmacological activities. We report here for the first time, the presence of high molecular weight compounds that participate in the envenomation cocktail used by these marine snails. Using a combination of proteomic and transcriptomic approaches, we identified glycosyl hydrolase proteins, of the hyaluronidase type (Hyal), from the dissected and injectable venoms (“injectable venom” stands for the venom variety obtained by milking of the snails. This is in contrast to the “dissected venom”, which was obtained from dissected snails by extraction of the venom glands) of a fish-hunting cone snail, Conus consors ( Pionoconus clade). The major Hyal isoform, Conohyal-Cn1, is expressed as a mixture of numerous glycosylated proteins in the 50 kDa molecular mass range, as observed in 2D gel and mass spectrometry analyses. Further proteomic analysis and venom duct mRNA sequencing allowed full sequence determination. Additionally, unambiguous segment location of at least three glycosylation sites could be determined, with glycans corresponding to multiple hexose (Hex) and N-acetylhexosamine (HexNAc) moieties. With respect to other known Hyals, Conohyal-Cn1 clearly belongs to the hydrolase-type of Hyals, with strictly conserved consensus catalytic donor and positioning residues. Potent biological activity of the native Conohyals could be confirmed in degrading hyaluronic acid. A similar Hyal sequence was also found in the venom duct transcriptome of C. adamsonii ( Textilia clade), implying a possible widespread recruitment of this enzyme family in fish-hunting cone snail venoms. These results provide the first detailed Hyal sequence characterized from a cone snail venom, and to a larger extent in the Mollusca phylum, thus extending our knowledge on this protein family and its evolutionary selection in marine snail venoms.
References
[1]
Olivera, B.M.; Teichert, R.W. Diversity of the neurotoxic Conus peptides: a model for concerted pharmacological discovery. Mol. Interv. 2007, 7, 251–260.
[2]
Favreau, P.; St？cklin, R. Marine snail venoms: use and trends in receptor and channel neuropharmacology. Curr. Opin. Pharmacol. 2009, 9, 594–601.
Newcomb, R.; Miljanich, G. Handbook of Neurotoxicology; Massaro, E.J., Ed.; Humana Press Inc: Totowa, NJ, USA, 2002; Volume 1, pp. 617–652. Chapter 28.
[6]
Mebs, D. Venomous and Poisonous Animals; Medpharm Scientific Publishers: Stuttgart, Germany, 2002.
[7]
Marsh, H. The caseinase activity of some vermivorous cone shell venoms. Toxicon 1971, 9, 63–67.
[8]
Balbin, J.A. Acetylcholinesterase from Conus geographus venom: partial purification and characterization. MS Thesis, Dept. of Biochemistry and Molecular Biology, University of the Philippines, Manila, Philippines, 1980.
[9]
Miranda, R.R. Partial purification and characterization of Conus textile venom phosphodiesterase I. MS Thesis, Dept. of Biochemistry and Molecular Biology, University of the Philippines, Manila, Philippines, 1982.
[10]
Milne, T.J.; Abbenante, G.; Tyndall, J.D.; Halliday, J.; Lewis, R.J. Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily. J. Biol. Chem. 2003, 278, 31105–31110.
[11]
Qian, J.; Guo, Z.Y.; Chi, C.W. Cloning and isolation of a Conus cysteine-rich protein homologous to Tex31 but without proteolytic activity. Acta Biochim. Biophys. Sin. (Shanghai) 2008, 40, 174–181, doi:10.1111/j.1745-7270.2008.00385.x.
[12]
Stanley, T.B.; Stafford, D.W.; Olivera, B.M.; Bandyopadhyay, P.K. Identification of a vitamin K-dependent carboxylase in the venom duct of a Conus snail. FEBS Lett. 1997, 407, 85–88.
[13]
Safavi-Hemami, H.; Bulaj, G.; Olivera, B.M.; Williamson, N.A.; Purcell, A.W. Identification of Conus peptidylprolyl cis-trans isomerases (PPIases) and assessment of their role in the oxidative folding of conotoxins. J. Biol. Chem. 2010, 285, 12735–12746.
[14]
Buczek, O.; Olivera, B.M.; Bulaj, G. Propeptide does not act as an intramolecular chaperone but facilitates protein disulfide isomerase-assisted folding of a conotoxin precursor. Biochemistry 2004, 43, 1093–1101.
[15]
Gowd, K.H.; Krishnan, K.S.; Balaram, P. Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation. Mol. Biosyst. 2007, 3, 554–566.
[16]
Price-Carter, M.; Gray, W.R.; Goldenberg, D.P. Folding of omega-conotoxins. 2. Influence of precursor sequences and protein disulfide isomerase. Biochemistry 1996, 35, 15547–15557.
[17]
Wang, Z.Q.; Han, Y.H.; Shao, X.X.; Chi, C.W.; Guo, Z.Y. Molecular cloning, expression and characterization of protein disulfide isomerase from Conus marmoreus. FEBS J. 2007, 274, 4778–4787.
[18]
Moller, C.; Mari, F. 9.3 KDa components of the injected venom of Conus purpurascens define a new five-disulfide conotoxin framework. Biopolymers 2011, 96, 158–165, doi:10.1002/bip.21406.
[19]
McIntosh, J.M.; Ghomashchi, F.; Gelb, M.H.; Dooley, D.J.; Stoehr, S.J.; Giordani, A.B.; Naisbitt, S.R.; Olivera, B.M. Conodipine-M, a novel phospholipase A2 isolated from the venom of the marine snail Conus magus. J. Biol. Chem. 1995, 270, 3518–3526.
[20]
Kini, R.M. Venom Phospholipase A2 Enzymes, Structure, Function and Mechanism; Kini, R.M., Ed.; John Wiley & Sons: Chichester, UK, 1997.
[21]
Stern, R.; Jedrzejas, M.J. Hyaluronidases: their genomics, structures, and mechanisms of action. Chem. Rev. 2006, 106, 818–839.
[22]
Terrat, Y.; Biass, D.; Dutertre, S.; Favreau, P.; Remm, M.; St？cklin, R.; Piquemal, D.; Ducancel, F. High-resolution picture of a venom gland transcriptome: Case study with the marine snail Conus consors. Toxicon 2011, 59, 34–46.
[23]
Dutertre, S.; Biass, D.; St？cklin, R.; Favreau, P. Dramatic intraspecimen variations within the injected venom of Conus consors: an unsuspected contribution to venom diversity. Toxicon 2010, 55, 1453–1462.
[24]
Sutherland, S.K.; Lane, W.R. Toxins and mode of envenomation of the common ringed or blue-banded octopus. Med. J. Aust. 1969, 1, 893–898.
[25]
Fry, B.G.; Scheib, H.; van der Weerd, L.; Young, B.; McNaughtan, J.; Ramjan, S.F.; Vidal, N.; Poelmann, R.E.; Norman, J.A. Evolution of an arsenal: structural and functional diversification of the venom system in the advanced snakes (Caenophidia). Mol. Cell. Proteomics 2008, 7, 215–246.
[26]
Harrison, R.A.; Ibison, F.; Wilbraham, D.; Wagstaff, S.C. Identification of cDNAs encoding viper venom hyaluronidases: cross-generic sequence conservation of full-length and unusually short variant transcripts. Gene 2007, 392, 22–33.
Feng, L.; Gao, R.; Meng, J.; Gopalakrishnakone, P. Cloning and molecular characterization of BmHYA1, a novel hyaluronidase from the venom of Chinese red scorpion Buthus martensi Karsch. Toxicon 2010, 56, 474–479.
[29]
Madokoro, M.; Ueda, A.; Kiriake, A.; Shiomi, K. Properties and cDNA cloning of a hyaluronidase from the stonefish Synanceia verrucosa venom. Toxicon 2011, 58, 285–292.
[30]
Magalhaes, M.R.; da Silva, N.J., Jr.; Ulhoa, C.J. A hyaluronidase from Potamotrygon motoro (freshwater stingrays) venom: isolation and characterization. Toxicon 2008, 51, 1060–1067.
[31]
Ng, H.C.; Ranganathan, S.; Chua, K.L.; Khoo, H.E. Cloning and molecular characterization of the first aquatic hyaluronidase, SFHYA1, from the venom of stonefish (Synanceja horrida). Gene 2005, 346, 71–81.
[32]
Gmachl, M.; Kreil, G. Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm. Proc. Natl. Acad. Sci. USA 1993, 90, 3569–3573.
[33]
Kolarich, D.; Leonard, R.; Hemmer, W.; Altmann, F. The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris. FEBS J. 2005, 272, 5182–5190.
[34]
Feng, L.; Gao, R.; Gopalakrishnakone, P. Isolation and characterization of a hyaluronidase from the venom of Chinese red scorpion Buthus martensi. Comp Biochem. Physiol. C. Toxicol. Pharmacol. 2008, 148, 250–257.
[35]
Pessini, A.C.; Takao, T.T.; Cavalheiro, E.C.; Vichnewski, W.; Sampaio, S.V.; Giglio, J.R.; Arantes, E.C. A hyaluronidase from Tityus serrulatus scorpion venom: isolation, characterization and inhibition by flavonoids. Toxicon 2001, 39, 1495–1504.
[36]
Nagaraju, S.; Devaraja, S.; Kemparaju, K. Purification and properties of hyaluronidase from Hippasa partita (funnel web spider) venom gland extract. Toxicon 2007, 50, 383–393.
[37]
Girish, K.S.; Shashidharamurthy, R.; Nagaraju, S.; Gowda, T.V.; Kemparaju, K. Isolation and characterization of hyaluronidase a "spreading factor" from Indian cobra (Naja naja) venom. Biochimie 2004, 86, 193–202.
[38]
Muchmore, D.B.; Vaughn, D.E. Review of the mechanism of action and clinical efficacy of recombinant human hyaluronidase coadministration with current prandial insulin formulations. J. Diabetes Sci. Technol. 2010, 4, 419–428.
[39]
Frost, G.I. Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration. Expert Opin. Drug Deliv. 2007, 4, 427–440.
[40]
Farr, C.; Menzel, J.; Seeberger, J.; Schweigle, B. Clinical pharmacology and possible applications of hyaluronidase with reference to Hylase "Dessau". Wien. Med. Wochenschr. 1997, 147, 347–355.
[41]
Tanaka, T.; Nakatani, T. New Therapeutic Strategies for Castration-Resistant Prostate Cancer. Recent Pat. Anticancer Drug Discov. 2011, 6, 373–383.
Mikesh, L.M.; Ueberheide, B.; Chi, A.; Coon, J.J.; Syka, J.E.; Shabanowitz, J.; Hunt, D.F. The utility of ETD mass spectrometry in proteomic analysis. Biochim. Biophys. Acta 2006, 1764, 1811–1822.
[44]
Mann, M.; Jensen, O.N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 2003, 21, 255–261.
[45]
Craig, A.G.; Bandyopadhyay, P.; Olivera, B.M. Post-translationally modified neuropeptides from Conus venoms. Eur. J. Biochem. 1999, 264, 271–275.
[46]
Le Gall, F.; Favreau, P.; Benoit, E.; Mattei, C.; Bouet, F.; Menou, J.L.; Menez, A.; Letourneux, Y.; Molgo, J. A new conotoxin isolated from Conus consors venom acting selectively on axons and motor nerve terminals through a Na+-dependent mechanism. Eur. J. Neurosci. 1999, 11, 3134–3142.
Chao, K.L.; Muthukumar, L.; Herzberg, O. Structure of human hyaluronidase-1, a hyaluronan hydrolyzing enzyme involved in tumor growth and angiogenesis. Biochemistry 2007, 46, 6911–6920.
[49]
Yan, B.; Zhang, W.; Ding, J.; Gao, P. Sequence pattern for the occurrence of N-glycosylation in proteins. J. Protein Chem. 1999, 18, 511–521.
[50]
Cantarel, B.L.; Coutinho, P.M.; Rancurel, C.; Bernard, T.; Lombard, V.; Henrissat, B. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009, 37, D233–D238.
[51]
Le, S.Q.; Gascuel, O. An improved general amino acid replacement matrix. Mol. Biol. Evol. 2008, 25, 1307–1320.
[52]
Csoka, A.B.; Frost, G.I.; Stern, R. The six hyaluronidase-like genes in the human and mouse genomes. Matrix Biol. 2001, 20, 499–508.
[53]
Holford, M.; Zhang, M.M.; Gowd, K.H.; Azam, L.; Green, B.R.; Watkins, M.; Ownby, J.P.; Yoshikami, D.; Bulaj, G.; Olivera, B.M. Pruning nature: Biodiversity-derived discovery of novel sodium channel blocking conotoxins from Conus bullatus. Toxicon 2009, 53, 90–98.
[54]
Safavi-Hemami, H.; Siero, W.A.; Gorasia, D.G.; Young, N.D.; Macmillan, D.; Williamson, N.A.; Purcell, A.W. Specialisation of the Venom Gland Proteome in Predatory Cone Snails Reveals Functional Diversification of the Conotoxin Biosynthetic Pathway. J. Proteome Res. 2011, 3904–3919.
[55]
Hopkins, C.; Grilley, M.; Miller, C.; Shon, K.J.; Cruz, L.J.; Gray, W.R.; Dykert, J.; Rivier, J.; Yoshikami, D.; Olivera, B.M. A new family of Conus peptides targeted to the nicotinic acetylcholine receptor. J. Biol. Chem. 1995, 270, 22361–22367.
[56]
Tastet, C.; Lescuyer, P.; Diemer, H.; Luche, S.; van Dorsselaer, A.; Rabilloud, T. A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins. Electrophoresis 2003, 24, 1787–1794.
[57]
Castellanos-Serra, L.; Vallin, A.; Proenza, W.; Le Caer, J.P.; Rossier, J. An optimized procedure for detection of proteins on carrier ampholyte isoelectric focusing and immobilized pH gradient gels with imidazole and zinc salts: its application to the identification of isoelectric focusing separated isoforms by in-gel proteolysis and mass spectrometry analysis. Electrophoresis 2001, 22, 1677–1685.
[58]
Di Ferrante, N. Turbidimetric measurement of acid mucopolysaccharides and hyaluronidase activity. J. Biol Chem. 1956, 220, 303–306.
[59]
Bordoli, L.; Kiefer, F.; Arnold, K.; Benkert, P.; Battey, J.; Schwede, T. Protein structure homology modeling using SWISS-MODEL workspace. Nat. Protoc. 2009, 4, 1–13.
[60]
Edgar, R.C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 2004, 5, 113.
[61]
Talavera, G.; Castresana, J. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst. Biol. 2007, 56, 564–577.
[62]
Darriba, D.; Taboada, G.L.; Doallo, R.; Posada, D. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 2011, 27, 1164–1165.
[63]
Guindon, S.; Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 2003, 52, 696–704.
[64]
Tamura, K.; Dudley, J.; Nei, M.; Kumar, S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 2007, 24, 1596–1599, doi:10.1093/molbev/msm092.
[65]
Kreil, G. Hyaluronidases--a group of neglected enzymes. Protein Sci. 1995, 4, 1666–1669.
