Venom gland transcriptomic of the snake Bothrops alternatus (urutu) and partial molecular and biochemical characterization of the dipeptidyl peptidase IV

Author(s)

Kiara Carolina Cardoso

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2011-11-18

Examining board members

Stephen Hyslop; Ana Maria Moura da Silva; Heloisa Selistre de Araujo; Marcelo Lancellotti; Marcos Tadeu Nolasco da Silva

• Advisor: Stephen Hyslop

Abstract

Transcriptomic studies of snake venom gland cDNA based on the analysis of expressed sequence tags (ESTs) have been useful in identifying the genes expressed in this organ in a variety of species, including the genus Bothrops, which is responsible for most venomous snakebites in Brazil. In this work, we used a transcriptomic approach to analyze the gene composition of the venom gland of Bothrops alternatus (urutu), a species found in southeastern and southern Brazil, Uruguay, northern Argentina e eastern Paraguay. We also cloned and partially characterized dipeptidylpeptidase IV (DPP IV), an enzyme that cleaves peptides with proline or alanine as the penultimate residue in the N-terminal region and has been identified in several snake venoms. A cDNA library constructed using conventional methods of cloning, sequencing and bioinformatic analysis yielded 5,350 ESTs that formed 838 contigs and 4512 singletons. Databank BLAST searches yielded 30% hits and 70% no-hits. Toxin-related transcripts accounted for 23% of the total transcripts and 78% of the hits. Gene ontology analysis detected genes related to general metabolism, transcription, translation, processing, polypeptide degradation, structural functions and cellular regulation. The main toxin groups identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases 'A IND. 2' ('PLA IND. 2'; 5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively class PIII, with few class PII and no class PI enzymes. The 'PLA IND. 2' were all acidic; no basic 'PLA IND. 2' were detected. Other toxins identified included L-amino acid oxidase, cysteine-rich secretory proteins, DPP IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and a dual specificity phosphatase (Dusp6), shared high sequence homology with similar proteins from other snakes. Single-nucleotide polymorphisms (SNPs), microsatellites, transposons and inverted repeats were also observed and may contribute to the toxin diversity of the gland. These results show that the venom gland of B. alternatus contains the major toxin classes identified in transcriptomic and proteomic studies of other Bothrops species. The predominance of class PIII metalloproteinases agrees with the hemorrhagic activity of this venom, while the low content of serine proteinases and C-type lectins could account for the less intense coagulopathy observed after envenoming by this species. The lack of basic 'PLA IND. 2' agrees with the lower myotoxicity of this venom compared to other Bothrops species. ... Note: The complete abstract is available with the full electronic digital thesis or dissertations (AU)