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1.
Toxicon ; 106: 20-9, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26363292

ABSTRACT

Throughout evolution, parasites have adapted in order to successfully intervene in the host defense, producing specific peptides and proteins. Interestingly, these peptides and proteins have been exploited as potential drug candidates against several diseases. Furthermore, biotechnology studies and cDNA libraries have remarkably contributed to identify potentially bioactive molecules. In this regard, herein, a cDNA library of salivary complexes from Haementeria vizottoi leeches was constructed, the transcriptome was characterized and a phylogenetic analysis was performed considering antistasin-like and antiplatelet-like proteins. Hundred twenty three transcripts were identified coding for putative proteins involved in animal feeding (representing about 10% of the expression level). These sequences showed similarities with myohemerythrins, carbonic anhydrases, anticoagulants, antimicrobials, proteases and protease inhibitors. The phylogenetic analysis, regarding antistasin-like and antiplatetlet-like proteins, revealed two main clades in the Rhynchobdellida leeches. As expected, the sequences from H. vizottoi have presented high similarities with those types of proteins. Thus, our findings could be helpful not only to identify new coagulation inhibitors, but also to better understand the biological composition of the salivary complexes.


Subject(s)
Anticoagulants/chemistry , Leeches/chemistry , Platelet Aggregation Inhibitors/chemistry , Salivary Glands/chemistry , Animals , Anticoagulants/isolation & purification , Computational Biology , Feeding Behavior , Gene Expression Profiling , Gene Library , Hemostasis , Leeches/genetics , Leeches/physiology , Models, Molecular , Phylogeny , Platelet Aggregation Inhibitors/isolation & purification , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, Protein
2.
BMC Genomics ; 13: 362, 2012 Aug 01.
Article in English | MEDLINE | ID: mdl-22853446

ABSTRACT

BACKGROUND: The scorpion Tityus stigmurus is widely distributed in Northeastern Brazil and known to cause severe human envenoming, inducing pain, hyposthesia, edema, erythema, paresthesia, headaches and vomiting. The present study uses a transcriptomic approach to characterize the gene expression profile from the non-stimulated venom gland of Tityus stigmurus scorpion. RESULTS: A cDNA library was constructed and 540 clones were sequenced and grouped into 153 clusters, with one or more ESTs (expressed sequence tags). Forty-one percent of ESTs belong to recognized toxin-coding sequences, with transcripts encoding antimicrobial toxins (AMP-like) being the most abundant, followed by alfa KTx- like, beta KTx-like, beta NaTx-like and alfa NaTx-like. Our analysis indicated that 34% of the transcripts encode "other possible venom molecules", which correspond to anionic peptides, hypothetical secreted peptides, metalloproteinases, cystein-rich peptides and lectins. Fifteen percent of ESTs are similar to cellular transcripts. Sequences without good matches corresponded to 11%. CONCLUSIONS: This investigation provides the first global view of gene expression of the venom gland from Tityus stigmurus under resting conditions. This approach enables characterization of a large number of venom gland component molecules, which belong either to known or non yet described types of venom peptides and proteins from the Buthidae family.


Subject(s)
Gene Expression Profiling , Scorpion Venoms/genetics , Scorpions/metabolism , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/metabolism , Expressed Sequence Tags , Gene Library , Insulin-Like Growth Factor Binding Proteins/chemistry , Insulin-Like Growth Factor Binding Proteins/metabolism , Lectins/chemistry , Lectins/metabolism , Molecular Sequence Data , Neurotoxins/chemistry , Neurotoxins/metabolism , Peptides/chemistry , Peptides/metabolism , Scorpion Venoms/classification , Scorpion Venoms/metabolism , Sequence Alignment
3.
J Proteomics ; 74(9): 1795-809, 2011 Aug 24.
Article in English | MEDLINE | ID: mdl-21515432

ABSTRACT

The venom proteomes of Micrurus altirostris and M. corallinus were analyzed by combining snake venomics and venom gland transcriptomic surveys. In both coral snake species, 3FTx and PLA(2) were the most abundant and diversified toxin families. 33 different 3FTxs and 13 PLA(2) proteins, accounting respectively for 79.5% and 13.7% of the total proteins, were identified in the venom of M. altirostris. The venom of M. corallinus comprised 10 3FTx (81.7% of the venom proteome) and 4 (11.9%) PLA(2) molecules. Transcriptomic data provided the full-length amino acid sequences of 18 (M. altirostris) and 10 (M. corallinus) 3FTxs, and 3 (M. altirostris) and 1 (M. corallinus) novel PLA(2) sequences. In addition, venom from each species contained single members of minor toxin families: 3 common (PIII-SVMP, C-type lectin-like, L-amino acid oxidase) and 4 species-specific (CRISP, Kunitz-type inhibitor, lysosomal acid lipase in M. altirostris; serine proteinase in M. corallinus) toxin classes. The finding of a lipase (LIPA) in the venom proteome and in the venom gland transcriptome of M. altirostris supports the view of a recruitment event predating the divergence of Elapidae and Viperidae more than 60 Mya. The toxin profile of both M. altirostris and M. corallinus venoms points to 3FTxs and PLA(2) molecules as the major players of the envenoming process. In M. altirostris venom, all major, and most minor, 3FTxs display highest similarity to type I α-neurotoxins, suggesting that these postsynaptically acting toxins may play the predominant role in the neurotoxic effect leading to peripheral paralysis, respiratory arrest, and death. M. corallinus venom posesses both, type I α-neurotoxins and a high-abundance (26% of the venom proteome) protein of subfamily XIX of 3FTxs, exhibiting similarity to bucandin from Malayan krait, Bungarus candidus, venom, which enhances acetylcholine release presynaptically. This finding may explain the presynaptic neurotoxicity of M. corallinus venom and the lack of this effect in M. altirostris venom. The anti-Micrurus (corallinus and frontalis) antivenom produced by Instituto Butantan quantitatively immunodepleted the minor toxins from M. altirostris and M. corallinus venoms but showed impaired crossreactivity towards their major 3FTx and PLA(2) molecules. The structural diversity of 3FTxs among Micrurus sp. may underlay the impaired cross-immunoreactivity of the Butantan antivenom towards M. altirostris and M. corallinus toxins, hampering the possibility to raise an antivenom against a simple venom mixture exhibiting paraspecific neutralization of other Micrurus venoms.


Subject(s)
Elapid Venoms/analysis , Elapidae/genetics , Proteome/analysis , Transcriptome , Animals , Antivenins/immunology , Cross Reactions/immunology , Elapid Venoms/chemistry , Elapid Venoms/enzymology , Gene Library
4.
J. Proteomics ; 74(9): 1795-1809, Apr 12 , 2011.
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP, SESSP-IBACERVO | ID: biblio-1063780

ABSTRACT

The venom proteomes of Micrurus altirostris and M. corallinus were analyzed by combining snake venomics and venom gland transcriptomic surveys. In both coral snake species, 3FTx and PLA2 were the most abundant and diversified toxin families. 33 different 3FTxs and 13 PLA2 proteins, accounting respectively for 79.5% and 13.7% of the total proteins, were identified in the venom of M. altirostris. The venom of M. corallinus comprised 10 3FTx (81.7% of the venom proteome) and 4 (11.9%) PLA2 molecules. Transcriptomic data provided the full-length amino acid sequences of 18 (M. altirostris) and 10 (M. corallinus) 3FTxs, and 3 (M. altirostris) and 1 (M. corallinus) novel PLA2 sequences. In addition, venom from each species contained single members of minor toxin families: 3 common (PIII-SVMP, C-type lectin-like, L-amino acid oxidase) and 4 species-specific (CRISP, Kunitz-type inhibitor, lysosomal acid lipase in M. altirostris; serine proteinase in M. corallinus) toxin classes.


Subject(s)
Animals , Elapidae/classification , Elapidae/genetics , Elapid Venoms/analysis , Snake Venoms/poisoning , Mass Spectrometry/methods , Proteomics/methods , Transcriptome/physiology
5.
BMC Genomics ; 10: 112, 2009 Mar 16.
Article in English | MEDLINE | ID: mdl-19291316

ABSTRACT

BACKGROUND: Micrurus corallinus (coral snake) is a tropical forest snake belonging to the family Elapidae. Its venom shows a high neurotoxicity associated with pre- and post-synaptic toxins, causing diaphragm paralysis, which may result in death. In spite of a relatively small incidence of accidents, serum therapy is crucial for those bitten. However, the adequate production of antiserum is hampered by the difficulty in obtaining sufficient amounts of venom from a small snake with demanding breeding conditions. In order to elucidate the molecular basis of this venom and to uncover possible immunogens for an antiserum, we generated expressed sequences tags (ESTs) from its venom glands and analyzed the transcriptomic profile. In addition, their immunogenicity was tested using DNA immunization. RESULTS: A total of 1438 ESTs were generated and grouped into 611 clusters. Toxin transcripts represented 46% of the total ESTs. The two main toxin classes consisted of three-finger toxins (3FTx) (24%) and phospholipases A(2) (PLA(2)s) (15%). However, 8 other classes of toxins were present, including C-type lectins, natriuretic peptide precursors and even high-molecular mass components such as metalloproteases and L-amino acid oxidases. Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions. Five antigenic candidates were selected (four 3FTx and one PLA(2)) and used for a preliminary study of DNA immunization. The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens. CONCLUSION: Besides an improvement in our knowledge of the composition of coral snake venoms, which are very poorly known when compared to Old World elapids, the expression profile suggests abundant and diversified components that may be used in future antiserum formulation. As recombinant production of venom antigens frequently fails due to complex disulfide arrangements, DNA immunization may be a viable alternative. In fact, the selected candidates provided an initial evidence of the feasibility of this approach, which is less costly and not dependent on the availability of the venom.


Subject(s)
Elapid Venoms/genetics , Elapidae/genetics , Expressed Sequence Tags , Gene Expression Profiling , Alternative Splicing , Amino Acid Sequence , Animals , Antigens/immunology , Antivenins , Cluster Analysis , Elapid Venoms/immunology , Female , Gene Library , Immune Sera/analysis , Immunoglobulin G/blood , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Protein Isoforms/genetics , Sequence Alignment , Sequence Analysis, DNA , Vaccines, DNA/immunology
6.
Toxicon ; 53(7-8): 743-53, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19249326

ABSTRACT

Sphingomyelinase D (SMase D) present in the venoms of Loxosceles spiders is the principal component responsible for local and systemic effects observed in the loxoscelism. By using "expressed sequencing tag", it was possible to identify, in a L. laeta venom gland library, clones containing inserts coding for proteins with similarity to SMase D. One of these clones was expressed and the recombinant protein compared with the previously characterized SMase I from L. laeta, in terms of their biological, biochemical and structural properties. The new recombinant protein, SMase II, possesses all the biological properties ascribed to the whole venom and SMase I. SMase II shares 40% and 77% sequence similarity with SMase I and Lb3, respectively; the latter, a SMase D isoform from L. boneti, catalytically inactive. Molecular modeling and molecular dynamics simulations were employed to understand the structural basis, especially the presence of an additional disulfide bridge, in an attempt to account for the observed differences in SMases D activity.


Subject(s)
Exocrine Glands/enzymology , Phosphoric Diester Hydrolases/metabolism , Spider Venoms/enzymology , Amino Acid Sequence , Blotting, Western , Buffers , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Erythrocytes/drug effects , Flow Cytometry , Hemolysis/drug effects , Humans , Indicators and Reagents , Models, Molecular , Molecular Sequence Data , Necrosis/chemically induced , Necrosis/pathology , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Recombinant Proteins/chemistry , Skin/pathology
7.
BMC Genomics ; 9: 279, 2008 Jun 12.
Article in English | MEDLINE | ID: mdl-18547439

ABSTRACT

BACKGROUND: The bite of spiders belonging to the genus Loxosceles can induce a variety of clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis, and persistent inflammation. In order to examine the transcripts expressed in venom gland of Loxosceles laeta spider and to unveil the potential of its products on cellular structure and functional aspects, we generated 3,008 expressed sequence tags (ESTs) from a cDNA library. RESULTS: All ESTs were clustered into 1,357 clusters, of which 16.4% of the total ESTs belong to recognized toxin-coding sequences, being the Sphingomyelinases D the most abundant transcript; 14.5% include "possible toxins", whose transcripts correspond to metalloproteinases, serinoproteinases, hyaluronidases, lipases, C-lectins, cystein peptidases and inhibitors. Thirty three percent of the ESTs are similar to cellular transcripts, being the major part represented by molecules involved in gene and protein expression, reflecting the specialization of this tissue for protein synthesis. In addition, a considerable number of sequences, 25%, has no significant similarity to any known sequence. CONCLUSION: This study provides a first global view of the gene expression scenario of the venom gland of L. laeta described so far, indicating the molecular bases of its venom composition.


Subject(s)
Spiders/genetics , Amino Acid Sequence , Animals , Expressed Sequence Tags , Female , Gene Expression , Gene Expression Profiling , Gene Library , Humans , Hyaluronoglucosaminidase/genetics , Molecular Sequence Data , Phosphoric Diester Hydrolases/genetics , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Spider Bites , Spider Venoms/genetics , Spiders/pathogenicity , Spiders/physiology
8.
Biochim Biophys Acta ; 1760(12): 1762-71, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17034951

ABSTRACT

The protein (LV-PA) from bushmaster (Lachesis muta muta) venom is a serine proteinase which specifically activates the inactive proenzyme plasminogen. LV-PA is a single chain glycoprotein with an apparent molecular mass of 33 kDa that fell to 28 kDa after treatment with N-Glycosidase F (PNGase F). Approximately 93% of its protein sequence was determined by automated Edman degradation of various fragments derived from a digestion with trypsin. A cDNA library of L. m. muta was constructed to generate expressed sequence tags (ESTs) and the plasminogen activator precursor cDNA was sequenced. The complete amino acid sequence of the enzyme was deduced from the cDNA sequence. LV-PA is composed of 234 residues and contains a single asparagine-linked glycosylation site, Asn-X-Ser, bearing sugars that account for approximately 10% of the enzyme's total molecular mass of 33 kDa. The sequence of LV-PA is highly similar to the plasminogen activators (PAs) TSV-PA from Trimeresurus stejnegeri venom and Haly-PA from Agkistrodon halys. Furthermore, the mature protein sequence of LV-PA exhibits significant similarity with other viperidae venom serine proteinases which affect many steps of hemostasis, ranging from the blood coagulation cascade to platelet function. The Michaelis constant (Km) and the catalytic rate constant (kcat) of LV-PA on four chromogenic substrates were obtained from Lineweaver-Burk plots. In addition, we used an indirect enzyme-linked immunoabsorbent assay (ELISA) to explore the phylogenetic range of immunological cross-reactivity (using antibodies raised against LV-PA) with analogous serine proteinases from two viperidae venoms and mammals.


Subject(s)
Peptide Hydrolases/metabolism , Plasminogen Activators/metabolism , Viper Venoms/enzymology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary , Enzyme-Linked Immunosorbent Assay , Glycosylation , Kinetics , Molecular Sequence Data , Peptide Hydrolases/chemistry , Peptide Hydrolases/genetics , Plasminogen Activators/chemistry , Plasminogen Activators/genetics , Sequence Homology, Amino Acid , Structure-Activity Relationship
9.
Toxicon ; 48(4): 437-61, 2006 Sep 15.
Article in English | MEDLINE | ID: mdl-16905169

ABSTRACT

Bothrops jararaca is a pit viper responsible for the majority of snake envenoming accidents in Brazil. As an attempt to describe the transcriptional activity of the venom gland, ESTs of a cDNA library constructed from B. jararaca venom gland were generated and submitted to bioinformatics analysis. The results showed a clear predominance of transcripts coding for toxins instead of transcripts coding for proteins involved in cellular functions. Among toxins, the most frequent transcripts were from metalloproteinases (52.6%), followed by serine-proteinases (28.5%), C-type lectins (8.3%) and bradykinin-potentiating peptides (BPPs) (6.2%). Results were similar to that obtained from the transcriptome analysis of B. insularis, a phylogenetically close sister of B. jararaca, though some differences were observed and are pointed out, such as a higher amount of the hypotensive BPPs in B. insularis transcriptome (19.7%). Another striking difference observed is that PIII and PII-classes of metalloproteinases are similarly represented in B. jararaca in contrast to B. insularis, in which a predominance of PIII-class metalloproteinase, which present a more intense hemorrhagic action, is observed. These features may, in part, explain the higher potency of B. insularis venom. The results obtained can help in proteome studies, and the clones can be used to directly probe the genetic material from other snake species or to investigate differences in gene expression pattern in response to factors such as diet, aging and geographic localization.


Subject(s)
Bothrops/metabolism , Crotalid Venoms/biosynthesis , Gene Expression Profiling , Animals , Gene Library , Lectins, C-Type/genetics , Metalloproteases/genetics , Phylogeny , Serine Endopeptidases/genetics , Teprotide/metabolism
10.
Toxicon ; 46(1): 31-8, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15876444

ABSTRACT

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.


Subject(s)
Bradykinin/metabolism , Crotalid Venoms/chemistry , Natriuretic Peptide, C-Type/analysis , Peptides/analysis , Viperidae/physiology , Amino Acid Sequence , Animals , Base Sequence , Protein Isoforms , Sequence Alignment , Sequence Homology, Amino Acid
11.
Gene ; 349: 173-85, 2005 Apr 11.
Article in English | MEDLINE | ID: mdl-15777719

ABSTRACT

A survey of the transcriptional profile of Haementeria depressa Ringuelet, 1972 (Annelida, Hirudinea) salivary complexes was produced through expressed sequence tag (EST). Sequences from 898 independent clones were assembled in 555 clusters, representing the transcript profile of this tissue. The repertoire of possible proteins involved in feeding and host interaction processes of the leech corresponded to 10.6% of all identified transcripts (67 clusters), being the carbonic anhydrases (30%), several coagulation inhibitors (25%) and hemerythrin-like molecules (19%), the major components. Among the 387 clusters matching cellular proteins, the majority represents molecules involved in gene and protein expression, reflecting a high specialization of this tissue for protein synthesis. Our H. depressa dbEST was also compared to those from other blood-feeding organisms, providing evidences that among the secreted proteins, the coagulation inhibitors present a profile very characteristic of this animal class.


Subject(s)
Expressed Sequence Tags , Gene Expression Profiling , Gene Expression , Leeches/genetics , Salivary Proteins and Peptides/metabolism , Amino Acid Sequence , Animals , Cluster Analysis , Coagulants/antagonists & inhibitors , Conserved Sequence , DNA, Complementary , Female , Gene Library , Molecular Sequence Data , Protein Structure, Tertiary , Salivary Proteins and Peptides/chemistry , Salivary Proteins and Peptides/genetics , Sequence Homology, Amino Acid , Transcription, Genetic
12.
Arch Biochem Biophys ; 432(1): 1-11, 2004 Dec 01.
Article in English | MEDLINE | ID: mdl-15519291

ABSTRACT

Lectins are carbohydrate-binding molecules that mediate a variety of biological processes. In this work, we identify and characterize a lectin from Bothrops insularis venom, with respect to its biochemical properties and theoretical structure. Initially, from a venom gland cDNA library, we cloned and sequenced a cDNA encoding a protein with high identity to snake venom lectins. A lectin molecule was purified to homogeneity from the venom by affinity column and gel filtration. This protein named BiL displayed hemagglutinating activity that was inhibited by galactose, lactose, and EDTA. Mass spectrometry analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that BiL is a disulfide-linked dimeric protein consisting of monomers with 16,206 m/z. The amino acid sequence, deduced from its cDNA sequence, was confirmed by Edman sequencing and by peptide mass fingerprint analysis. BiL shows similarity to other C-type lectin family members. Modeling studies provide insights into BiL dimeric structure and its structural determinants for carbohydrate and calcium binding.


Subject(s)
Bothrops/metabolism , Crotalid Venoms/chemistry , Crotalid Venoms/genetics , Lectins, C-Type/chemistry , Lectins, C-Type/genetics , Amino Acid Sequence , Animals , Base Sequence , Calcium/metabolism , Carbohydrates/chemistry , Chromatography, Gel , Cloning, Molecular , DNA, Complementary/metabolism , Dimerization , Disulfides/chemistry , Edetic Acid/pharmacology , Electrophoresis, Polyacrylamide Gel , Galactose/chemistry , Gene Library , Lectins/chemistry , Mass Spectrometry , Methylation , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Polysaccharides/chemistry , Protein Binding , Protein Structure, Tertiary , Sepharose/chemistry , Sequence Homology, Amino Acid , Software , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Time Factors , Toxins, Biological/chemistry
13.
Toxicon ; 44(5): 571-5, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15450933

ABSTRACT

Vascular endothelial growth factors (VEGFs) are among the most important angiogenic proteins found on vertebrates. In the last years, some reports of the occurrence of such proteins in snake venoms are rising the importance of this family of proteins as toxins, since they appear to be involved in many features of Viperidae envenoming, such as hypotension and venom spread through increase in vascular permeability. Here we describe the occurrence of snake venom VEGF in Bothrops erythromelas, a clinical important snake from Northeast of Brazil, through immunodetection and cloning of its cDNA and briefly provide an overview comparison of all recent described svVEGF sequences.


Subject(s)
Bothrops/genetics , Snake Venoms/genetics , Vascular Endothelial Growth Factor A/genetics , Animals , Blotting, Western , Brazil , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Humans , Immunologic Techniques , Molecular Sequence Data , Sequence Homology, Amino Acid , Snake Venoms/metabolism , Snake Venoms/toxicity , Vascular Endothelial Growth Factor A/metabolism
14.
Biochim Biophys Acta ; 1648(1-2): 90-8, 2003 May 30.
Article in English | MEDLINE | ID: mdl-12758151

ABSTRACT

The EF-hand protein family is comprised of many proteins with conserved Ca(2+)-binding motifs with important biological roles in intracellular communication. During the generation of Expressed Sequence Tags (ESTs) from the venom glands of the Viperidae snake Bothrops insularis, we identified a cDNA coding for a putative Ca(2+) binding protein with four EF-hand motifs, named here calglandulin. The deduced amino acid sequence displayed the greatest sequence similarity with calmodulin (59%), followed by troponin-C (52%). The encoded polypeptide was first expressed in Escherichia coli as a 6XHis-tagged fusion protein. The expressed protein was purified by Ni(2+)-charged affinity chromatography and circular dichroism (CD) spectroscopy confirmed the prevalence of alpha-helix as observed in calmodulin/calmodulin-like proteins. A polyclonal antiserum was generated in mice using this recombinant calglandulin. To investigate the tissue-specific biological occurrence of this protein, this antiserum was used in Western blot experiments, which revealed an immunoreactive band in samples of venom gland extracts from different snakes, but not in the crude venom or in brain, heart and other tissues. This exclusive occurrence suggests a specialized function of calglandulin in snake venom glands.


Subject(s)
Bothrops/genetics , Crotalid Venoms/genetics , Amino Acid Sequence , Animals , Base Sequence , Bothrops/metabolism , Cloning, Molecular , Crotalid Venoms/chemistry , Crotalid Venoms/metabolism , Escherichia coli , Molecular Sequence Data , Organ Specificity , Phylogeny , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Sequence Alignment
15.
Gene ; 299(1-2): 279-91, 2002 Oct 16.
Article in English | MEDLINE | ID: mdl-12459276

ABSTRACT

In order to produce a global panorama of the transcriptional activity of snake venom glands and to correlate with its venom composition, we constructed a DNA complementary to RNA library from the venom glands of the Viperidae snake Bothrops insularis for the generation of expressed sequence tags (ESTs). Sequences from 610 independent clones were grouped in 297 clusters, revealing the putative identification of 210 distinct gene products. Toxin sequences correspond to 56% of all transcripts (85 clusters), being the metalloproteinases (23%) and the bradykinin-potentiating peptides (11%) the major components. This approach revealed a new highly expressed toxin similar to vascular endothelial growth factor, which was recently reported (J. Biol. Chem. 276 (2001) 39836). Among the 125 clusters matching cellular proteins, the major part represents molecules involved in gene and protein expression, notably in disulfide bond assembly, reflecting a high specialization of this tissue for toxin synthesis. An unusual representation of retrotransposon-like sequences was also found and could be related to the occurrence and diversity of many paralogous forms of toxins in the venom gland. Our B. insularis dbEST allowed the identification of the most common classes of toxins present in Viperidae venoms, which parallels the complex hemorrhagic effects evoked by the venom on the prey. In addition, it provides the first comprehensive set of reptilian gene sequences described so far.


Subject(s)
Bothrops/genetics , Crotalid Venoms/genetics , Exocrine Glands/metabolism , Amino Acid Sequence , Animals , Base Composition , Base Sequence , DNA, Complementary/chemistry , DNA, Complementary/genetics , Enzyme Precursors/genetics , Expressed Sequence Tags , Gene Expression , Gene Library , Genetic Variation , Lectins/genetics , Metalloendopeptidases/genetics , Molecular Sequence Data , Multigene Family/genetics , Natriuretic Peptide, C-Type/genetics , Phospholipases A/genetics , Protein Precursors/genetics , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Serine Endopeptidases/genetics
16.
Eur J Biochem ; 269(24): 6052-62, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12473101

ABSTRACT

Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human alpha1B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood.


Subject(s)
Blood Proteins/chemistry , Blood Proteins/genetics , Blood Proteins/isolation & purification , Crotalid Venoms/antagonists & inhibitors , Crotalid Venoms/enzymology , Glycoproteins , Immunoglobulins , Metalloendopeptidases/antagonists & inhibitors , Protease Inhibitors/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , Blood Proteins/metabolism , Blood Proteins/pharmacology , Bothrops , Cloning, Molecular , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Gene Library , Humans , Hydrolysis , Isoelectric Focusing , Liver/metabolism , Mice , Molecular Sequence Data , Opossums , Phospholipases A/metabolism , Phospholipases A2 , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Structure, Tertiary , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
17.
Biochem Biophys Res Commun ; 298(5): 638-45, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12419302

ABSTRACT

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement-dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using "Expressed Sequencing Tag" strategy of aleatory clones from, L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases from Loxosceles intermedia venom [1]. Clone H17 was expressed as a fusion protein containing a 6x His-tag at its N-terminus and yielded a 33kDa protein. The recombinant protein was endowed with all biological properties ascribed to the whole L. laeta venom and sphingomyelinases from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities. Antiserum raised against the recombinant protein recognised a 32-kDa protein in crude L. laeta venom and was able to block the dermonecrotic reaction caused by whole L. laeta venom. This study demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.


Subject(s)
Spider Venoms/genetics , Spiders/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Complement Activation/drug effects , DNA, Complementary/genetics , Erythrocytes/drug effects , Gene Expression , Glycophorins/drug effects , Hemolysis/drug effects , Humans , In Vitro Techniques , Molecular Sequence Data , Necrosis , Recombinant Proteins/genetics , Recombinant Proteins/toxicity , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/toxicity , Spider Bites/etiology , Spider Venoms/enzymology , Spider Venoms/toxicity , Spiders/enzymology , Spiders/pathogenicity
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