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Snake venom glutaminyl cyclase
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Toxicon 48 (2006) 278–286 www.elsevier.com/locate/toxicon
Snake venom glutaminyl cyclase Joanna Pawlak, R. Manjunatha Kini Department of Biological Sciences, Faculty of Science, National University of Singapore, Science Drive 4, Singapore 117543, Singapore Received 25 March 2006; received in revised form 24 May 2006; accepted 30 May 2006 Available online 14 June 2006
Abstract Glutaminyl cyclase (QC) catalyzes N-terminal glutamine cyclization of many endocrine peptides and is typically abundant in brain tissue. As three-finger toxins in the venoms of colubrid snakes Boiga dendrophila and Boiga irregularis contain N-terminal pyroglutamate, we searched for QC in venom glands of both snakes. Here we report cDNA sequences of QC from brain and venom gland tissues of Boiga species. We propose that QC expressed in snake venom gland tissue plays a role in the N-terminal pyroglutamate formation of several snake venom toxins, indirectly contributing to venom potency. r 2006 Elsevier Ltd. All rights reserved. Keywords: Glutaminyl cyclase; Pyroglutamic acid; Snake venom; Three-finger toxins
1. Introduction Snake venom is a complex mixture of bioactive peptides and proteins, which integrated with delivery system, create one of the most sophisticated weapons in the natural world. Snake venom includes enzymatic (e.g. phospholipases, metalloproteinases, serine proteinases, phosphodiesterases, acetylocholinesterase, L-amino acid oxidases) and non-enzymatic polypeptides (e.g. three-finger toxins, lectins, natriuretic peptides, CRISPs, waprins, desintegrins, sarafotoxins) (Kini, 2002). Amongst non-enzymatic components, three-finger toxins (3FTXs) form a broad and well-recognized superfamily. All members of this family share small stable three-finger-like scaffold, but are bestowed with a broad array of functions (e.g. antagonism of various Corresponding author. Tel.: 65 6516 5235; fax: 65 6779 2486.
E-mail address: [email protected] (R. Manjunatha Kini). 0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.05.013
subtypes of nicotinic acetylcholine receptors, cardiotoxicity, agonism of muscarinic receptors, inhibition of L-type calcium channel and acetylocholinesterase) (Tsetlin, 1999; Kini, 2002). Hence, 3FTXs are extensively used as tools in biomedical research, diagnostics and therapeutics. Previously, they were thought to be limited to the venoms of elapid (cobras, kraits, mambas) and hydrophid (sea snakes) snakes (Kini, 2002). Recently, we reported the first three-finger neurotoxin (a-colubritoxin) from colubrid snake Coelognathus radiatus (Swissprot ]P83490) (Fry et al., 2003) and subsequently determined 3FTX sequences from other colubrids: Boiga dendrophila and Boiga irregularis (GenBank ]s DQ366293, DQ304538, DQ304539). Interestingly, unlike elapid, hydrophid and recently reported viperid 3FTXs (Junqueira-de-Azevedo et al., 2006), all 3FTXs isolated so far from colubrid snakes, possess a characteristic long N-terminal which is blocked by pyroglutamate (pGlu). In
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nature, spontaneous deamination of N-terminal glutaminyl precursor to pGlu occurs very slowly under physiological conditions and in vivo this posttranslational modification is catalyzed by the glutaminyl cyclases (EC 2.3.2.5; QCs) (Busby et al., 1987; Fischer and Spiess, 1987). Therefore, the presence of pGlu at the N-terminal of 3FTXs in the venom of colubrid snakes indicated the expression of QC in their venom gland. QCs are 33–40 kDa acyltransferases, highly conserved from yeast to humans (Huang et al., 2005). They were identified in plant and animal sources (Busby et al., 1987; Fischer and Spiess, 1987; Oberg et al., 1998; Huang et al., 2005) and they are especially abundant in vertebrate neuroendocrine tissues, such as hypothalamus and pituitary (Busby et al., 1987). Cyclization of N-terminal glutamine is an important posttranslational event in the processing of numerous bioactive proteins [12.3% proteins with signal peptides (Liao et al., 2003)] including neuropeptides, hormones and cytokines during their maturation in the secretory pathway (Huang et al., 2005). The N-terminal pGlu protects them from exopeptidase degradation and/or enables them to have proper conformation for binding to the receptors (Van Coillie et al., 1998; Hinke et al., 2000). Present paper provides the evidence for the presence of QC in the venom gland of colubrid snakes (Boiga) and discusses putative role of QC in the snake venom. 2. Materials and method 2.1. Total RNA isolation Boiga dendrophila (Sulawesi) and Boiga irregularis (Guam) venom glands and B. irregularis brain tissue were kindly provided by Dr. Stephen P. Mackessy, University of Northern Colorado, CO, USA. Specimens were sacrificed by decapitation. Venom glands and brain were dissected and kept in RNAlater solution in 80 1C until use. Total RNA extraction was performed with rotor homogenizer and RNeasy mini kit from Qiagen (Valencia, CA, USA). Amount of RNA was calculated according to 260 nm absorbance of the sample. 2.2. Reverse transcription—Polymerase chain reaction (RT-PCR) QC sequences from different sources (human, mouse and frog) were aligned in order to design
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internal forward GSP1f 50 -GCTGTGCCTTGTGC AATGAT-30 and reverse GSP1r 50 -GTATGCCA TACTCTAGGGAAAGG-30 primers from the most conserved regions. Using total RNA isolated from venom glands as templates, RT-PCR was performed to amplify partial cDNAs of QCs. Onestep RT-PCR (Qiagen) was carried out using 100 ng of total RNA as follows: RT at 50 1C for 30 min, activation of hot start Taq polymerase at 95 1C for 15 min and 30 cycles of 3-step PCR (94 1C for 1 min, 50 1C for 1 min, 72 1C for 1 min) with the final extension at 72 1C for 10 min. PCR products were subjected to 1% agarose gel electrophoresis and visualized by ethidium bromide staining. As the obtained partial sequences showed high identity to cDNA of QC from Agkistrodon blomhoffi (BAB69586), another forward primer was designed based on crotalid’s 50 UTR sequence GSP2f (50 -TGAATCTGCCTC CCTCTGAGG-30 ) and used in RT-PCR, together with reverse primer GSP2r 50 -GGGACCATCGCA CAAAAGCT-30 . This set of primers was also used to fish out cDNA of QC from the brain of B. irregularis. 2.3. 30 Rapid amplification of cDNA ends (RACE) The 30 RACE cDNA libraries were constructed using Invitrogen (Carlsbad, CA, USA) 30 RACE kit for rapid amplification of cDNA ends according to the manufacturer’s protocol. cDNA was synthesized from 1.0 mg of total RNA using SuperScript II reverse transcriptase and 30 RACE adapter primer [50 -GGCCACGCGTCGACTAGTAC(T)17-30 ]. Reverse transcription reaction product was treated with RNase H (2 U) for 30 min at 37 1C and further used as a template in PCR (95 1C for 15 min followed by 30 cycles of 3-step PCR: 94 1C for 1 min, 50 1C for 1 min, 72 1C for 1 min and the final extension at 72 1C for 10 min) with two different forward primers GSP3f 50 -GACGACCATATTC CATTTTTGAGA-30 , GSP4f 50 TGTTTGTTGGA GCCACCGACT 30 and reverse 30 -RACE abridged universal amplification primer 50 -GGCCACGC GTCGACTAGTAC-30 . The PCR products obtained were visualized using ethidium bromide staining following agarose gel (1%) electrophoresis. Using sequence data generated by RT-PCR and 30 RACE reactions, new sets of gene-specific primers were designed to generate the full-length cDNA of QC from both snake species and to confirm the sequence.
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2.4. Cloning and sequencing of PCR products
Sequence analysis was carried out using BLAST program at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov) and ExPASy proteomics tools at www.expasy.org. Sequence alignments and neighbor-joining tree construction were carried out using DNAMAN version 4.15 (Lynnon Biosoft).
deduced protein sequence shows the highest (96%) identity to Agkistrodon QC (BAB69586) and quite high (63%) identity to human QC (NP_036545). All functional residues in the active site as well as three residues (Asp164, Glu208, His308) involved in binding to the zinc ion of the catalytic center, are conserved in Boiga QCs. Based on the multiple sequence alignment and the crystal structure of human QC (Huang et al., 2005), we predict that secondary structure of Boiga QCs have mixed a=b fold with an open sandwich topology, similar to human QC (Fig. 2). In addition to the full length clones, a short variant of QC cDNA was found in the venom gland and brain of B. irregularis, but not in B. dendrophila venom gland. This variant lacks 180 nucleotides, leading to putative in-frame deletion mutant shorter by 60 amino acid residues (Fig. 1), which constitute b4 strand and a7 helix in human QC (Fig. 2). The fragment absent in the short variant aligns well with 167 nucleotides long, fourth exon of the bovine QC gene (GeneID 281437). The 30 end of the exon 4 exactly matches the end of deletion sequence. However, at the 50 end, the deletion segment is 13 nucleotides longer than bovine exon 4 and thus the deleted sequence does not correspond to exon 4 alone. As with other genes, we believe that in the genes of QCs intron-exon boundaries are also conserved. Therefore, the short variant may not be a product of simple alternate splicing.
3. Results
3.2. Signal peptide and secretion
3.1. cDNA sequence of brain and venom QCs
Human, bovine as well as other QCs are secreted proteins and their precursors contain a signal peptide. We subjected snake QC sequence for signal peptide identification. SignalP 3.0 algorithm (http:// www.cbs.dtu.dk/services/SignalP/) confirmed the presence of signal peptide, but predicted three possible cleavage sites. Neural (eukaryotic) network method predicted that most likely cleavage occurs between residues 23 and 24 (CLA-LP) (Fig. 1), while hidden Markov (eukaryotic) models method predicted maximal cleavage site probability between residues 28 and 29 (GFP-QH). The latter putative cleavage may require self-glutamyl cyclization. Apart from these sites, both methods predicted the cleavage between residues 33 and 34 (VGG-RE) but with lower probability. As signal peptide cleavage site is ambiguous, the length of mature enzyme can be 345, 340 or 335 amino acid residues and thus we cannot precisely establish the molecular weight
All amplification products were ligated with pDrive vector (Qiagen) and transformed by heat shock method into DH5a strain of competent E. coli cells. Selection of the transformants (blue/white colony screening) was performed on LB-agar plates containing 100 mg/ml ampicillin and supplemented with IPTG and X-gal. Sizes of the inserts were estimated by EcoRI digestion followed by 1% agarose gel electrophoresis and ethidium bromide visualization. All sequencing reactions were carried out on the ABI PRISMs 3100 automated DNA sequencer, using the ABI PRISM BigDye terminator cycle sequencing ready reaction kit purchased from Applied Biosystems (Foster City, CA, USA), according to the manufacturer’s instructions. All clones were sequenced in both directions using T7 and SP6 sequencing primers and gene specific primers. 2.5. Sequence analysis and phylogenetic tree
The cDNA encoding QCs present in the venom glands of B. irregularis (DQ404533) and B. dendrophila (DQ404534) were obtained by RT-PCR followed by 30 RACE, providing direct evidence that QC is expressed in the snake venom gland. For comparison, cDNA encoding QC from the brain of B. irregularis was sequenced and the results showed that QC cDNAs from the brain and venom glands of B. irregularis are identical. All cDNA sequences encode for putative open reading frames of 1107 nucleotides, coding for 368 amino acids (Fig. 1). cDNA sequences from B. irregularis and B. dendrophila show 98% nucleotide identity to each other and have mutations either in untranslated regions (21 mutations and three deletions) or synonymous (six mutations). Consequently, both are 100% identical at the protein level. Their
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Fig. 1. Pairwise alignment of cDNA sequences encoding venom QCs from Boiga snakes. Differences in nucleotide sequences are highlighted in gray. Polyadenylation signals (AATAAA) are underlined with single line and stop codon (TAA) is indicated by an asterisk. The deduced amino acid sequence is shown in single letter code beneath nucleotide sequences. Nucleotide and amino acid numbers are shown in the column on the right starting from translational start site. Putative signal peptide predicted by neuronal networks method is boxed. The corresponding missing nucleotide segment in short variant of QC from B. irregularis is double underlined. As the cDNA sequence of the brain QC from B. irregularis is identical to its venom QC, it is not included in the alignment.
(39526–38536 Da). Although we expect this enzyme to be present in the venom, its mass was not detected in LC-MS profile (Fry et al., 2003) or SDSPAGE gel of Boiga venoms (Mackessy, 2002; Mackessy et al., 2006). Thus QCs are found either in only small quantities or their masses vary significantly due to posttranslational modifications (like glycosylation). 3.3. Posttranslational modifications Typically snake venom proteins have high content of cysteine residues (10%) and multiple disulfide bridges. However, QC from snake venom
possesses only two cysteine residues (Cys143 and Cys169) forming one disulfide bridge (Huang et al., 2005). This sole disulfide is conserved throughout all species (Fig. 2) and is important for the catalytic activity (Schilling et al., 2002). N-glycosylation is another significant modification observed in QCs, which may play a role in stabilization of the protein conformation or its functional activity (Rudd et al., 1994; Wang et al., 1996). The deduced amino acid sequence of human QC reveals two potential Nglycosylation sites (Asn49 and Asn296) (Song et al., 1994), while Boiga QCs have three potential N-glycosylation sites (Asn53, Asn292 and Asn352). The first site is conserved in mammals but is absent
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Fig. 2. Multiple sequence alignment of deduced amino acid sequences of QC from B. dendrophila and B. irregularis with several representative animal QCs. The sequences have been taken from following sources: Boiga dendrophila (this paper) DQ404534; Boiga irregularis (this paper) Q404533; Agkistrodon blomhoffi BAB69586; Bothrops jararaca BAA34290; Homo sapiens NP_036545; Mus musculus XP_128770; Rattus norvegicus XP_233812; Bos taurus NP_803472; Gallus gallus XP_419527; Xenopus laevis AAH80397, Caenorhabditis elegans CAB08740; Drosophila melanogaster AAV36900, Apis mellifera XP_395412. The residues that have 100% identity are colored in black and those with 470% identity are shaded in gray. Three residues responsible for zinc binding are marked with triangle above the sequence. Three putative N-glycosylation sites in snake QCs are indicated by hexagon under the sequence. The secondary structural elements of a-helices and b-strands according to human crystal structure (Huang et al., 2005) are also shown.
in chicken, frog and lower organisms (Fig. 2). The second site seems to be characteristic for snakes only, while the third site is also found in chicken QC. Interestingly, only snake QCs possess glycine residue at the C-terminal end (Fig. 2), which might undergo amidation. 3.4. Phylogenetic analyses of QCs A phylogenetic tree constructed using consensus bootstrapping separated well into the vertebrate and invertebrate clades. The vertebrate taxa were properly classified into tetrapod and non-tetrapod lineages indicating the authenticity of the phylogenetic tree. Further, the snake QC cluster divided into crotalid and colubrid QC groups. Hence, the phylogentic tree (Fig. 3) as well as multiple sequence
alignments (Fig. 2) show that QC sequences are highly conserved (67% identity) throughout the phylogeny, from yeast to mammals. 4. Discussion QC is a tissue-specific, differentially expressed enzyme, with transcription and translation correlated to the need for peptides with N-terminal pGlu (Sykes et al., 1999). The highest expression of QC was reported in pituitary and various brain regions (Pohl et al., 1991). Only very small levels were detected in some peripheral tissues like thymus and kidney but not in lung or liver (Pohl et al., 1991). We identified the presence of QC cDNAs in the venom gland tissue of two colubrid snakes, B. dendrophila and B. irregularis. Venom gland is a
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0.05
Homo sapiens
79
Canis familiaris Bos taurus
97
Mus musculus 100
Rattus norvegicus
100
Gallus gallus 95 100
95
100
Agkistrodon blomhoffi Bothrops jararaca Boiga dendrophila
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100
Boiga irregularis Xenopus laevis
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Danio rerio 99
Apis mellifera Drosophila melanogaster
44
Caenorhabditis elegans Saccharomyces cerevisiae
Fig. 3. Phylogenetic relationship among QCs. The neighbor-joining tree represents bootstrap values after 1000 replicates. Tree was plotted using DNAMAN. Sources of the sequences are same as in Fig. 2, except that three sequences from: Canis familiaris XP_532934, Danio rerio XP_689638 and Saccharomyces cerevisiae P43599 were added.
unique QC expression site associated only with snakes. Apart from the QC sequence of both Boiga species, other two sequences are known from crotalid snakes: Bothrops jararaca (BAA34290) and Agkistrodon blomhoffi (BAB69586). Although these sequences are reported in the GenBank database, so far there was no published literature on any snake venom QC. Snake venom proteins are secreted and contain a signal peptide for protein translocation through endoplasmic reticulum (Blobel and Dobberstein, 1975). The deduced protein sequences (obtained from genome and/or mRNA sequencing) indicate that QCs also contain signal peptides, as their N-termini (Pohl et al., 1991) show key features of signal sequence: a short positively charged region for penetrability, followed by central hydrophobic region extending across the membranes and the polar C-terminal for cleavage (Von Heijne, 1986). Signal peptides identified in colubrid and crotalid QCs corroborate well with the above observations,
although signal peptide of snake QCs have notably more charged residues towards N-terminus. Interestingly, three different signal peptide cleavage sites were predicted at positions 23–24, 28–29 and 33–34 in Boiga QCs (Fig. 1). All three sites are in agreement with the (3,1) rule (Von Heijne, 1986), by which the third last residue of signal peptide cannot be aromatic, charged or large/polar and the last residue of signal sequence should be small for the cleavage to occur. As the signal peptide region is not conserved, it is not possible to extrapolate the cleavage position of Boiga QCs based on other QCs. Bovine QC is the only one in which the N-terminal sequence has been determined by protein chemistry and the mature protein starts with Gly32 (Fischer and Spiess, 1987; Pohl et al., 1991). It is not clear whether bovine QC is atypical. Unlike other QCs, it may be synthesized as prepropeptide with putative signal peptide of 27 or 28 residues followed by short propeptide region. The propeptide segment is cleaved after dibasic
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residues Arg30–Arg31 (Pohl et al., 1991). However, such dibasic sites are not found in other QCs. Thus, the signal peptide cleavage site in Boiga QCs is yet to be determined. QCs from human (NCBI GeneID: 25797), bovine (281437), mouse (70536) and dog (403861) are encoded by a single gene with protein coding region of 1.1 kb. We obtained QC with similar size of open reading frame (1107 bp) as those of mammalian origin. In addition, we also obtained a short variant of QC from the brain and venom glands of B. irregularis. It shows 100% sequence identity with the full length QC, but for the missing segment of 180 bases. Although it is tempting to speculate that this short variant could be an alternate splice product, the missing segment is close to but not equivalent to the fourth exon of the bovine QC gene (GeneID 281437) (see Results). QC has been shown to be a metal dependent transferase (Schilling et al, 2002) and the short variant encodes for a protein lacking residue Glu208, important for the metal binding (Huang et al., 2005). Thus short variant may not be functional. Its genetic origin and physiological relevance is unclear. Study of B. irregularis QC gene organization, establishing exon/intron boundaries and splicing sites is needed to unequivocally decipher the origin of the short form of QC. Snake venom proteins have been recruited from different tissue types and manifold body proteins, to create venom proteome (Fry, 2005). For example, venom prothrombin activator has evolved from liver blood coagulation factor X (Reza et al., 2005). Similarly, venom phospholipase A2, 3FTXs and cobra venom factor have pancreatic phospholipase A2 (EC 3.1.1.4), lynx1/SLUR and complement C3 as their respective ancestors (Fry, 2005). Thus these venom proteins evolved through gene duplications and accelerated evolution in the protein-coding regions (Nakashima et al., 1995; Nobuhisa et al., 1996; Deshimaru et al., 1996; Ohno et al., 1998; Kini and Chan, 1999); only features that are essential for protein folding and structural integrity are conserved while functional residues are manipulated. Interestingly, the cDNA sequences from the brain and venom glands of B. irregularis encoding QC are identical (Fig. 1) and hence QC appears to be a single gene, which is recruited for expression in the venom gland without duplication. Snake venoms are one of the most concentrated enzyme sources surpassing pancreatic juice or liver
secretions, but the majority of snake venom enzymes are hydrolases, which play a crucial role in prey immobilization and digestion (Mebs, 1998). Only a few non-toxic enzymes have been identified as the components of the venom (e.g. ribonucleases, oligopeptidases, phosphodiesterases, 50 -nucleotidases, phosphatases) which might aid in initiation of prey digestion. It is hypothesized that some of these enzymes facilitate venom action by systemic distribution of other venom components or activation of precursors present in the inactive form in pre-secreted venom to prevent tissue damage and self-poisoning (Mebs, 1998). The existence of enzyme, like QC in the snake venom is intriguing. Venom QCs may not act as toxins themselves, but they catalyze the maturation of toxins and help in their protection from exopeptidase degradation and/or developing proper conformation. Thus venom QCs may indirectly contribute to venom toxicity. In crotalid venoms, several toxins are known to possess N-terminal pGlu, e.g. metalloproteases (Takeya et al., 1989), bradykinin-potentiating peptides (Chi et al., 1985), crotoxin (Aird et al., 1990) and Mojave toxin (Bieber et al., 1990). So far, among 3FTXs (Mebs and Claus, 1991), only colubrid 3FTXs are known to possess pGlu at their N-terminal. The N-terminally located glutamine in their precursor acts as a novel substrate for QC. The presence of both QC as well as pGlu-containing toxins in crotalid and colubrid venoms indeed reinforces that pyroglutamation is an important and required posttranslational modification, which might be crucial towards proper metabolism, integrity and activity of several venom components (Liao et al., 2003). In conclusion, QCs in the venom gland may play crucial role in cyclization of N-terminal glutamine in several snake venom toxins. Such posttranslational modifications often affect the biological activity and/or stability against proteolysis. Thus venom QCs may indirectly contribute to venom toxicity.
Acknowledgments We thank Dr. Stephen P. Mackessy for providing venom glands and brain tissues. This work was supported by a grant from the Biomedical Research Council, Agency for Science, Technology and Research, Singapore.
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References Aird, S.D., Yates III, J.R., Martino, P.A., Shabanowitz, J., Hunt, D.F., Kaiser, I.I., 1990. The amino acid sequence of the acidic subunit B-chain of crotoxin. Biochim. Biophys. Acta 1040, 217–224. Bieber, A.L., Becker, R.R., McParland, R., Hunt, D.F., Shabanowitz, J., Yates III, J.R., Martino, P.A., Johnson, G.R., 1990. The complete sequence of the acidic subunit from Mojave toxin determined by Edman degradation and mass spectrometry. Biochim. Biophys. Acta 1037, 413–421. Blobel, G., Dobberstein, B., 1975. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J. Cell Biol. 67, 835–851. Busby Jr., W.H., Quackenbush, G.E., Humm, J., Youngblood, W.W., Kizer, J.S., 1987. An enzyme(s) that converts glutaminyl-peptides into pyroglutamyl-peptides. Presence in pituitary, brain, adrenal medulla, and lymphocytes. J. Biol. Chem. 262, 8532–8536. Chi, C.W., Wang, S.Z., Xu, L.G., Wang, M.Y., Lo, S.S., Huang, W.D., 1985. Structure-function studies on the bradykinin potentiating peptide from Chinese snake venom (Agkistrodon halys Pallas). Peptides 6 (Suppl 3), 339–342. Deshimaru, M., Ogawa, T., Nakashima, K., Nobuhisa, I., Chijiwa, T., Shimohigashi, Y., Fukumaki, Y., Niwa, M., Yamashina, I., Hattori, S., Ohno, M., 1996. Accelerated evolution of crotalinae snake venom gland serine proteases. FEBS Lett. 397, 83–88. Fischer, W.H., Spiess, J., 1987. Identification of a mammalian glutaminyl cyclase converting glutaminyl into pyroglutamyl peptides. Proc. Natl. Acad. Sci. USA 84, 3628–3632. Fry, B.G., 2005. From genome to ‘‘venome’’: molecular origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences and related body proteins. Genome Res. 15, 403–420. Fry, B.G., Lumsden, N.G., Wuster, W., Wickramaratna, J.C., Hodgson, W.C., Kini, R.M., 2003. Isolation of a neurotoxin (alpha-colubritoxin) from a nonvenomous colubrid: evidence for early origin of venom in snakes. J. Mol. Evol. 57, 446–452. Fry, B.G., Wuster, W., Ryan Ramjan, S.F., Jackson, T., Martelli, P., Kini, R.M., 2003. Analysis of Colubroidea snake venoms by liquid chromatography with mass spectrometry: evolutionary and toxinological implications. Rapid Commun. Mass Spectrom. 17, 2047–2062. Hinke, S.A., Pospisilik, J.A., Demuth, H.U., Mannhart, S., Kuhn-Wache, K., Hoffmann, T., Nishimura, E., Pederson, R.A., McIntosh, C.H., 2000. Dipeptidyl peptidase IV (DPIV/ CD26) degradation of glucagon. Characterization of glucagon degradation products and DPIV-resistant analogs. J. Biol. Chem. 275, 3827–3834. Huang, K.F., Liu, Y.L., Cheng, W.J., Ko, T.P., Wang, A.H., 2005. Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. Proc. Natl. Acad. Sci. USA 102, 13117–13122. Junqueira-de-Azevedo, I.L., Ching, A.T., Carvalho, E., Faria, F., Nishiyama Jr, M.Y., Ho, P.L., Diniz, M.R., 2006. Lachesis muta (Viperidae) cDNAs reveal diverging pitviper molecules and scaffolds typical of cobra (Elapidae) venoms: implications in snake toxin repertoire evolution. Genetics 173, 877–889.
285
Kini, R.M., 2002. Molecular moulds with multiple missions: functional sites in three-finger toxins. Clin. Exp. Pharmacol. Physiol 29, 815–822. Kini, R.M., Chan, Y.M., 1999. Accelerated evolution and molecular surface of venom phospholipase A2 enzymes. J. Mol. Evol. 48, 125–132. Liao, Y.D., Wang, S.C., Leu, Y.J., Wang, C.F., Chang, S.T., Hong, Y.T., Pan, Y.R., Chen, C., 2003. The structural integrity exerted by N-terminal pyroglutamate is crucial for the cytotoxicity of frog ribonuclease from Rana pipiens. Nucleic Acids Res. 31, 5247–5255. Mackessy, S.P., 2002. Biochemistry and pharmacology of colubrid snake venoms. J. Toxicol.—Toxin Rev. 21, 43–83. Mackessy, S.P., Sixberry, N.M., Heyborne, W.H., Fritts, T., 2006. Venom of the Brown Treesnake, Boiga irregularis: ontogenetic shifts and taxa-specific toxicity. Toxicon 47, 537–548. Mebs, D., 1998. Enzymes in snake venoms: an overview. In: Bailey, T.D. (Ed.), Enzymes from Snake Venom. Alaken Inc., Fort Collins, CO, pp. 1–10. Mebs, D., Claus, I., 1991. Amino acid sequences and toxicities of snake venom components. In: Harvey, A.L. (Ed.), Snake Toxins. Pergamon Press Inc, NY, pp. 425–447. Nakashima, K., Nobuhisa, I., Deshimaru, M., Nakai, M., Ogawa, T., Shimohigashi, Y., Fukumaki, Y., Hattori, M., Sakaki, Y., Hattori, S., 1995. Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes. Proc. Natl. Acad. Sci. USA 92, 5605–5609. Nobuhisa, I., Nakashima, K., Deshimaru, M., Ogawa, T., Shimohigashi, Y., Fukumaki, Y., Sakaki, Y., Hattori, S., Kihara, H., Ohno, M., 1996. Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes. Gene 172, 267–272. Oberg, K.A., Ruysschaert, J.M., Azarkan, M., Smolders, N., Zerhouni, S., Wintjens, R., Amrani, A., Looze, Y., 1998. Papaya glutamine cyclase, a plant enzyme highly resistant to proteolysis, adopts an all-beta conformation. Eur. J. Biochem. 258, 214–222. Ohno, M., Menez, R., Ogawa, T., Danse, J.M., Shimohigashi, Y., Fromen, C., Ducancel, F., Zinn-Justin, S., Le Du, M.H., Boulain, J.C., Tamiya, T., Menez, A., 1998. Molecular evolution of snake toxins: is the functional diversity of snake toxins associated with a mechanism of accelerated evolution? Prog. Nucleic Acid Res. Mol. Biol. 59, 307–364. Pohl, T., Zimmer, M., Mugele, K., Spiess, J., 1991. Primary structure and functional expression of a glutaminyl cyclase. Proc. Natl. Acad. Sci USA 88, 10059–10063. Reza, A., Swarup, S., Kini, R.M., 2005. Two parallel prothrombin activator systems in Australian rough-scaled snake, Tropidechis carinatus. Structural comparison of venom prothrombin activator with blood coagulation factor X. Thromb. Haemost. 93, 40–47. Rudd, P.M., Joao, H.C., Coghill, E., Fiten, P., Saunders, M.R., Opdenakker, G., Dwek, R.A., 1994. Glycoforms modify the dynamic stability and functional activity of an enzyme. Biochemistry 33, 17–22. Schilling, S., Hoffmann, T., Rosche, F., Manhart, S., Wasternack, C., Demuth, H.U., 2002. Heterologous expression and characterization of human glutaminyl cyclase: evidence for a disulfide bond with importance for catalytic activity. Biochemistry 41, 10849–10857.
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Song, I., Chuang, C.Z., Bateman Jr., R.C., 1994. Molecular cloning, sequence analysis and expression of human pituitary glutaminyl cyclase. J. Mol. Endocrinol. 13, 77–86. Sykes, P.A., Watson, S.J., Temple, J.S., Bateman Jr., R.C., 1999. Evidence for tissue-specific forms of glutaminyl cyclase. FEBS Lett. 455, 159–161. Takeya, H., Arakawa, M., Miyata, T., Iwanaga, S., Omori-Satoh, T., 1989. Primary structure of H2-proteinase, a non-hemorrhagic metalloproteinase, isolated from the venom of the habu snake, Trimeresurus flavoviridis. J. Biochem. (Tokyo) 106, 151–157. Tsetlin, V., 1999. Snake venom alpha-neurotoxins and other ‘three-finger’ proteins. Eur. J. Biochem. 264, 281–286.
Van Coillie, E., Proost, P., Van I, A., Struyf, S., Polfliet, M., De I, M., Harvey, D.J., Van Damme, J., Opdenakker, G., 1998. Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV. Biochemistry 37, 12672–12680. Von Heijne, G., 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683–4690. Wang, C., Eufemi, M., Turano, C., Giartosio, A., 1996. Influence of the carbohydrate moiety on the stability of glycoproteins. Biochemistry 35, 7299–7307.
Toxicon 48 (2006) 278–286 www.elsevier.com/locate/toxicon
Snake venom glutaminyl cyclase Joanna Pawlak, R. Manjunatha Kini Department of Biological Sciences, Faculty of Science, National University of Singapore, Science Drive 4, Singapore 117543, Singapore Received 25 March 2006; received in revised form 24 May 2006; accepted 30 May 2006 Available online 14 June 2006
Abstract Glutaminyl cyclase (QC) catalyzes N-terminal glutamine cyclization of many endocrine peptides and is typically abundant in brain tissue. As three-finger toxins in the venoms of colubrid snakes Boiga dendrophila and Boiga irregularis contain N-terminal pyroglutamate, we searched for QC in venom glands of both snakes. Here we report cDNA sequences of QC from brain and venom gland tissues of Boiga species. We propose that QC expressed in snake venom gland tissue plays a role in the N-terminal pyroglutamate formation of several snake venom toxins, indirectly contributing to venom potency. r 2006 Elsevier Ltd. All rights reserved. Keywords: Glutaminyl cyclase; Pyroglutamic acid; Snake venom; Three-finger toxins
1. Introduction Snake venom is a complex mixture of bioactive peptides and proteins, which integrated with delivery system, create one of the most sophisticated weapons in the natural world. Snake venom includes enzymatic (e.g. phospholipases, metalloproteinases, serine proteinases, phosphodiesterases, acetylocholinesterase, L-amino acid oxidases) and non-enzymatic polypeptides (e.g. three-finger toxins, lectins, natriuretic peptides, CRISPs, waprins, desintegrins, sarafotoxins) (Kini, 2002). Amongst non-enzymatic components, three-finger toxins (3FTXs) form a broad and well-recognized superfamily. All members of this family share small stable three-finger-like scaffold, but are bestowed with a broad array of functions (e.g. antagonism of various Corresponding author. Tel.: 65 6516 5235; fax: 65 6779 2486.
E-mail address: [email protected] (R. Manjunatha Kini). 0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.05.013
subtypes of nicotinic acetylcholine receptors, cardiotoxicity, agonism of muscarinic receptors, inhibition of L-type calcium channel and acetylocholinesterase) (Tsetlin, 1999; Kini, 2002). Hence, 3FTXs are extensively used as tools in biomedical research, diagnostics and therapeutics. Previously, they were thought to be limited to the venoms of elapid (cobras, kraits, mambas) and hydrophid (sea snakes) snakes (Kini, 2002). Recently, we reported the first three-finger neurotoxin (a-colubritoxin) from colubrid snake Coelognathus radiatus (Swissprot ]P83490) (Fry et al., 2003) and subsequently determined 3FTX sequences from other colubrids: Boiga dendrophila and Boiga irregularis (GenBank ]s DQ366293, DQ304538, DQ304539). Interestingly, unlike elapid, hydrophid and recently reported viperid 3FTXs (Junqueira-de-Azevedo et al., 2006), all 3FTXs isolated so far from colubrid snakes, possess a characteristic long N-terminal which is blocked by pyroglutamate (pGlu). In
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nature, spontaneous deamination of N-terminal glutaminyl precursor to pGlu occurs very slowly under physiological conditions and in vivo this posttranslational modification is catalyzed by the glutaminyl cyclases (EC 2.3.2.5; QCs) (Busby et al., 1987; Fischer and Spiess, 1987). Therefore, the presence of pGlu at the N-terminal of 3FTXs in the venom of colubrid snakes indicated the expression of QC in their venom gland. QCs are 33–40 kDa acyltransferases, highly conserved from yeast to humans (Huang et al., 2005). They were identified in plant and animal sources (Busby et al., 1987; Fischer and Spiess, 1987; Oberg et al., 1998; Huang et al., 2005) and they are especially abundant in vertebrate neuroendocrine tissues, such as hypothalamus and pituitary (Busby et al., 1987). Cyclization of N-terminal glutamine is an important posttranslational event in the processing of numerous bioactive proteins [12.3% proteins with signal peptides (Liao et al., 2003)] including neuropeptides, hormones and cytokines during their maturation in the secretory pathway (Huang et al., 2005). The N-terminal pGlu protects them from exopeptidase degradation and/or enables them to have proper conformation for binding to the receptors (Van Coillie et al., 1998; Hinke et al., 2000). Present paper provides the evidence for the presence of QC in the venom gland of colubrid snakes (Boiga) and discusses putative role of QC in the snake venom. 2. Materials and method 2.1. Total RNA isolation Boiga dendrophila (Sulawesi) and Boiga irregularis (Guam) venom glands and B. irregularis brain tissue were kindly provided by Dr. Stephen P. Mackessy, University of Northern Colorado, CO, USA. Specimens were sacrificed by decapitation. Venom glands and brain were dissected and kept in RNAlater solution in 80 1C until use. Total RNA extraction was performed with rotor homogenizer and RNeasy mini kit from Qiagen (Valencia, CA, USA). Amount of RNA was calculated according to 260 nm absorbance of the sample. 2.2. Reverse transcription—Polymerase chain reaction (RT-PCR) QC sequences from different sources (human, mouse and frog) were aligned in order to design
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internal forward GSP1f 50 -GCTGTGCCTTGTGC AATGAT-30 and reverse GSP1r 50 -GTATGCCA TACTCTAGGGAAAGG-30 primers from the most conserved regions. Using total RNA isolated from venom glands as templates, RT-PCR was performed to amplify partial cDNAs of QCs. Onestep RT-PCR (Qiagen) was carried out using 100 ng of total RNA as follows: RT at 50 1C for 30 min, activation of hot start Taq polymerase at 95 1C for 15 min and 30 cycles of 3-step PCR (94 1C for 1 min, 50 1C for 1 min, 72 1C for 1 min) with the final extension at 72 1C for 10 min. PCR products were subjected to 1% agarose gel electrophoresis and visualized by ethidium bromide staining. As the obtained partial sequences showed high identity to cDNA of QC from Agkistrodon blomhoffi (BAB69586), another forward primer was designed based on crotalid’s 50 UTR sequence GSP2f (50 -TGAATCTGCCTC CCTCTGAGG-30 ) and used in RT-PCR, together with reverse primer GSP2r 50 -GGGACCATCGCA CAAAAGCT-30 . This set of primers was also used to fish out cDNA of QC from the brain of B. irregularis. 2.3. 30 Rapid amplification of cDNA ends (RACE) The 30 RACE cDNA libraries were constructed using Invitrogen (Carlsbad, CA, USA) 30 RACE kit for rapid amplification of cDNA ends according to the manufacturer’s protocol. cDNA was synthesized from 1.0 mg of total RNA using SuperScript II reverse transcriptase and 30 RACE adapter primer [50 -GGCCACGCGTCGACTAGTAC(T)17-30 ]. Reverse transcription reaction product was treated with RNase H (2 U) for 30 min at 37 1C and further used as a template in PCR (95 1C for 15 min followed by 30 cycles of 3-step PCR: 94 1C for 1 min, 50 1C for 1 min, 72 1C for 1 min and the final extension at 72 1C for 10 min) with two different forward primers GSP3f 50 -GACGACCATATTC CATTTTTGAGA-30 , GSP4f 50 TGTTTGTTGGA GCCACCGACT 30 and reverse 30 -RACE abridged universal amplification primer 50 -GGCCACGC GTCGACTAGTAC-30 . The PCR products obtained were visualized using ethidium bromide staining following agarose gel (1%) electrophoresis. Using sequence data generated by RT-PCR and 30 RACE reactions, new sets of gene-specific primers were designed to generate the full-length cDNA of QC from both snake species and to confirm the sequence.
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2.4. Cloning and sequencing of PCR products
Sequence analysis was carried out using BLAST program at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov) and ExPASy proteomics tools at www.expasy.org. Sequence alignments and neighbor-joining tree construction were carried out using DNAMAN version 4.15 (Lynnon Biosoft).
deduced protein sequence shows the highest (96%) identity to Agkistrodon QC (BAB69586) and quite high (63%) identity to human QC (NP_036545). All functional residues in the active site as well as three residues (Asp164, Glu208, His308) involved in binding to the zinc ion of the catalytic center, are conserved in Boiga QCs. Based on the multiple sequence alignment and the crystal structure of human QC (Huang et al., 2005), we predict that secondary structure of Boiga QCs have mixed a=b fold with an open sandwich topology, similar to human QC (Fig. 2). In addition to the full length clones, a short variant of QC cDNA was found in the venom gland and brain of B. irregularis, but not in B. dendrophila venom gland. This variant lacks 180 nucleotides, leading to putative in-frame deletion mutant shorter by 60 amino acid residues (Fig. 1), which constitute b4 strand and a7 helix in human QC (Fig. 2). The fragment absent in the short variant aligns well with 167 nucleotides long, fourth exon of the bovine QC gene (GeneID 281437). The 30 end of the exon 4 exactly matches the end of deletion sequence. However, at the 50 end, the deletion segment is 13 nucleotides longer than bovine exon 4 and thus the deleted sequence does not correspond to exon 4 alone. As with other genes, we believe that in the genes of QCs intron-exon boundaries are also conserved. Therefore, the short variant may not be a product of simple alternate splicing.
3. Results
3.2. Signal peptide and secretion
3.1. cDNA sequence of brain and venom QCs
Human, bovine as well as other QCs are secreted proteins and their precursors contain a signal peptide. We subjected snake QC sequence for signal peptide identification. SignalP 3.0 algorithm (http:// www.cbs.dtu.dk/services/SignalP/) confirmed the presence of signal peptide, but predicted three possible cleavage sites. Neural (eukaryotic) network method predicted that most likely cleavage occurs between residues 23 and 24 (CLA-LP) (Fig. 1), while hidden Markov (eukaryotic) models method predicted maximal cleavage site probability between residues 28 and 29 (GFP-QH). The latter putative cleavage may require self-glutamyl cyclization. Apart from these sites, both methods predicted the cleavage between residues 33 and 34 (VGG-RE) but with lower probability. As signal peptide cleavage site is ambiguous, the length of mature enzyme can be 345, 340 or 335 amino acid residues and thus we cannot precisely establish the molecular weight
All amplification products were ligated with pDrive vector (Qiagen) and transformed by heat shock method into DH5a strain of competent E. coli cells. Selection of the transformants (blue/white colony screening) was performed on LB-agar plates containing 100 mg/ml ampicillin and supplemented with IPTG and X-gal. Sizes of the inserts were estimated by EcoRI digestion followed by 1% agarose gel electrophoresis and ethidium bromide visualization. All sequencing reactions were carried out on the ABI PRISMs 3100 automated DNA sequencer, using the ABI PRISM BigDye terminator cycle sequencing ready reaction kit purchased from Applied Biosystems (Foster City, CA, USA), according to the manufacturer’s instructions. All clones were sequenced in both directions using T7 and SP6 sequencing primers and gene specific primers. 2.5. Sequence analysis and phylogenetic tree
The cDNA encoding QCs present in the venom glands of B. irregularis (DQ404533) and B. dendrophila (DQ404534) were obtained by RT-PCR followed by 30 RACE, providing direct evidence that QC is expressed in the snake venom gland. For comparison, cDNA encoding QC from the brain of B. irregularis was sequenced and the results showed that QC cDNAs from the brain and venom glands of B. irregularis are identical. All cDNA sequences encode for putative open reading frames of 1107 nucleotides, coding for 368 amino acids (Fig. 1). cDNA sequences from B. irregularis and B. dendrophila show 98% nucleotide identity to each other and have mutations either in untranslated regions (21 mutations and three deletions) or synonymous (six mutations). Consequently, both are 100% identical at the protein level. Their
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Fig. 1. Pairwise alignment of cDNA sequences encoding venom QCs from Boiga snakes. Differences in nucleotide sequences are highlighted in gray. Polyadenylation signals (AATAAA) are underlined with single line and stop codon (TAA) is indicated by an asterisk. The deduced amino acid sequence is shown in single letter code beneath nucleotide sequences. Nucleotide and amino acid numbers are shown in the column on the right starting from translational start site. Putative signal peptide predicted by neuronal networks method is boxed. The corresponding missing nucleotide segment in short variant of QC from B. irregularis is double underlined. As the cDNA sequence of the brain QC from B. irregularis is identical to its venom QC, it is not included in the alignment.
(39526–38536 Da). Although we expect this enzyme to be present in the venom, its mass was not detected in LC-MS profile (Fry et al., 2003) or SDSPAGE gel of Boiga venoms (Mackessy, 2002; Mackessy et al., 2006). Thus QCs are found either in only small quantities or their masses vary significantly due to posttranslational modifications (like glycosylation). 3.3. Posttranslational modifications Typically snake venom proteins have high content of cysteine residues (10%) and multiple disulfide bridges. However, QC from snake venom
possesses only two cysteine residues (Cys143 and Cys169) forming one disulfide bridge (Huang et al., 2005). This sole disulfide is conserved throughout all species (Fig. 2) and is important for the catalytic activity (Schilling et al., 2002). N-glycosylation is another significant modification observed in QCs, which may play a role in stabilization of the protein conformation or its functional activity (Rudd et al., 1994; Wang et al., 1996). The deduced amino acid sequence of human QC reveals two potential Nglycosylation sites (Asn49 and Asn296) (Song et al., 1994), while Boiga QCs have three potential N-glycosylation sites (Asn53, Asn292 and Asn352). The first site is conserved in mammals but is absent
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Fig. 2. Multiple sequence alignment of deduced amino acid sequences of QC from B. dendrophila and B. irregularis with several representative animal QCs. The sequences have been taken from following sources: Boiga dendrophila (this paper) DQ404534; Boiga irregularis (this paper) Q404533; Agkistrodon blomhoffi BAB69586; Bothrops jararaca BAA34290; Homo sapiens NP_036545; Mus musculus XP_128770; Rattus norvegicus XP_233812; Bos taurus NP_803472; Gallus gallus XP_419527; Xenopus laevis AAH80397, Caenorhabditis elegans CAB08740; Drosophila melanogaster AAV36900, Apis mellifera XP_395412. The residues that have 100% identity are colored in black and those with 470% identity are shaded in gray. Three residues responsible for zinc binding are marked with triangle above the sequence. Three putative N-glycosylation sites in snake QCs are indicated by hexagon under the sequence. The secondary structural elements of a-helices and b-strands according to human crystal structure (Huang et al., 2005) are also shown.
in chicken, frog and lower organisms (Fig. 2). The second site seems to be characteristic for snakes only, while the third site is also found in chicken QC. Interestingly, only snake QCs possess glycine residue at the C-terminal end (Fig. 2), which might undergo amidation. 3.4. Phylogenetic analyses of QCs A phylogenetic tree constructed using consensus bootstrapping separated well into the vertebrate and invertebrate clades. The vertebrate taxa were properly classified into tetrapod and non-tetrapod lineages indicating the authenticity of the phylogenetic tree. Further, the snake QC cluster divided into crotalid and colubrid QC groups. Hence, the phylogentic tree (Fig. 3) as well as multiple sequence
alignments (Fig. 2) show that QC sequences are highly conserved (67% identity) throughout the phylogeny, from yeast to mammals. 4. Discussion QC is a tissue-specific, differentially expressed enzyme, with transcription and translation correlated to the need for peptides with N-terminal pGlu (Sykes et al., 1999). The highest expression of QC was reported in pituitary and various brain regions (Pohl et al., 1991). Only very small levels were detected in some peripheral tissues like thymus and kidney but not in lung or liver (Pohl et al., 1991). We identified the presence of QC cDNAs in the venom gland tissue of two colubrid snakes, B. dendrophila and B. irregularis. Venom gland is a
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0.05
Homo sapiens
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Canis familiaris Bos taurus
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Mus musculus 100
Rattus norvegicus
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Gallus gallus 95 100
95
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Agkistrodon blomhoffi Bothrops jararaca Boiga dendrophila
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Boiga irregularis Xenopus laevis
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Danio rerio 99
Apis mellifera Drosophila melanogaster
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Caenorhabditis elegans Saccharomyces cerevisiae
Fig. 3. Phylogenetic relationship among QCs. The neighbor-joining tree represents bootstrap values after 1000 replicates. Tree was plotted using DNAMAN. Sources of the sequences are same as in Fig. 2, except that three sequences from: Canis familiaris XP_532934, Danio rerio XP_689638 and Saccharomyces cerevisiae P43599 were added.
unique QC expression site associated only with snakes. Apart from the QC sequence of both Boiga species, other two sequences are known from crotalid snakes: Bothrops jararaca (BAA34290) and Agkistrodon blomhoffi (BAB69586). Although these sequences are reported in the GenBank database, so far there was no published literature on any snake venom QC. Snake venom proteins are secreted and contain a signal peptide for protein translocation through endoplasmic reticulum (Blobel and Dobberstein, 1975). The deduced protein sequences (obtained from genome and/or mRNA sequencing) indicate that QCs also contain signal peptides, as their N-termini (Pohl et al., 1991) show key features of signal sequence: a short positively charged region for penetrability, followed by central hydrophobic region extending across the membranes and the polar C-terminal for cleavage (Von Heijne, 1986). Signal peptides identified in colubrid and crotalid QCs corroborate well with the above observations,
although signal peptide of snake QCs have notably more charged residues towards N-terminus. Interestingly, three different signal peptide cleavage sites were predicted at positions 23–24, 28–29 and 33–34 in Boiga QCs (Fig. 1). All three sites are in agreement with the (3,1) rule (Von Heijne, 1986), by which the third last residue of signal peptide cannot be aromatic, charged or large/polar and the last residue of signal sequence should be small for the cleavage to occur. As the signal peptide region is not conserved, it is not possible to extrapolate the cleavage position of Boiga QCs based on other QCs. Bovine QC is the only one in which the N-terminal sequence has been determined by protein chemistry and the mature protein starts with Gly32 (Fischer and Spiess, 1987; Pohl et al., 1991). It is not clear whether bovine QC is atypical. Unlike other QCs, it may be synthesized as prepropeptide with putative signal peptide of 27 or 28 residues followed by short propeptide region. The propeptide segment is cleaved after dibasic
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residues Arg30–Arg31 (Pohl et al., 1991). However, such dibasic sites are not found in other QCs. Thus, the signal peptide cleavage site in Boiga QCs is yet to be determined. QCs from human (NCBI GeneID: 25797), bovine (281437), mouse (70536) and dog (403861) are encoded by a single gene with protein coding region of 1.1 kb. We obtained QC with similar size of open reading frame (1107 bp) as those of mammalian origin. In addition, we also obtained a short variant of QC from the brain and venom glands of B. irregularis. It shows 100% sequence identity with the full length QC, but for the missing segment of 180 bases. Although it is tempting to speculate that this short variant could be an alternate splice product, the missing segment is close to but not equivalent to the fourth exon of the bovine QC gene (GeneID 281437) (see Results). QC has been shown to be a metal dependent transferase (Schilling et al, 2002) and the short variant encodes for a protein lacking residue Glu208, important for the metal binding (Huang et al., 2005). Thus short variant may not be functional. Its genetic origin and physiological relevance is unclear. Study of B. irregularis QC gene organization, establishing exon/intron boundaries and splicing sites is needed to unequivocally decipher the origin of the short form of QC. Snake venom proteins have been recruited from different tissue types and manifold body proteins, to create venom proteome (Fry, 2005). For example, venom prothrombin activator has evolved from liver blood coagulation factor X (Reza et al., 2005). Similarly, venom phospholipase A2, 3FTXs and cobra venom factor have pancreatic phospholipase A2 (EC 3.1.1.4), lynx1/SLUR and complement C3 as their respective ancestors (Fry, 2005). Thus these venom proteins evolved through gene duplications and accelerated evolution in the protein-coding regions (Nakashima et al., 1995; Nobuhisa et al., 1996; Deshimaru et al., 1996; Ohno et al., 1998; Kini and Chan, 1999); only features that are essential for protein folding and structural integrity are conserved while functional residues are manipulated. Interestingly, the cDNA sequences from the brain and venom glands of B. irregularis encoding QC are identical (Fig. 1) and hence QC appears to be a single gene, which is recruited for expression in the venom gland without duplication. Snake venoms are one of the most concentrated enzyme sources surpassing pancreatic juice or liver
secretions, but the majority of snake venom enzymes are hydrolases, which play a crucial role in prey immobilization and digestion (Mebs, 1998). Only a few non-toxic enzymes have been identified as the components of the venom (e.g. ribonucleases, oligopeptidases, phosphodiesterases, 50 -nucleotidases, phosphatases) which might aid in initiation of prey digestion. It is hypothesized that some of these enzymes facilitate venom action by systemic distribution of other venom components or activation of precursors present in the inactive form in pre-secreted venom to prevent tissue damage and self-poisoning (Mebs, 1998). The existence of enzyme, like QC in the snake venom is intriguing. Venom QCs may not act as toxins themselves, but they catalyze the maturation of toxins and help in their protection from exopeptidase degradation and/or developing proper conformation. Thus venom QCs may indirectly contribute to venom toxicity. In crotalid venoms, several toxins are known to possess N-terminal pGlu, e.g. metalloproteases (Takeya et al., 1989), bradykinin-potentiating peptides (Chi et al., 1985), crotoxin (Aird et al., 1990) and Mojave toxin (Bieber et al., 1990). So far, among 3FTXs (Mebs and Claus, 1991), only colubrid 3FTXs are known to possess pGlu at their N-terminal. The N-terminally located glutamine in their precursor acts as a novel substrate for QC. The presence of both QC as well as pGlu-containing toxins in crotalid and colubrid venoms indeed reinforces that pyroglutamation is an important and required posttranslational modification, which might be crucial towards proper metabolism, integrity and activity of several venom components (Liao et al., 2003). In conclusion, QCs in the venom gland may play crucial role in cyclization of N-terminal glutamine in several snake venom toxins. Such posttranslational modifications often affect the biological activity and/or stability against proteolysis. Thus venom QCs may indirectly contribute to venom toxicity.
Acknowledgments We thank Dr. Stephen P. Mackessy for providing venom glands and brain tissues. This work was supported by a grant from the Biomedical Research Council, Agency for Science, Technology and Research, Singapore.
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References Aird, S.D., Yates III, J.R., Martino, P.A., Shabanowitz, J., Hunt, D.F., Kaiser, I.I., 1990. The amino acid sequence of the acidic subunit B-chain of crotoxin. Biochim. Biophys. Acta 1040, 217–224. Bieber, A.L., Becker, R.R., McParland, R., Hunt, D.F., Shabanowitz, J., Yates III, J.R., Martino, P.A., Johnson, G.R., 1990. The complete sequence of the acidic subunit from Mojave toxin determined by Edman degradation and mass spectrometry. Biochim. Biophys. Acta 1037, 413–421. Blobel, G., Dobberstein, B., 1975. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J. Cell Biol. 67, 835–851. Busby Jr., W.H., Quackenbush, G.E., Humm, J., Youngblood, W.W., Kizer, J.S., 1987. An enzyme(s) that converts glutaminyl-peptides into pyroglutamyl-peptides. Presence in pituitary, brain, adrenal medulla, and lymphocytes. J. Biol. Chem. 262, 8532–8536. Chi, C.W., Wang, S.Z., Xu, L.G., Wang, M.Y., Lo, S.S., Huang, W.D., 1985. Structure-function studies on the bradykinin potentiating peptide from Chinese snake venom (Agkistrodon halys Pallas). Peptides 6 (Suppl 3), 339–342. Deshimaru, M., Ogawa, T., Nakashima, K., Nobuhisa, I., Chijiwa, T., Shimohigashi, Y., Fukumaki, Y., Niwa, M., Yamashina, I., Hattori, S., Ohno, M., 1996. Accelerated evolution of crotalinae snake venom gland serine proteases. FEBS Lett. 397, 83–88. Fischer, W.H., Spiess, J., 1987. Identification of a mammalian glutaminyl cyclase converting glutaminyl into pyroglutamyl peptides. Proc. Natl. Acad. Sci. USA 84, 3628–3632. Fry, B.G., 2005. From genome to ‘‘venome’’: molecular origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences and related body proteins. Genome Res. 15, 403–420. Fry, B.G., Lumsden, N.G., Wuster, W., Wickramaratna, J.C., Hodgson, W.C., Kini, R.M., 2003. Isolation of a neurotoxin (alpha-colubritoxin) from a nonvenomous colubrid: evidence for early origin of venom in snakes. J. Mol. Evol. 57, 446–452. Fry, B.G., Wuster, W., Ryan Ramjan, S.F., Jackson, T., Martelli, P., Kini, R.M., 2003. Analysis of Colubroidea snake venoms by liquid chromatography with mass spectrometry: evolutionary and toxinological implications. Rapid Commun. Mass Spectrom. 17, 2047–2062. Hinke, S.A., Pospisilik, J.A., Demuth, H.U., Mannhart, S., Kuhn-Wache, K., Hoffmann, T., Nishimura, E., Pederson, R.A., McIntosh, C.H., 2000. Dipeptidyl peptidase IV (DPIV/ CD26) degradation of glucagon. Characterization of glucagon degradation products and DPIV-resistant analogs. J. Biol. Chem. 275, 3827–3834. Huang, K.F., Liu, Y.L., Cheng, W.J., Ko, T.P., Wang, A.H., 2005. Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. Proc. Natl. Acad. Sci. USA 102, 13117–13122. Junqueira-de-Azevedo, I.L., Ching, A.T., Carvalho, E., Faria, F., Nishiyama Jr, M.Y., Ho, P.L., Diniz, M.R., 2006. Lachesis muta (Viperidae) cDNAs reveal diverging pitviper molecules and scaffolds typical of cobra (Elapidae) venoms: implications in snake toxin repertoire evolution. Genetics 173, 877–889.
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Kini, R.M., 2002. Molecular moulds with multiple missions: functional sites in three-finger toxins. Clin. Exp. Pharmacol. Physiol 29, 815–822. Kini, R.M., Chan, Y.M., 1999. Accelerated evolution and molecular surface of venom phospholipase A2 enzymes. J. Mol. Evol. 48, 125–132. Liao, Y.D., Wang, S.C., Leu, Y.J., Wang, C.F., Chang, S.T., Hong, Y.T., Pan, Y.R., Chen, C., 2003. The structural integrity exerted by N-terminal pyroglutamate is crucial for the cytotoxicity of frog ribonuclease from Rana pipiens. Nucleic Acids Res. 31, 5247–5255. Mackessy, S.P., 2002. Biochemistry and pharmacology of colubrid snake venoms. J. Toxicol.—Toxin Rev. 21, 43–83. Mackessy, S.P., Sixberry, N.M., Heyborne, W.H., Fritts, T., 2006. Venom of the Brown Treesnake, Boiga irregularis: ontogenetic shifts and taxa-specific toxicity. Toxicon 47, 537–548. Mebs, D., 1998. Enzymes in snake venoms: an overview. In: Bailey, T.D. (Ed.), Enzymes from Snake Venom. Alaken Inc., Fort Collins, CO, pp. 1–10. Mebs, D., Claus, I., 1991. Amino acid sequences and toxicities of snake venom components. In: Harvey, A.L. (Ed.), Snake Toxins. Pergamon Press Inc, NY, pp. 425–447. Nakashima, K., Nobuhisa, I., Deshimaru, M., Nakai, M., Ogawa, T., Shimohigashi, Y., Fukumaki, Y., Hattori, M., Sakaki, Y., Hattori, S., 1995. Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes. Proc. Natl. Acad. Sci. USA 92, 5605–5609. Nobuhisa, I., Nakashima, K., Deshimaru, M., Ogawa, T., Shimohigashi, Y., Fukumaki, Y., Sakaki, Y., Hattori, S., Kihara, H., Ohno, M., 1996. Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes. Gene 172, 267–272. Oberg, K.A., Ruysschaert, J.M., Azarkan, M., Smolders, N., Zerhouni, S., Wintjens, R., Amrani, A., Looze, Y., 1998. Papaya glutamine cyclase, a plant enzyme highly resistant to proteolysis, adopts an all-beta conformation. Eur. J. Biochem. 258, 214–222. Ohno, M., Menez, R., Ogawa, T., Danse, J.M., Shimohigashi, Y., Fromen, C., Ducancel, F., Zinn-Justin, S., Le Du, M.H., Boulain, J.C., Tamiya, T., Menez, A., 1998. Molecular evolution of snake toxins: is the functional diversity of snake toxins associated with a mechanism of accelerated evolution? Prog. Nucleic Acid Res. Mol. Biol. 59, 307–364. Pohl, T., Zimmer, M., Mugele, K., Spiess, J., 1991. Primary structure and functional expression of a glutaminyl cyclase. Proc. Natl. Acad. Sci USA 88, 10059–10063. Reza, A., Swarup, S., Kini, R.M., 2005. Two parallel prothrombin activator systems in Australian rough-scaled snake, Tropidechis carinatus. Structural comparison of venom prothrombin activator with blood coagulation factor X. Thromb. Haemost. 93, 40–47. Rudd, P.M., Joao, H.C., Coghill, E., Fiten, P., Saunders, M.R., Opdenakker, G., Dwek, R.A., 1994. Glycoforms modify the dynamic stability and functional activity of an enzyme. Biochemistry 33, 17–22. Schilling, S., Hoffmann, T., Rosche, F., Manhart, S., Wasternack, C., Demuth, H.U., 2002. Heterologous expression and characterization of human glutaminyl cyclase: evidence for a disulfide bond with importance for catalytic activity. Biochemistry 41, 10849–10857.
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Song, I., Chuang, C.Z., Bateman Jr., R.C., 1994. Molecular cloning, sequence analysis and expression of human pituitary glutaminyl cyclase. J. Mol. Endocrinol. 13, 77–86. Sykes, P.A., Watson, S.J., Temple, J.S., Bateman Jr., R.C., 1999. Evidence for tissue-specific forms of glutaminyl cyclase. FEBS Lett. 455, 159–161. Takeya, H., Arakawa, M., Miyata, T., Iwanaga, S., Omori-Satoh, T., 1989. Primary structure of H2-proteinase, a non-hemorrhagic metalloproteinase, isolated from the venom of the habu snake, Trimeresurus flavoviridis. J. Biochem. (Tokyo) 106, 151–157. Tsetlin, V., 1999. Snake venom alpha-neurotoxins and other ‘three-finger’ proteins. Eur. J. Biochem. 264, 281–286.
Van Coillie, E., Proost, P., Van I, A., Struyf, S., Polfliet, M., De I, M., Harvey, D.J., Van Damme, J., Opdenakker, G., 1998. Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV. Biochemistry 37, 12672–12680. Von Heijne, G., 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683–4690. Wang, C., Eufemi, M., Turano, C., Giartosio, A., 1996. Influence of the carbohydrate moiety on the stability of glycoproteins. Biochemistry 35, 7299–7307.