**2. Exploring the genes from the griffon vulture (***Gyps fulvus***) leukocytes**

Given that the vulture is protected in Spain, we have used an *ex-vivo* approach. We have generated a cDNA library from from vulture peripheral blood monuclear cells (PBMC) and screened it, either with specific probes or randomly, to search for molecules in‐ volved in the immune recognition of pathogens and in the mechanism of resistance to toxins. In order to search for molecules involved in the immune recognition of patho‐ gens and in the mechanism of resistance to toxins, we screened the cDNA library ran‐ domly. Several clones were identified and sequenced from the screening of the cDNA library from vulture´s leukocytes. A total of 49 open reading frames (ORFs) were identi‐ fied by BLAST analysis from 100 plaques approximately. The identification and function of each ORF are summarized in the Table 1.


response. The rapid identification of Toll orthologues in invertebrates and mammals sug‐ gests that these genes must be present in other vertebrates (Takeda, 2005). During the re‐ cent years, members of the multigene family of TLRs have been recognised as key players in the recognition of microbes during host defence (Hopkinsn & Sriskandan, 2005). Recognition of pathogens by immune receptors leads to activation of macrophag‐ es, dendritic cells, and lymphocytes. Signals are then communicated to enhance expres‐ sion of target molecules such as cytokines and adhesion molecules, depending on activation of various inducible transcription factors, among which the family NF-kappaB transcription factors plays a critical role. The involvement of nuclear factor-kappa B (NFκB) in the expression of numerous cytokines and adhesion molecules has supported its role as an evolutionarily conserved coordinating element in organism's response to situa‐ tions of infection, stress, and injury. In many species, pathogen recognition, whether mediated via the Toll-like receptors or via the antigen-specific T- and B-cell receptors, in‐ itiates the activation of distinct signal transduction pathways that activate NF-κB (Ghosh et al., 1998). TLR-mediated NF-κB activation is also an evolutionarily conserved event

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

that occurs in phylogenetically distinct species ranging from insects to mammals.

motif) ligand 1.

Applications

242

of each ORF are summarized in the Table 1.

Botulinun toxins are the most deadly neurotoxins known to man and animals. When an animal dies from botulism or other causes, the carcass provides the growth conditions necessary for *C. botulinun* to thrive and produce high levels of toxins. Certain species of carrion-eater birds and mammals are able to feed upon such carcasses with no apparent ill effects. Turkey vultures (*Cathartes aura*), have been shown to be highly resistant to bot‐ ulinun toxins (Kalmbach, 1993; Pates, 1967, cited by Oishi et al., 1979). The mechanism by which these species are protected against botulinun toxin was investigated by explor‐ ing the genes from the griffon vulture (*Gyps fulvus*) leukocytes, particularly with the identification of ORFs with homology to the Ras-related botulinun toxin substrate 2 (RAC2), ADP-ribosylation factor 1 (a GTP-binding protein that functions as an allosteric activator of the cholera toxin catalytic subunit); a ras-related protein Rabb-11-B-like, and other ORFs with homology to some chemical mediators, such as IL-8, Chemokine (C-C

**2. Exploring the genes from the griffon vulture (***Gyps fulvus***) leukocytes**

Given that the vulture is protected in Spain, we have used an *ex-vivo* approach. We have generated a cDNA library from from vulture peripheral blood monuclear cells (PBMC) and screened it, either with specific probes or randomly, to search for molecules in‐ volved in the immune recognition of pathogens and in the mechanism of resistance to toxins. In order to search for molecules involved in the immune recognition of patho‐ gens and in the mechanism of resistance to toxins, we screened the cDNA library ran‐ domly. Several clones were identified and sequenced from the screening of the cDNA library from vulture´s leukocytes. A total of 49 open reading frames (ORFs) were identi‐ fied by BLAST analysis from 100 plaques approximately. The identification and function

> protein that functions as an allosteric activator of the cholera toxin catalytic subunit); a rasrelated protein Rabb-11-B-like; some chemical mediators, such as IL-8, Chemokine (C-C mo‐ tif) ligand 1, etc. These sequences were deposited in the Genbank under accession numbers

Identification of Key Molecules Involved in the Protection of Vultures Against Pathogens and Toxins

http://dx.doi.org/10.5772/54191

245

The *ras* and *ras-related* genes represent a superfamily coding for low molecular weight GTPases (Bourne et al., 1991). These proteins, which share significant homology in the four regions shown for the H-ras protein to be involved in the binding and hydrolysis of GTP, regulate a diverse number of cellular processes including growth and differentiation, vesicu‐ lar trafficking, and cytoskeleton organization. GTPases are in an active state when GTP is bound and are inactive when GDP is bound, and a variety of additional proteins have been identified that regulate the switch between active and inactive states. ADP-ribosylation of cellular proteins by a number of bacterial toxins (*i.e.* cholera, pertussis, pseudomonas exo‐ toxins A., and diphtheria) is the primary mechanism for their toxicity (Eidels et al., 1983). Botulinun toxins C1 and D contain an ADP-ribosyltransferase activity that is able to ADPribosylate 21-26 kDa eukaryotic proteins. The ORF found with high homology to the Rasrelated botulinun toxin substrate 2 (RAC2) led us to hypothesize that this ORF is candidate for such a regulatory function in the vulture and it may be involved in the protection of vul‐

indicated in Table 1.

tures against toxins.

**Figure 1.** Pie-chart showing number of ORFs with different functions.

cific probes for TLR1 and IκBα.

**3. Strategy for cloning of vulture TLR1 and IκBα**

In order to identify key components of the vulture system for sensing of pathogens, we screened a cDNA library from vulture peripheral blood monuclear cells (PBMC) using spe‐

Since the majority of toll-like receptors are expressed in leukocytes and lymphoid tissues in human and other vertebrates, we decided to use vulture PBMC as the source of RNA to ob‐ tain a specific probes for TLR1 and IκBα and to construct a cDNA library. Using this strat‐ egy we cloned cDNAs encoding for griffon vulture (*Gyps fulvus*) orthologues of mammalian


**Table 1.** ORFs found from the random screening of the vulture leukocyte cDNA library. Assignment of the function of each ORF was performed based on the information shown at the Universal Protein Resource (UniProt) web page (http://www.uniprot.org/) and it was assigned as immune system (IS), Catalytic activity (CA), Cell motility (CM), Regulatory protein (RP) and Other (OT).

Interestingly, we found ORFs with homology to the Ras-related botulinun toxin substrate 2 (RAC2); the interferon regulatory factor I (IRF1), ADP-ribosylation factor 1 (a GTP-binding protein that functions as an allosteric activator of the cholera toxin catalytic subunit); a rasrelated protein Rabb-11-B-like; some chemical mediators, such as IL-8, Chemokine (C-C mo‐ tif) ligand 1, etc. These sequences were deposited in the Genbank under accession numbers indicated in Table 1.

The *ras* and *ras-related* genes represent a superfamily coding for low molecular weight GTPases (Bourne et al., 1991). These proteins, which share significant homology in the four regions shown for the H-ras protein to be involved in the binding and hydrolysis of GTP, regulate a diverse number of cellular processes including growth and differentiation, vesicu‐ lar trafficking, and cytoskeleton organization. GTPases are in an active state when GTP is bound and are inactive when GDP is bound, and a variety of additional proteins have been identified that regulate the switch between active and inactive states. ADP-ribosylation of cellular proteins by a number of bacterial toxins (*i.e.* cholera, pertussis, pseudomonas exo‐ toxins A., and diphtheria) is the primary mechanism for their toxicity (Eidels et al., 1983). Botulinun toxins C1 and D contain an ADP-ribosyltransferase activity that is able to ADPribosylate 21-26 kDa eukaryotic proteins. The ORF found with high homology to the Rasrelated botulinun toxin substrate 2 (RAC2) led us to hypothesize that this ORF is candidate for such a regulatory function in the vulture and it may be involved in the protection of vul‐ tures against toxins.

**Figure 1.** Pie-chart showing number of ORFs with different functions.

protein L23a variant 1, JX889411 2',3'-cyclicnucleotide 3' phosphodiesterase (EC 3.1.4.37) (CNP), JX889397

Applications

244

Ribosomal protein S6 (RPS6), JX889418

Hippocalcin-like protein 1 (Protein Rem-1), JX889389

Sel-1 suppressor of lin-12-like

Arf-GAP domain and FG repeatscontaining protein 1-like, JX889409

TNF receptorassociated factor 6 (TRAF-6) JX889385

Sorting nexin-5, JX889390

F-box protein 34 (FBXO34), JX889403

Low density lipoprotein receptor-related protein 5 (LRP5) JX889414

Coronin, actin binding protein 1C JX889404

tumor protein, translationallycontrolled 1 (TPT1) JX889417

SH3 domain binding glutamic acid-rich protein like (SH3BGRL) JX889401

GATA-binding factor 2 (GATA-2) (Transcription factor NF-E1b) JX889387

Cytochrome b5 JX889381

iron sulfur cluster assembly 1 homolog mitochondria JX889395

Regulatory protein (RP) and Other (OT).

Catalytic activity: Nucleoside 2',3'-cyclic phosphate + H(2)O = nucleoside 2'-phosphate CA

May play an important role in controlling cell growth and proliferation through the selective

May play a role in Notch signaling. May be involved in the endoplasmic reticulum quality control (ERQC) system also called ER-associated degradation (ERAD) involved in ubiquitin-

Required for vesicle docking or fusion during acrosome biogenesis. May play a role in RNA trafficking or localization. In case of infection by HIV-1, acts as a cofactor for viral ISRev and promotes movement of Rev-responsive element-containing RNAs from the nuclear

E3 ubiquitin ligase that, together with UBE2N and UBE2V1, mediates the synthesis of 'Lys-63'-linked-polyubiquitin chains conjugated to proteins, such as IKBKG, AKT1 and AKT2. Seems to also play a role in dendritic cells (DCs) maturation and/or activation. Represses c-Myb-mediated transactivation, in B lymphocytes. Adapter protein that seems to play a role in signal transduction initiated via TNF receptor, IL-1 receptor and IL-17 receptor.

Substrate-recognition component of the SCF (SKP1-CUL1-F-box protein)-type E3 ubiquitin

Component of the Wnt-Fzd-LRP5-LRP6 complex that triggers beta-catenin signaling through inducing aggregation of receptor-ligand complexes into ribosome-sized signalsomes.

May be involved in several stages of intracellular trafficking. OT

May be involved in cytokinesis, motility, and signal transduction. CM

Involved in calcium binding and microtubule stabilization. CM

Acts as a transcriptional regulator of PAX6. Acts as a transcriptional activator of PF4 in complex with PBX1 or PBX2. Required for hematopoiesis, megakaryocyte lineage

Transcriptional activator, which regulates endothelin-1 gene expression in endothelial cells.

Membrane bound hemoprotein which function as an electron carrier for several membrane

**Table 1.** ORFs found from the random screening of the vulture leukocyte cDNA library. Assignment of the function of each ORF was performed based on the information shown at the Universal Protein Resource (UniProt) web page (http://www.uniprot.org/) and it was assigned as immune system (IS), Catalytic activity (CA), Cell motility (CM),

Interestingly, we found ORFs with homology to the Ras-related botulinun toxin substrate 2 (RAC2); the interferon regulatory factor I (IRF1), ADP-ribosylation factor 1 (a GTP-binding

Acts as a co-chaperone in iron-sulfur cluster assembly in mitochondria OT

dependent degradation of misfolded endoplasmic reticulum proteins.

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

May be involved in the calcium-dependent regulation of rhodopsin phosphorylation OT

RP

IS

OT

IS

OT

IS

RP

RP

OT

translation of particular classes of mRNA.

periphery to the cytoplasm.

development and vascular patterning.

Binds to the consensus sequence 5'-AGATAG-3'

bound oxygenases, including fatty acid desaturases

ligase complex
