Identification of a Novel Family of Snake Venom Proteins Veficolins from Cerberus rynchops Using a Venom Gland Transcriptomics and Proteomics Approach G. OmPraba, †,‡ Alex Chapeaurouge, †,§ Robin Doley, †,# K. Rama Devi, ⊥ P. Padmanaban, ⊥ C. Venkatraman, ⊥ D. Velmurugan, ‡ Qingsong Lin, | and R. Manjunatha Kini* ,†,∇ Protein Sciences Laboratory, Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543, Centre for Advanced Studies in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India, Laborato ´rio de Toxinologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ 21045-900, Brazil, Zoological Survey of India, Marine Biological Station, Chennai 600 028, India, Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543, and Department of Biochemistry and Molecular Biology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0614 Received November 13, 2009 Cerberus rynchops (dog-faced water snake) belongs to Homalopsidae of Colubroidea (rear-fanged snakes). So far, venom compositions of snakes of the Homalopsidae family are not known. To determine the venom composition of C. rynchops, we have used both transcriptomics and proteomics approaches. The venom gland transcriptome revealed 104 ESTs and the presence of three known snake protein families, namely, metalloprotease, CRISP, and C-type lectin. In addition, we identified two proteins that showed sequence homology to ficolin, a mammalian protein with collagen-like and fibrinogen- like domains. We named them as ryncolin 1 and ryncolin 2 (rynchops ficolin) and this new family of snake venom proteins as veficolins (venom ficolins). On the basis of its structural similarity to ficolin, we speculate that ryncolins may induce platelet aggregation and/or initiate complement activation. To determine the proteome, the whole C. rynchops venom was trypsinized and fractionated by reverse phase HPLC followed by MALDI-MS/MS analysis of the tryptic peptides. Analysis of the tandem mass spectrometric data indicated the presence of all protein families compared to the translated cDNA library. Overall, our combined approach of transcriptomics and proteomics revealed that C. rynchops venom is among the least complex snake venom characterized to date despite the presence of a new family of snake venom proteins. Keywords: Venom transcriptome • venomics • Venom proteome • snake venom toxicity • innate immunity Introduction Snake venoms are complex mixtures of biologically active peptides and proteins which bind specifically to exogenous targets, such as receptors and ion channels. Thus, snake venom proteins have not only significantly contributed to the under- standing of basic physiological systems, like the characteriza- tion of acetylcholine receptors by R-bungarotoxin 1,2 and the complement system by cobra venom factor (CVF), 3 but also led to the development of original diagnostic agents 4 as well as therapeutic agents (for example, antihypertensive drug captopril). 5,6 With the advent of new technologies, we are now able to identify new toxins from smaller amounts of venoms or venom gland tissues. Such studies will foster our under- standing of function and evolution of venomous systems and accelerate developments in drug discovery. Cerberus rynchops is a nocturnal Colubrid snake encountered in mangroves and brackish rivers in Asia and Australia. 7 It belongs to Homalopsidae family, and to date, venom composi- tions of snakes of Homalopsidae family are not well character- ized. Homalopsids feed on frogs, fish, and tadpoles and are phylogenetically of particular interest since recent studies indicate that they represent a basal colubrid family. 8,9 To identify toxin components and understand the possible phar- macological effects, we have investigated the venom composi- tion of C. rynchops using a combined transcriptomic and proteomic approach. Our results indicate that the venom of C. rynchops contains only four different protein families, which can be classified as metalloprotease, C-type lectin, CRISP, and a novel snake venom family referred to as veficolins (venom * To whom correspondence should be addressed. R. Manjunatha Kini, Protein Sciences Laboratory, Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543. E-mail: dbskinim@nus.edu.sg. † Protein Sciences Laboratory, National University of Singapore. ‡ University of Madras. § Laborato ´rio de Toxinologia, Instituto Oswaldo Cruz. # Current address: Department of Molecular Biology and Biotechnology, Tezpur University, Assam, India. ⊥ Zoological Survey of India, Marine Biological Station. | Department of Biological Sciences, National University of Singapore. ∇ Virginia Commonwealth University. 1882 Journal of Proteome Research 2010, 9, 1882–1893 10.1021/pr901044x  2010 American Chemical Society Published on Web 02/16/2010