Toxicon 43 (2004) 751–759 www.elsevier.com/locate/toxicon Identification of crotasin, a crotamine-related gene of Crotalus durissus terrificus G. Ra´dis-Baptistaa,*, T. Kubob, N. Oguiurac, A.R.B. Prieto da Silvaa, M.A.F. Hayashid, E.B. Oliveirae, T. Yamanea aMolecular Toxinology Laboratory, Butantan Institute, Av. Vital Brazil 1500, Sa˜o Paulo 05503-900, Brazil bMolecular Neurophysiology Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba Central 6, Tsukuba 305-8566, Japan cLaboratory of Herpetology, Butantan Institute, Av. Vital Brazil 1500, Sa˜o Paulo 05503-900, Brazil dLaboratory of Biochemistry and Biophysics, Av. Vital Brazil 1500, Sa˜o Paulo 05503-900, Brazil eDepartment of Biochemistry, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Ribeira˜ Preto 14049-900, Brazil Received 25 November 2003; accepted 25 February 2004 Abstract Crotamine is a cationic peptide (4.9 kDa, pI 9.5) of South American rattlesnake, Crotalus durissus terrificus’ venom. Its presence varies according to the subspecies or the geographical locality of a given species. At the genomic level, we observed the presence of 1.8 kb gene, Crt-p1, in crotamine-positive specimens and its absence in crotamine-negative ones. In this work, we described a crotamine-related 2.5 kb gene, crotasin (Cts-p2), isolated from crotamine-negative specimens. Reverse transcription coupled to polymerase chain reaction indicates that Cts-p2 is abundantly expressed in several snake tissues, but scarcely expressed in the venom gland. The genome of crotamine-positive specimen contains both Crt-p1 and Cts-p2 genes. The present data suggest that both crotamine and crotasin have evolved by duplication of a common ancestor gene, and the conservation of their three disulfide bonds indicates that they might adopt the same fold as b-defensin. The physiological function of the crotasin is not yet known. q 2004 Elsevier Ltd. All rights reserved. Keywords: Animal toxin; Molecular toxinology; Molecular evolution; Toxin gene; Expression pattern; Gene duplication; Paralogous gene; Intron phase; Accelerated evolution; Protein fold 1. Introduction Crotamine belongs to a closely related group of myotoxin-a like proteins that are widely distributed among Vellard (1937) reported the dependence of the venoms of the rattlesnake species, Crotalus, but its presence physiopathological effect of Crotalus durissus venom on depends on the subspecies or the geographical locality of a its geographical origin, reflecting the variability of the given species (Schenberg, 1959; Bober et al., 1988; Ownby, venom composition. In 1956, Gonc¸alves isolated the 1998). Crotamine-positive subspecies of C. d. terrificus first small basic myotoxin from the venom of South inhabit regions extending roughly from western part of the American rattlesnake, C. d. terrificus, and described state of Sa˜o Paulo (west of the 49th meridian) into northern the venom polymorphism in relation to crotamine and western part of the state of Parana´ and 218 south (Bober (Gonc¸alves, 1956). et al., 1988). Dry crude venom yields approximately 10% of crotamine by weight, although intra-specific variation may * Corresponding author. Tel.: þ55-11-3726-1127; fax: þ55-11- occur. Crotamine shares a 92.9% peptide sequence similarity 3726-1501. with crotamine-a from Crotalus viridis viridis,prairie E-mail address: [email protected] (G. Ra´dis-Baptista). rattlesnake, of Western USA (Ownby, 1998). 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.02.023 752 G. Ra´dis-Baptista et al. / Toxicon 43 (2004) 751–759 Crotamine causes similar pathological effects as the 2. Materials and methods most studied myotoxin-a does (Cameron and Tu, 1978) that is, induction of localized myonecrosis of skeletal 2.1. Snake specimens muscle cell with a low onset of action (Fletcher et al., 1996). It has been reported that crotamine increases the Specimens of C. d. terrificus crotamine-positive and voltage-activated Naþ current causing a high depolariz- negative were obtained from the Herpetology Laboratory, ation of the muscle fiber membrane at the junctional Instituto Butantan, Sa˜o Paulo, Brazil. Crotamine-positive region of the myocyte membrane, which is prevented by specimens (Cdt 001, Cdt 002, Cdt 9705 and Cdt 9706) were tetrodotoxin (Matavel et al., 1998). On the other hand, captured in western region of Sa˜o Paulo State. Crotamine- myotoxin-a that differs from crotamine by only three negative ones (Cdt 93015, Cdt 9315-2, Cdt 00081 and Cdt amino acids (92.9% amino acid sequence homology) 00083) were from eastern region of the State. seems to prevent the Ca2þ uptake by interacting to the sarcoplasmic reticulum Ca2þ-ATPase (Utaisincharoen 2.2. Preparation of genomic DNAs et al., 1991) or by releasing calcium ions from the calsequestrin stocks (Okhura et al., 1995) or through Snakes were anesthetized with carbon dioxide and ryanodine receptors (Yudkowisky et al., 1994). However, decapitated. Livers were excised and quickly frozen in the basic mechanism by which these toxins, crotamine liquid nitrogen. For DNA isolation, frozen livers were and myotoxin-a; act is still unresolved. ground to a powder under liquid nitrogen with a mortar and Our previous comparative studies on Northern blot pestle and purified in the presence of proteinase K and SDS, analysis of poly(Aþ) RNA from venom glands of a according to Ausubel et al. (1995). crotamine-positive specimen and total RNA and poly(Aþ) RNA from a crotamine-negative rattlesnake, C. d. 2.3. Construction of the C. d. terrificus crotamine-negative terrificus, revealed that crotamine transcripts were not genomic library expressed in the crotamine-negative specimens (Ra´dis- Baptista et al., 1999). In addition, Oguiura et al. (2000) A genomic phage library from C. d. terrificus’s liver crotamine-negative (specimen No. 93015) was constructed devised an enzyme-linked immunosorbent assay method using the Lambda-FIXII/Xho I partial fill-in Kit, essentially to detect crotamine in crude venom, and correlating the as described by the manufacturer (Stratagene, La Jolla, CA). pattern of expression of crotamine in the venom of C. d. terrificus variants. 2.4. Isolation sequencing and analysis of crotasin The crotamine gene, from C. d. terrificus, has 1.8 kb gene (Cts-p2) organized into three exons separated by a long phase-1 (900 bp) and a short phase-2 (140 bp) introns. Exon I Based on highly conserved crotamine gene sequence includes the 50-untranslated region and codes for the first (Ra´dis-Baptista et al., 2003), two oligonucleotides PRO-S2 19 amino acids of the signal peptide. Exon II codifies 42 (50-ACACTGCAAGTCCTGAGCCT-30) and NOR-RV amino acids, three belonging to the signal peptide and 39 (50-TAGCAGGCGGCACTCTCAGGT-30) were synthe- to the mature crotamine. Exon III codes for the 0 0 sized, which correspond to 5 -UTR and 3 -UTR of the last three amino acids of the mature toxin, including 0 Crt-p1 gene, respectively. The DNA, isolated from liver of the terminal lysine, and the 3 -untranslated region. The crotamine-negative specimen Cdt 93015, was used in a long crotamine gene, Cts-p1, is localized to the distance polymerase chain reaction (LD-PCR) protocol. telomeric region of the chromosome 2 long arm of C. This crotamine-related gene, crotasin (Cts-p2), was d. terrificus crotamine-positive karyotype (Ra´dis-Baptista amplified using 1 mg of genomic DNA or 107 –108 phage et al., 2003). particles (pfu) of amplified genomic library, 10 pmol of Southern blotting analysis of genomic DNA per- each primer, and 25 ml of ExLONGase enzyme mix formed in our laboratory indicated the presence of (Invitrogen Life Technologies, San Diego), as described crotamine related sequences in crotamine-negative gen- by the manufacturer. The LD-PCR products were purified ome. This fact hinted the probable presence of a from the gel slice and cloned into pCR2.1-TOPO (Invitro- defective crotamine gene or a related gene in crota- gen Life Technologies, San Diego). mine-negative specimens, and the cause of absence of The gene was sequenced with ABI Prism Big Dye crotamine expression in the venom of crotamine-negative Terminator (Perkin–Elmer, Foster City, CA) in an auto- specimen might be operating at the DNA level. mated sequencer (ABI Prism 373 or 377, Perkin–Elmer) In the present work, we report the genomic sequences using synthetic oligonucleotides designed from the Crt-p1 and analysis of crotamine gene, Crt-p1, present only in sequences (Ra´dis-Baptista et al., 2003). crotamine-positive specimens, and crotamine-related gene Theclonedcrotasingene,Cts-p2 (accession no. crotasin gene, Cts-p2, present in both crotamine-positive AF250212), were compared against crotamine gene, and negative specimens. Crt-p1 (AF223646 and AF223947), and crotamine mRNA G. Ra´dis-Baptista et al. / Toxicon 43 (2004) 751–759 753 sequences (AF04674 and AF53075) on the GenBank at 3. Results and discussion NCBI (http://www.ncbi.nlm.nih.gov), using the BLAST algorithm (Altschul et al., 1997) and using the Biocomput- We have previously shown that crotamine gene tran- ing software Lasergene (DNAStar, Inc., Madison, WI). scripts are present only in mRNA pool isolated from venom glands of crotamine-positive specimens and not in crota- 2.5. RT-PCR for analysis of Cts-p2 expression mine-negative rattlesnakes, C. d. terrificus. Several mRNA precursors, coding for two crotamine isoforms, were also To verify the expression pattern of Cts-p2 in crotamine- isolated (Ra´dis-Baptista et al., 1999). Recently, we have negative rattlesnake’s organs, reverse transcription and PCR reported the structure and chromosomal localization of ´ amplification were carried out in one tube reaction, using the crotamine gene in C. d. terrificus (Radis-Baptista et al., Super-Script One-Step PCR Kit (Invitrogen Life Technol- 2003). However, Southern blot analysis of genomic DNA, ogies, San Diego, CA).