**4. Structural analysis of vulture TLR1 and IκBα sequences**

Sequences were analyzed using the analysis software LaserGene (DNAstar, London, UK) and the analysis tools provided at the expasy web site (http://www.expasy.org). PEST regions are sequences rich in Pro, Glu, Asp, Ser and Thr, which have been pro‐ posed to constitute protein instability determinants. The analysis of the PEST region for the putative protein was made using the webtool PESTfind at http:// www.at.embnet.org/toolbox/pestfind. The potential phosphorylation sites were calculat‐ ed using the NetPhos 2.0 prediction server at http://www.cbs.dtu.dk/services/NetPhos. The prediction of the potential attachment of small ubiquitin-related modifier (SUMO) was made using the webtool SUMOplot™.

The alignment of vulture TIR domain sequences with TLR-1 from other species and of the vulture IκBα sequences with IκBα from other species was done using the program ClustalW v1.83 with Blosum62 as the scoring matrix and gap opening penalty of 1.53. Griffon vulture TLR-1 and IκBα sequences were deposited in the Genbank under accession numbers DQ480086 and EU161944, respectively.

### **4.1. Vulture TLR1**

TLR1 (CD281) and for the alpha inhibitor of NF-κB (IκBα). The tissue and cell expression pattern of vulture TLR1 and IκBα were analyzed by real-time RT-PCR and correlated with

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

To obtain specific probes for vulture TLR1 and IκBα, total RNA was isolated from vulture PBMC and from cells and tissues using the Ultraspec isolation reagent (Biotecx Laboratories, Houston TX, USA). Ten micrograms of total RNA was heated at 65 °C for 5 min, quenched on ice for 5 min and subjected to first strand cDNA synthesis. The RNA was reverse tran‐ scribed using an oligo dT12 primer by incubation with 200 U RNase H- reverse transcriptase (Invitrogen, Barcelona, Spain) at 25°C for 10 min, then at 42°C for 90 min in the presence of 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 30 U RNase-inhibitor and 1mM

For the vulture TLR probe, a partial fragment of 567 bp showing sequence similarity to hu‐ man TLR-1 was amplified by PCR from vulture PBMC cDNA using two oligonucleotide pri‐ mers TLR1/2Fw (5'-GAT TTC TTC CAG AGC TG–3') and TLR1/3Rv (5'-CAA AGA TGG ACT TGT AAC TCT TCT CAA TG -3'), which were designed based on regions of high ho‐ mology among the sequences of human and mouse TLR1 (GenBank, accession numbers NM\_003263 and NM\_030682, respectively). Cycling conditions were 94°C for 30 s, 52°C for

For the vulture IκBα probe, a partial fragment of 336 bp showing sequence similarity to hu‐ man and chicken IκBα was amplified by PCR from vulture PBMC cDNA using two oligonu‐ cleotide primers IκBα-Fw (5'-CCT GAA CTT CCA GAA CAA C-3') and IκBα-Rv (5'-GAT GTA AAT GCT CAG GAG CCA TG-3'), which were designed based on regions of high ho‐ mology among the sequences of human and chicken IκBα (GenBank, accession numbers M69043 and S55765, respectively). Cycling conditions were 94°C for 30 s, 52°C for 30 s and

The obtained PCR products were cloned into pGEM-T easy vector using a TA cloning kit (Promega, Barcelona, Spain) and sequenced bidirectionally to confirm their respective spe‐ cificities. These fragments were DIG-labelled following the recommendation of the manu‐ facturer (Roche, Barcelona, Spain) and used as probes to screen 500 000 plaque colonies of

Total RNA (500 μg) was extracted from PBMC (pooled from 6 birds) using the Ultraspec iso‐ lation reagent (Biotecx). mRNA (20 μg) was extracted by Dynabeads (Dynal biotech-Invitro‐ gen, Barcelona, Spain) and used in the construction of a cDNA library in Lambda ZAP vector (Stratagene, La Jolla, CA, USA) by directional cloning into EcoRI and XhoI sites. The cDNA library was plated by standard protocols at 50 000 plaque forming units (pfu) per plate and grown on a lawn of XL1-Blue E. coli for 6-8 h. Screening of the library was per‐ formed with DIG labelled probes. Plaques were transferred onto Hybond-N+ membranes

the ability to respond to various pathogenic challenges.

dNTPs, in a total volume of 20 μl.

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30 s and 72°C for 1.5 min, for 30 cycles.

72°C for 1.5 min, for 30 cycles.

the vulture-PBMC cDNA library.

**3.2. cDNA library construction and screening**

**3.1. Design of specific probes for vulture TLR1 and IκBα**

The screening of the vulture PBMC cDNA library for TLR1 yielded seven clones with identi‐ cal open reading frame (ORF) sequences. The fact that the screening of 500,000 vulture cDNA clones resulted in 7 identical sequences suggested that this TLR receptor is broadly represented in PBMC, possibly illustrating its important role in pathogen recognition during vulture innate immune response. This result was consistent with the real time RT-PCR anal‐ ysis of TLR1 transcripts in vulture cells.

The largest clone (2,355 bp) contained an ORF that encoded a 650 amino acid putative vulture orthologue to TLR1, flanked by 319 bp 5'UTR and a 83 bp 3'UTR that contained a potential polyadenylation signal, AATAAA, 21 bp upstream of the poly (A) tail (Fig. 2). The predicted molecular weight of the putative vulture TLR1 was of 74.6 KDa. The predicted protein sequence had a signal peptide, an extracellular portion, a short trans‐ membrane region and a cytoplasmic segment (Fig. 2). In assigning names to the vulture TLR, we looked at the closest orthologue in chicken and followed the nomenclature that was proposed for this species (Yilmaz et al., 2005). Therefore, the discovered sequence

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The comparison of the deduced amino acid sequence of vulture TLR1 with the sequence of chicken, pig, cattle, human and mouse TLR1 indicated that the deduced protein had a high‐ er degree of similarity to chicken (64% of amino acid similarity) than to pig (51%), cattle (51%), human (51%) and mouse (48%) sequences (Fig. 3). Protein sequence similarity was

*4.1.1. Amino acid sequence comparison of vulture TLR1 with other species*

**Figure 3.** Alignment of amino acid sequences of TLR1 from different species.

Amino acid sequence of vulture TLR1 was aligned with the orthologous sequence of chicken (*Gallus gallus*), pig (*Sus scrofa*), cattle (*Bos taurus*), human (*Homo sapiens*) and mouse (*Mus musculus*) based on amino acid identity and structural similarity. Identical amino acid resi‐

was identified as vulture TLR1.

different on different TLR domains (Fig. 3).


**Figure 2.** Nucleotide and deduced amino acid sequence of vulture TLR1. Complete sequence of the full-length Vulture TLR obtained from the cDNA library (GenBank accession number: DQ480086). Translated amino acid sequence is also shown under nucleotide sequence. Numbers to the right of each row refer to nucleotide or amino acid position. The cleavage site for the putative signal peptide is indicated by an arrow. LRRs domains are shaded. Potential N-glycosyla‐ tion sites are circled. The predicted transmembrane segment is underlined. The initiation codon (atg) and the polyade‐ nilation site are underlined. The translational stop site is indicated by an asterisk. The cysteines critical for the maintenance of the structure of LRR-CT are in bold.

The largest clone (2,355 bp) contained an ORF that encoded a 650 amino acid putative vulture orthologue to TLR1, flanked by 319 bp 5'UTR and a 83 bp 3'UTR that contained a potential polyadenylation signal, AATAAA, 21 bp upstream of the poly (A) tail (Fig. 2). The predicted molecular weight of the putative vulture TLR1 was of 74.6 KDa. The predicted protein sequence had a signal peptide, an extracellular portion, a short trans‐ membrane region and a cytoplasmic segment (Fig. 2). In assigning names to the vulture TLR, we looked at the closest orthologue in chicken and followed the nomenclature that was proposed for this species (Yilmaz et al., 2005). Therefore, the discovered sequence was identified as vulture TLR1.

#### *4.1.1. Amino acid sequence comparison of vulture TLR1 with other species*

cccagttctcagaagcatgcttcacaaatacggatcatactatgtgacttacacgcttatc 61 aggcaaaagtctctgaagtttcccataaaggatattctgaagaaagtttgaaggtactca 121 taaataatttgactgaatgccaggatataggaaggagaaagaaaattaagcacatgtgga 181 agaattgtatccttctttcacctagtccctggatattgatgaaattttgtcctaagaaga 241 aataacgacttgaaggattagaacaaaggtggacagataagagaagtattgagcatctcc 301 aaggaaacagaaaccagtatgacagaaaatatgagatctctcagaaacttttttctttac 361 **M** T E N M R S L R N F F L Y 14 aagtgtctgtttgcattaactttttggaattgtgtcagcctgtctgtggaaaatgaactc 421 K C L F A L T F W N C V S L S V E N E L 34 ttcacatctgtttctaacgaagatggttctgacaaaaaaatcaagagcctgccactcctc 481 F T S V S N E D G S D K K I K S L P L L 54 tatacaaatagtcatcagtccaaagctaattttgactgggttgtgatacaaaatactaca 541 Y T N S H Q S K A N F D W V V I Q N T T 74 gaaagcctatcgttgtcagaaatcacaaatgacaatgtaaaaaaattagtagcattatta 601 E S L S L S E I T N D N V K K L V A L L 94 tctaatttcagacaaggctccaggttacaaaatctgacactgacaaatgtgtcagttgac 661 S N F R Q G S R L Q N L T L T N V S V D 114 tggaatgctcttattgaaacttttcagactgtatggcactcacccattgaatacttcagt 721 W N A L I E T F Q T V W H S P I E Y F S 134 gttaacggtgtaacacaattgtcggacatcgaaagctatgactttgactattcaggtacg 781 V N G V T Q L S D I E S Y D F D Y S G T 154 tctatgaaagcggtcacaatgaagaaagttttaatcacagatctgtacttctcacagaat 841 S M K A V T M K K V L I T D L Y F S Q N 174 gacctatacaaaatatttgcagacatgaatattgcagccttgacaatagctgaatcagag 901 D L Y K I F A D M N I A A L T I A E S E 194 atgatacatatgctgtgtccttcgtctgacagtccctttagatacttaaattttttaaag 961 M I H M L C P S S D S P F R Y L N F L K 214 aacgatttaacagatctgctttttcaaaaatgtgacaaattaattcaactggagacatta 1021 N D L T D L L F Q K C D K L I Q L E T L 234 atcttgccgaagaataaatttgagagcctttccaaggtaagcttcatgactagccgtatg 1081 I L P K N K F E S L S K V S F M T S R M 254 aaatcactgaaatacctggacatcagcagcaacttgctgagtcacgatggagctgatgtg 1141 K S L K Y L D I S S N L L S H D G A D V 274 caatgccaatgggctgagtctctgacagagttggacctgtcctcaaatcagttgacggat 1201 Q C Q W A E S L T E L D L S S N Q L T D 294 gccgtgtttgagtgcttgccagtcaacatcagaaaactcaacctccaaaacaatcacatc 1261 A V F E C L P V N I R K L N L Q N N H I 314 accagtgtccccaagggaatggctgagctgaaatccttgaaagagctgaacctggcatcg 1321 T S V P K G M A E L K S L K E L N L A S 334 aacaggctggctgacctgccggggtgcagtggctttacgtcgctggagttcctgaacgta 1381 N R L A D L P G C S G F T S L E F L N V 354 gagatgaattcgatcctcaccccatctgccgacttcttccagagctgcccacaggtcagg 1441 E M N S I L T P S A D F F Q S C P Q V R 374 gagctgcaagccgggcacaacccattcaagtgttcctgtgaactgcaagactttatccgt 1501 E L Q A G H N P F K **C** S **C** E L Q D F I R 394 ctggcgaggcagtctggggggaagctgtttggctggccagcggcgtatgtgtgcgagtac 1561 L A R Q S G G K L F G W P A A Y V **C** E Y 414 ccggaagacttgcaaggaacgcagctgaaggacttccacctgactgaactggcttgcaac 1621 P E D L Q G T Q L K D F H L T E L A **C** N 434 acggtgctcttgctggtgacagctctgctgctgacgctggtgctggtggctgtcgtggcc 1681 T V L L L V T A L L L T L V L V A V V A 454 tttctgtgcatctacttggatgtgccgtggtacgtgcggatgacgtggcagtggacgcag 1741 F L C I Y L D V P W Y V R M T W Q W T Q 474 acaaagcggagggcttggcacagccaccccgaagagcaggagaccattctgcagtttcac 1801 T K R R A W H S H P E E Q E T I L Q F H 494 gcgttcatttcctacagcgagcgcgattcgttgtgggtgaagaacgagctgatcccgaac 1861 A F I S Y S E R D S L W V K N E L I P N 514 ctggagaagggggagggctgtgtacaactgtgccagcacgagaggaactttatccccggc 1921 L E K G E G C V Q L C Q H E R N F I P G 534 aagagcattgtggagaacatcattaactgcattgagaagagctacaggtcgatctttgtg 1981 K S I V E N I I N C I E K S Y R S I F V 554 ttgtctcccaactttgtgcagagcgagtggtgtcactatgagctgtactttgcccatcac 2041 L S P N F V Q S E W C H Y E L Y F A H H 574 aaattattcagtgagaattccaacagcttaatcctcattttactggagccgatccctccg 2101 K L F S E N S N S L I L I L L E P I P P 594 tacattatccctgccaggtatcacaagctgaaggctctcatggcaaagcgaacctacctg 2161 Y I I P A R Y H K L K A L M A K R T Y L 614 gagtggccaaaggagaggagcaagcatccccttttctgggctaacctgagggcagctatt 2221 E W P K E R S K H P L F W A N L R A A I 634 agcattaacctgctaatggctgatggaaagaggtgtggggaaacagattaagaatctttc 2281 S I N L L M A D G K R C G E T D \* 650 taatggagtttcttccattttttcttggtgaagcaataaatgctttatgatttccaaaaa 2341

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aaaaaaaaaaaaaa

maintenance of the structure of LRR-CT are in bold.

**Figure 2.** Nucleotide and deduced amino acid sequence of vulture TLR1. Complete sequence of the full-length Vulture TLR obtained from the cDNA library (GenBank accession number: DQ480086). Translated amino acid sequence is also shown under nucleotide sequence. Numbers to the right of each row refer to nucleotide or amino acid position. The cleavage site for the putative signal peptide is indicated by an arrow. LRRs domains are shaded. Potential N-glycosyla‐ tion sites are circled. The predicted transmembrane segment is underlined. The initiation codon (atg) and the polyade‐ nilation site are underlined. The translational stop site is indicated by an asterisk. The cysteines critical for the

The comparison of the deduced amino acid sequence of vulture TLR1 with the sequence of chicken, pig, cattle, human and mouse TLR1 indicated that the deduced protein had a high‐ er degree of similarity to chicken (64% of amino acid similarity) than to pig (51%), cattle (51%), human (51%) and mouse (48%) sequences (Fig. 3). Protein sequence similarity was different on different TLR domains (Fig. 3).

**Figure 3.** Alignment of amino acid sequences of TLR1 from different species.

Amino acid sequence of vulture TLR1 was aligned with the orthologous sequence of chicken (*Gallus gallus*), pig (*Sus scrofa*), cattle (*Bos taurus*), human (*Homo sapiens*) and mouse (*Mus musculus*) based on amino acid identity and structural similarity. Identical amino acid resi‐

dues to vulture TLR1 from the aligned sequences are shaded. Gaps were introduced for op‐ timal alignment of the sequences and are indicated by dashes (-). GenBank or Swiss protein accession numbers are: DQ480086, Q5WA51, Q59HI9, Q706D2, Q5FWG5 and Q6A0E8, re‐ spectively.

For the TLRs, it is assumed that the structure of the ectodomain has evolved more quickly than the structure of the TIR (Johnson et al., 2003). Similarly to other TLR receptors, the de‐ gree of homology of vulture TLR1 was higher in the transmembrane and cytoplasmic do‐ mains than in the extracellular domain.

The vulture TLR1 with 650 amino acids is probably the TLR with the shortest length and the smallest predicted MW (74.6 kDa). Recently, a chicken isoform of TLR1 (Ch-TLR1 type 2) was identified *in silico* and predicted to have a similar number of residues than vulture TLR1 (Yilmaz et al., 2005). However, this receptor also contains an additional transmembrane re‐ gion in its N-terminal end, and the pattern of expression in tissues is also different from that ChTLR1 type 1 (Yilmaz et al., 2005).

Comparison of the structure obtained from the SMART analysis (at expasy web server) of the amino acid sequence from human, bovine, pig, mouse, chicken and vulture TLR1. Each diagram shows a typical structure of a member of the toll-like receptor family. Vulture TLR1 consists of an ectodomain containing five leucine rich repeats (LRRs) followed by an addi‐ tional leucine rich repeat C terminal (LRR-CT) motif. The Vulture TLR has a transmembrane segment and a cytoplasmic tail which contains the TIR domain. Genbank or swiss accession number for proteins are DQ480086 (vulture), Q5WA51 (chicken), Q59HI9 (pig), Q706D2 (bo‐ vine), Q5FWG5 (human) and Q6A0E8 (mouse).


**Table 2.** Structural features of TLR1 receptor from Griffon vulture (*G. fulvus*), Chicken (*G. gallus*), pig (*S. scrofa*), cattle (*B. taurus*) human (*H. sapiens*) and mouse (*M. musculus*) amino acid sequences. The theoretical molecular weight, number of LRRs, and of glycosilation sites was calculated using the software available at the expasy web server (http://www.expasy.org). Genbank or Swiss accession number for proteins are DQ480086 (*G. fulvus*), Q5WA51 (*G. gallus*), Q59HI9 (*S. scrofa*), Q706D2 (*B. taurus*), Q5FWG5 (*H. sapiens*) and Q6A0E8 (*M. musculus*).

**Figure 4.** Schematic structure of TLR1 from various species

In general, the structure of vulture TLR1 shows similarity to chicken and mammalian TLR1 (Table 2). However, vulture TLR1 exhibits some structural features that could influence its functional role as pathogen receptor (Fig. 4). For example, it is possible that the smaller size

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Identification of Key Molecules Involved in the Protection of Vultures Against Pathogens and Toxins http://dx.doi.org/10.5772/54191 251

**Figure 4.** Schematic structure of TLR1 from various species

dues to vulture TLR1 from the aligned sequences are shaded. Gaps were introduced for op‐ timal alignment of the sequences and are indicated by dashes (-). GenBank or Swiss protein accession numbers are: DQ480086, Q5WA51, Q59HI9, Q706D2, Q5FWG5 and Q6A0E8, re‐

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

For the TLRs, it is assumed that the structure of the ectodomain has evolved more quickly than the structure of the TIR (Johnson et al., 2003). Similarly to other TLR receptors, the de‐ gree of homology of vulture TLR1 was higher in the transmembrane and cytoplasmic do‐

The vulture TLR1 with 650 amino acids is probably the TLR with the shortest length and the smallest predicted MW (74.6 kDa). Recently, a chicken isoform of TLR1 (Ch-TLR1 type 2) was identified *in silico* and predicted to have a similar number of residues than vulture TLR1 (Yilmaz et al., 2005). However, this receptor also contains an additional transmembrane re‐ gion in its N-terminal end, and the pattern of expression in tissues is also different from that

Comparison of the structure obtained from the SMART analysis (at expasy web server) of the amino acid sequence from human, bovine, pig, mouse, chicken and vulture TLR1. Each diagram shows a typical structure of a member of the toll-like receptor family. Vulture TLR1 consists of an ectodomain containing five leucine rich repeats (LRRs) followed by an addi‐ tional leucine rich repeat C terminal (LRR-CT) motif. The Vulture TLR has a transmembrane segment and a cytoplasmic tail which contains the TIR domain. Genbank or swiss accession number for proteins are DQ480086 (vulture), Q5WA51 (chicken), Q59HI9 (pig), Q706D2 (bo‐

**Structural feature** *G fulvus G gallus S scrofa B taurus H sapiens M musculus*

**Amino acid residues** 650 818 796 727 786 795

*Number of LRRs* 5 5 5 5 4 6

*N-glycosylation sites* 3 5 4 6 7 8

*Predicted MW(KDa)* 74.60 94.46 90.94 83.04 90.29 90.67

*Length of ectodomain* 409 569 560 521 560 558

**Table 2.** Structural features of TLR1 receptor from Griffon vulture (*G. fulvus*), Chicken (*G. gallus*), pig (*S. scrofa*), cattle (*B. taurus*) human (*H. sapiens*) and mouse (*M. musculus*) amino acid sequences. The theoretical molecular weight, number of LRRs, and of glycosilation sites was calculated using the software available at the expasy web server (http://www.expasy.org). Genbank or Swiss accession number for proteins are DQ480086 (*G. fulvus*), Q5WA51 (*G.*

*gallus*), Q59HI9 (*S. scrofa*), Q706D2 (*B. taurus*), Q5FWG5 (*H. sapiens*) and Q6A0E8 (*M. musculus*).

spectively.

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mains than in the extracellular domain.

ChTLR1 type 1 (Yilmaz et al., 2005).

vine), Q5FWG5 (human) and Q6A0E8 (mouse).

In general, the structure of vulture TLR1 shows similarity to chicken and mammalian TLR1 (Table 2). However, vulture TLR1 exhibits some structural features that could influence its functional role as pathogen receptor (Fig. 4). For example, it is possible that the smaller size of vulture TLR1, the lower number of N-glycosylation sites and the grouping of its LRRs in the proximal half of its ectodomain have functional implications.

The predicted molecular weight of the putative vulture IκBα was of 35170 Da. Structurally, the vulture I kappa B alpha molecule could be divided into three sections: a 70-amino-acid N terminus with no known function, a 205-residue midsection composed of five ankyrinlike repeats, and a very acidic 42-amino-acid C terminus that resembles a PEST sequence. Examination of the Griffon vulture sequence revealed the features characteristic of an IκB molecule (Fig. 6) The putative vulture IκBα protein was composed of a N-terminal regulato‐ ry domain, a central ankyrin repeat domain (ARD), required for its interaction with NF-κB, and a putative PEST-like sequence in the C-terminus (Fig. 6), which is similar to IκBα pro‐ teins from other organisms (Jaffray et al., 1995). Together with the N-terminal regulatory do‐ main and the central ARD domain, the presence of an acidic C-terminal PEST region rich in the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T) is characteristic of IκBα inhibitors (Luque & Gelinas, 1998). PEST regions have been found in the C-terminus of avian IκBα (Krishnan et al., 1995) and mammalian IκBα and it was also present in the vul‐ ture IκBα sequence (Fig. 6). Particularly, the PEST sequence of IκBα seems to be critical for

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Structure obtained from the SMART analysis (at expasy web server) of the amino acid se‐ quence from vulture IκBα. Each box shows a typical structure of a member of the IκBα in‐ hibitor. Vulture IκBα consists of an N-terminus regulatory domain, a central ankyrin domain containing five ankyrin repeats followed by an additional PEST-like motif. Number

Classical activation of NF-kappaB involves phosphorylation, polyubiquitination and subse‐ quent degradation of IκB (Figure. Several residues are known to be important in the N-ter‐ minal regulatory domain (Luque & Gelinas, 1998, Luque et al., 2000). In nonstimulated cells, NF-kappa B dimers are maintained in the cytoplasm through interaction with inhibitory

its calpain-dependent degradation (Shumway et al., 1999).

**Figure 6.** Schematic structure of vulture IκBα.

proteins, the IκBs (Fig. 7).

shows the amino acid flanking the relevant domains.

The set of Toll proteins for humans and insects each contain widely divergent LRR regions, and this is viewed as providing the potential to discriminate between different ligands. Per‐ haps these features provide vulture TLR1 some advantages on pathogen recognition. TLR glycosylation is also likely to influence receptor surface representation, trafficking and pat‐ tern recognition (Weber et al., 2004).

#### **4.2. Vulture IκBα**

The screening of the vulture PBMC cDNA library yielded one clone that contained an ORF that encoded a 313 amino acid putative vulture orthologue to IκBα, flanked by 15 bp 5'UTR and a 596 bp 3'UTR (Fig. 5).


**Figure 5.** Nucleotide and deduced amino acid sequence of vulture IκBα. Complete sequence of the full-length vulture IκBα obtained from the cDNA library (GenBank accession number: EU161944). Translated amino acid sequence is also shown under nucleotide sequence. Numbers to the right of each row refer to nucleotide or amino acid position. An‐ kyrin domains are shaded. The PEST region is underlined. The ATTTA domain is in bold. Phosphorylation sites Ser-35 and Ser-39 are circled. The translational stop site is indicated by an asterisk.

The predicted molecular weight of the putative vulture IκBα was of 35170 Da. Structurally, the vulture I kappa B alpha molecule could be divided into three sections: a 70-amino-acid N terminus with no known function, a 205-residue midsection composed of five ankyrinlike repeats, and a very acidic 42-amino-acid C terminus that resembles a PEST sequence. Examination of the Griffon vulture sequence revealed the features characteristic of an IκB molecule (Fig. 6) The putative vulture IκBα protein was composed of a N-terminal regulato‐ ry domain, a central ankyrin repeat domain (ARD), required for its interaction with NF-κB, and a putative PEST-like sequence in the C-terminus (Fig. 6), which is similar to IκBα pro‐ teins from other organisms (Jaffray et al., 1995). Together with the N-terminal regulatory do‐ main and the central ARD domain, the presence of an acidic C-terminal PEST region rich in the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T) is characteristic of IκBα inhibitors (Luque & Gelinas, 1998). PEST regions have been found in the C-terminus of avian IκBα (Krishnan et al., 1995) and mammalian IκBα and it was also present in the vul‐ ture IκBα sequence (Fig. 6). Particularly, the PEST sequence of IκBα seems to be critical for its calpain-dependent degradation (Shumway et al., 1999).

of vulture TLR1, the lower number of N-glycosylation sites and the grouping of its LRRs in

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

The set of Toll proteins for humans and insects each contain widely divergent LRR regions, and this is viewed as providing the potential to discriminate between different ligands. Per‐ haps these features provide vulture TLR1 some advantages on pathogen recognition. TLR glycosylation is also likely to influence receptor surface representation, trafficking and pat‐

The screening of the vulture PBMC cDNA library yielded one clone that contained an ORF that encoded a 313 amino acid putative vulture orthologue to IκBα, flanked by 15 bp 5'UTR

cggagccctgccgctatgatcagcgcccgccgcctcgtcgagccgccggttatggagggc 60

tacgagcaagcgaagaaagagcgccagggcggcttcccgctcgacgaccgccacgacagc 120 Y E Q A K K E R Q G G F P L D D R H D S 35 ggcttggactccatgaaggaggaagagtaccggcagctggtgaaggagctggaggacata 180 G L D S M K E E E Y R Q L V K E L E D I 55 cgcctgcagccccgcgagccgcccgcctgggcgcagcagctgacggaggacggagacact 240 R L Q P R E P P A W A Q Q L T E D G D T 75 tttctccacttggcgattattcacgaggaaaaagccctgagcctggaggtgatccggcag 300 F L H L A I I H E E K A L S L E V I R Q 95 gcggccggggaccgtgctttcctgaacttccagaacaacctcagccagactcctcttcac 360 A A G D R A F L N F Q N N L S Q T P L H 115 ctggcagtgatcaccgatcagcctgaaattgccgagcatcttctgaaggccggatgcgac 420 L A V I T D Q P E I A E H L L K A G C D 135 ctggaactcagggacttccgaggaaacacccccctgcatattgcctgccagcagggctcc 480 L E L R D F R G N T P L H I A C Q Q G S 155 ctcaggagcgtcagcgtcctcacgcagtactgccagccgcaccacctcctcgctgtcctg 540 L R S V S V L T Q Y C Q P H H L L A V L 175 caggcaaccaactacaacgggcatacatgtctccatttggcatctattcaaggatacctg 600 Q A T N Y N G H T C L H L A S I Q G Y L 195 cctattgtcgaatacttgctgtccttgggagcagatgtaaatgctcaggagccatgcaat 660 A I V E Y L L S L G A D V N A Q E P C N 215 ggcagaacggcactacatttggctgtcgacctgcagaattcagacctggtgtcgcttctg 720 G R T A L H L A V D L Q N S D L V S L L 235 gtgaaacatggggcggacgtgaacaaagtgacctaccaaggctattccccctatcagctc 780 V K H G A D V N K V T Y Q G Y S P Y Q L 255 acatggggaagagacaactccagcatacaggaacagctgaagcagctgaccacagccgac 840 T W G R D N S S I Q E Q L K Q L T T A D 275 ctgcagatgttgccagaaagtgaggacgaggagagcagtgaatcggagcctgaattcaca 900 L Q M L P E S E D E E S S E S E P E F T 295 gaggatgaacttatatacgatgactgccttattggaggacgacagctggcattttaaagc 960 E D E L I Y D D C L I G G R Q L A F \* 313 agagctatctgtgaaaagaagtgactgtgtacatatgtatagaaaaaggactgacttc**at** 1020 **tta**aaaagaaagtcgcaatgcaaagggaaaaaccaggagggaaatactacactgcccagc 1080 aaggagcacataattgtaacaggttctggcctgtgtttaaatacaggagtgggatgtgta 1140 acatcagtagggatctgtgattattcacaccacctgataaagagccacatagccaatctt 1200 ctcagccctacaaaggtaacagactacacatccaacctgctggttacagagagctatctt 1260 gtggtgttaagtaccacgaggaatgcgtgtcgcctcgtggcaaggcaggctcataccaac 1320 ccccccatcttctcggagactgcgtgttaatctgcgttgggctggtggtgctccctggcc 1380 ttactgaccggcctcagctgctcttggtggggtgtcccaggtggaggagtcaaaccaagg 1440 gactggtgacctcctgactgttagaagaaagtagcaataatgttaactgtgggcattgga 1500 aactgtgtgtttcacaccatgtgtgtcataattgctacactttttagcaattg 1553

**Figure 5.** Nucleotide and deduced amino acid sequence of vulture IκBα. Complete sequence of the full-length vulture IκBα obtained from the cDNA library (GenBank accession number: EU161944). Translated amino acid sequence is also shown under nucleotide sequence. Numbers to the right of each row refer to nucleotide or amino acid position. An‐ kyrin domains are shaded. The PEST region is underlined. The ATTTA domain is in bold. Phosphorylation sites Ser-35

and Ser-39 are circled. The translational stop site is indicated by an asterisk.

M I S A R R L V E P P V M E G 15

the proximal half of its ectodomain have functional implications.

tern recognition (Weber et al., 2004).

**4.2. Vulture IκBα**

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252

and a 596 bp 3'UTR (Fig. 5).

Structure obtained from the SMART analysis (at expasy web server) of the amino acid se‐ quence from vulture IκBα. Each box shows a typical structure of a member of the IκBα in‐ hibitor. Vulture IκBα consists of an N-terminus regulatory domain, a central ankyrin domain containing five ankyrin repeats followed by an additional PEST-like motif. Number shows the amino acid flanking the relevant domains.

Classical activation of NF-kappaB involves phosphorylation, polyubiquitination and subse‐ quent degradation of IκB (Figure. Several residues are known to be important in the N-ter‐ minal regulatory domain (Luque & Gelinas, 1998, Luque et al., 2000). In nonstimulated cells, NF-kappa B dimers are maintained in the cytoplasm through interaction with inhibitory proteins, the IκBs (Fig. 7).

Vulture MISARRLVEPPVMEGYEQA-KKERQGGFPL-DDRHDSGLDSMKEEEYRQLVKELEDIRLQP Chicken MLSAHRPAEPPAVEGCEPP-RKERQGGLLPPDDRHDSGLDSMKEEEYRQLVRELEDIRLQP Human MFQAAERPQEWAMEGPRDGLKKER---LL--DDRHDSGLDSMKDEEYEQMVKELQEIRLEP Mouse MFQPAGHGQDWAMEGPRDGLKKER---LV--DDRHDSGLDSMKDEEYEQMVKELREIRLQP Pig MFQPAEPGQEWAMEGPRDALKKER---LL--DDRHDSGLDSMKDEEYEQMVKELREIRLEP Rat MFQPAGHGQDWAMEGPRDGLKKER---LV--DDRHDSGLDSMKDEDYEQMVKELREIRLQP

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Vulture REPP----AWAQQLTEDGDTFLHLAIIHEEKALSLEVIRQAAGDRAFLNFQNNLSQTPLH Chicken REPPARPHAWAQQLTEDGDTFLHLAIIHEEKALSLEVIRQAAGDAAFLNFQNNLSQTPLH Human QEVPRGSEPWKQQLTEDGDSFLHLAIIHEEKALTMEVIRQVKGDLAFLNFQNNLQQTPLH Mouse QEAPLAAEPWKQQLTEDGDSFLHLAIIHEEKPLTMEVIGQVKGDLAFLNFQNNLQQTPLH Pig QEAPRGAEPWKQQLTEDGDSFLHLAIIHEEKALTMEVVRQVKGDLAFLNFQNNLQQTPLH Rat QEAPLAAEPWKQQLTEDGDSFLHLAIIHEEKTLTMEVIGQVKGDLAFLNFQNNLQQTPLH

Vulture LAVITDQPEIAEHLLKAGCDLELRDFRGNTPLHIACQQGSLRSVSVLTQYCQPHHLLAVL Chicken LAVITDQAEIAEHLLKAGCDLDVRDFRGNTPLHIACQQGSLRSVSVLTQHCQPHHLLAVL Human LAVITNQPEIAEALLGAGCDPELRDFRGNTPLHLACEQGCLASVGVLTQSCTTPHLHSIL Mouse LAVITNQPGIAEALLKAGCDPELRDFRGNTPLHLACEQGCLASVAVLTQTCTPQHLHSVL Pig LAVITNQPEIAEALLEAGCDPELRDFRGNTPLHLACEQGCLASVGVLTQPRGTQHLHSIL Rat LAVITNQPGIAEALLKAGCDPELRDFRGNTPLHLACEQGCLASVAVLTQTCTPQHLHSVL

Vulture QATNYNGHTCLHLASIQGYLAIVEYLLSLGADVNAQEPCNGRTALHLAVDLQNSDLVSLL Chicken QATNYNGHTCLHLASIQGYLAVVEYLLSLGADVNAQEPCNGRTALHLAVDLQNSDLVSLL Human KATNYNGHTCLHLASIHGYLGIVELLVSLGADVNAQEPCNGRTALHLAVDLQNPDLVSLL Mouse QATNYNGHTCLHLASIHGYLAIVEHLVTLGADVNAQEPCNGRTALHLAVDLQNPDLVSLL Pig QATNYNGHTCLHLASIHGYLGIVELLVSLGADVNAQEPCNGRTALHLAVDLQNPDLVSLL Rat QATNYNGHTCLHLASIHGYLGIVEHLVTLGADVNAQEPCNGRTALHLAVDLQNPDLVSLL

Vulture VKHGADVNKVTYQGYSPYQLTWGRDNSSIQEQLKQLTTADLQMLPESEDEESSESEP--- Chicken VKHGPDVNKVTYQGYSPYQLTWGRDNASIQEQLKLLTTADLQILPESEDEESSESEP--- Human LKCGADVNRVTYQGYSPYQLTWGRPSTRIQQQLGQLTLENLQMLPESEDEESYDTESEFT Mouse LKCGADVNRVTYQGYSPYQLTWGRPSTRIQQQLGQLTLENLQMLPESEDEESYDTES--- Pig LKCGADVNRVTYQGYSPYQLTWGRPSTRIQQQLGQLTLENLQMLPESEDEESYDTES--- Rat LKCGADVNRVTYQGYSPYQLTWGRPSTRIQQQLGQLTLENLQTLPESEDEESYDTES---

Amino acid sequence of vulture IκBα was aligned with the orthologous sequence of chicken (*Gallus gallus*), pig (*Sus scrofa*), cattle (*Bos taurus*), human (*Homo sapiens*) and mouse (*Mus musculus*) based on amino acid identity and structural similarity. Identical amino acid resi‐ dues to vulture IκBα from the aligned sequences are shaded. Gaps were introduced for opti‐ mal alignment of the sequences and are indicated by dashes (-). SUMOlation sites are squared and phosphorylation sites are circled. GenBank or Swiss protein accession numbers are: DQ480086, Q5WA51, Q59HI9, Q706D2, Q5FWG5 and Q6A0E8, respectively. Griffon vulture IκBα sequence was deposited in the Genbank under accession number EU161944.

A common characteristic of the IκB proteins is the presence of ankyrin repeats, which inter‐ act with the Rel-homology domain of NF-κB (Aoki et al., 1996; Luque & Gelinas, 1998). In the vulture sequence, five ankyrin repeats were detected using the Simple Modular Archi‐ tecture Research Tool (SMART) at EMBL (Table 2). Five ankyrin repeats also exist in human and other vertebrates IκBα (Jaffray et al., 1995). It is possible that individual repeats have

Overall identity Chicken 91% Human 73% Mouse 74% Pig 73% Rat 73%

 

Vulture EFTEDELIYDDCLIGGRQLAF Chicken EFTEDELMYDDCCIGGRQLTF Human EFTEDELPYDDCVFGGQRLTL Mouse EFTEDELPYDDCVFGGQRLTL Pig EFTEDELPYDDCVLGGQRLTL Rat EFTEDELPYDDCVFGGQRLTL

**Figure 8.** Alignment of amino acid sequences of IκBα from different species.

**Figure 7.** Activation of the NFκB pathway by TLRs. Ligand binding of TLR results in direct or indirect recruitment of a series of TIR-domain containing adapters, which in turn phosphorylates IκBs, causing degradation of the inhibitor and translocation of the transcription factor to the nucleus, where it initiates the transcription of genes encoding chemo‐ kines and pro-inflammatory cytokines.

In response to cell stimulation, mainly by proinflammatory cytokines, a multisubunit pro‐ tein kinase, the I kappa B kinase (IKK), is rapidly activated and phosphorylates two critical serines in the N-terminal regulatory domain of the I kappa Bs. Phosphorylated IκBs are rec‐ ognized by a specific E3 ubiquitin ligase complex on neighboring lysine residues, which tar‐ gets them for rapid degradation by the 26S proteasome, which frees NFκ-B and leads to its translocation to the nucleus, where it regulates gene transcription (Karin & Ben-Neriah, 2000). It has been demonstrated that phosphorylation of the N-terminus residues Ser-32 and Ser-36 is the signal that leads to inducer-mediated degradation of IκBα in mammals (Brown et al., 1997; Good et al., 1996).

As can be observed in the alignment of Figure 8, the griffon vulture equivalent residues seem to be Ser-35 and Ser-39, which are part of the conserved sequence DSGLDS (Luque et al., 2000; Pons et al., 2007). This observation suggests that the phosphorylation of these ser‐ ine residues could trigger the IκBα inducer-mediated degradation in vulture in a similar manner to that in mammals. Unlike ubiquitin modification, which requires phosphorylation of S32 and S36, the small ubiquitin-like modifier (SUMO) modification of IκBα is inhibited by phosphorylation. Thus, while ubiquitination targets proteins for rapid degradation, SU‐ MO modification acts antagonistically to generate proteins resistant to degradation (Dester‐ ro et al., 1998; Mabb & Miyamoto, 2007). This SUMO modification occurs primarily on K21 (Mabb & Miyamoto, 2007). This residue was also conserved in the IκBα sequence from hu‐ man, mouse, pig, rat and vulture, but not from chicken (Fig. 8).

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**Figure 8.** Alignment of amino acid sequences of IκBα from different species.

**Figure 7.** Activation of the NFκB pathway by TLRs. Ligand binding of TLR results in direct or indirect recruitment of a series of TIR-domain containing adapters, which in turn phosphorylates IκBs, causing degradation of the inhibitor and translocation of the transcription factor to the nucleus, where it initiates the transcription of genes encoding chemo‐

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

In response to cell stimulation, mainly by proinflammatory cytokines, a multisubunit pro‐ tein kinase, the I kappa B kinase (IKK), is rapidly activated and phosphorylates two critical serines in the N-terminal regulatory domain of the I kappa Bs. Phosphorylated IκBs are rec‐ ognized by a specific E3 ubiquitin ligase complex on neighboring lysine residues, which tar‐ gets them for rapid degradation by the 26S proteasome, which frees NFκ-B and leads to its translocation to the nucleus, where it regulates gene transcription (Karin & Ben-Neriah, 2000). It has been demonstrated that phosphorylation of the N-terminus residues Ser-32 and Ser-36 is the signal that leads to inducer-mediated degradation of IκBα in mammals (Brown

As can be observed in the alignment of Figure 8, the griffon vulture equivalent residues seem to be Ser-35 and Ser-39, which are part of the conserved sequence DSGLDS (Luque et al., 2000; Pons et al., 2007). This observation suggests that the phosphorylation of these ser‐ ine residues could trigger the IκBα inducer-mediated degradation in vulture in a similar manner to that in mammals. Unlike ubiquitin modification, which requires phosphorylation of S32 and S36, the small ubiquitin-like modifier (SUMO) modification of IκBα is inhibited by phosphorylation. Thus, while ubiquitination targets proteins for rapid degradation, SU‐ MO modification acts antagonistically to generate proteins resistant to degradation (Dester‐ ro et al., 1998; Mabb & Miyamoto, 2007). This SUMO modification occurs primarily on K21 (Mabb & Miyamoto, 2007). This residue was also conserved in the IκBα sequence from hu‐

man, mouse, pig, rat and vulture, but not from chicken (Fig. 8).

kines and pro-inflammatory cytokines.

Applications

254

et al., 1997; Good et al., 1996).

Amino acid sequence of vulture IκBα was aligned with the orthologous sequence of chicken (*Gallus gallus*), pig (*Sus scrofa*), cattle (*Bos taurus*), human (*Homo sapiens*) and mouse (*Mus musculus*) based on amino acid identity and structural similarity. Identical amino acid resi‐ dues to vulture IκBα from the aligned sequences are shaded. Gaps were introduced for opti‐ mal alignment of the sequences and are indicated by dashes (-). SUMOlation sites are squared and phosphorylation sites are circled. GenBank or Swiss protein accession numbers are: DQ480086, Q5WA51, Q59HI9, Q706D2, Q5FWG5 and Q6A0E8, respectively. Griffon vulture IκBα sequence was deposited in the Genbank under accession number EU161944.

A common characteristic of the IκB proteins is the presence of ankyrin repeats, which inter‐ act with the Rel-homology domain of NF-κB (Aoki et al., 1996; Luque & Gelinas, 1998). In the vulture sequence, five ankyrin repeats were detected using the Simple Modular Archi‐ tecture Research Tool (SMART) at EMBL (Table 2). Five ankyrin repeats also exist in human and other vertebrates IκBα (Jaffray et al., 1995). It is possible that individual repeats have remained conserved because of their important structural and functional roles in regulating NF-κB.

RT-PCR was performed on a SmartCycler® II thermal cycler (Cepheid, Sunnyvale, CA, USA) using the QuantiTect® SYBR® Green RT-PCR Kit (Quiagen, Valencia, CA, USA), fol‐ lowing the recommendations of the manufacturer. We used primers GfTLR-Fw (5'-GCT TGC CAG TCA ACA TCA GA-3') and GfTLR-Rv (5'-GAA CTC CAG CGA CGT AAA GC-3'), which amplify a fragment of 158 bp of vulture TLR1 and primers IκBα -L (5'- CTG CAG GCA ACC AAC TAC AA -3') and IκBα –R (5'- TGA ATT CTG CAG GTC GAC AG-3'), which amplify a fragment of 165 b of vulture IκBα. Cycling conditions were: 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min, for 40 cycles. As an internal control, RT-PCR was performed on the same RNAs using the primers BA-Fw (5'-CTA TCC AGG CTG TGC TGT CC-3') and BA-Rv (5'-TGA GGT AGT CTG TCA GGT CAG G-3'), which amplify a fragment of 165 bp from the conserved housekeeping gene beta-actin. Control reactions were done using the same procedures, but without RT to control for DNA contamination in the RNA prepara‐ tions, and without RNA added to control contamination of the PCR reaction. Amplification efficiencies were validated and normalized against vulture beta actin, (GenBank accession number DQ507221) using the comparative Ct method. Experiments were repeated for at least three times with similar results. Tissues used for the study were artery, liver, lung, bur‐ sa cloacalis, heart, small intestine, peripheral blood mononuclear cells (PBMC), large intes‐

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The level of TLR1 mRNA was higher in kidney, small intestine and PBMC (Fig. 9).

**Figure 9.** Relative expression of TLR1 and IκBα mRNA transcripts in vulture cells and tissues.

and calculated by the delta Ct method.

Real time RT-PCR was used to examine the relative amount of TLR1 (right) and IκBα (left) transcripts in vulture cells and tissues. The data were normalised using the beta-actin gene

Moderate vulture TLR1 mRNA levels were observed in Bursa cloacalis and large intestine, whereas the lowest TLR1 mRNA levels were found in liver, heart and artery (Fig. 9). It has been reported that the patterns of TLR tissue expression are variable, even among closely related species (Zarember & Godowski 2002). Likewise, the intensity and the anatomic loca‐

tine and kidney.

Compared with other species, vulture IκBα exhibited the lowest number of predicted SU‐ MOlation sites (Table 3).


**Table 3.** Structural features of IκBα from Griffon vulture (*G. fulvus*), Chicken (*G. gallus*), human (*H. sapiens*), pig (*S. scrofa*), rat (*R. norvegicus*) and mouse (*M. musculus*) amino acid sequences. The theoretical molecular weight, number of ankyrin repeats, SUMOlation and of phosphorylation sites was calculated using the software available at the expasy web server (http://www.expasy.org). Genbank or Swiss accession number for proteins are EU161944 (*G. fulvus*), Q91974 (*G. gallus*), P25963 (*H. sapiens*), Q08353 (*S. scrofa*), Q63746 (*R. norvegicu*s), and Q9Z1E3 (*M. musculus*).

#### *4.2.1. Amino acid sequence comparison of vulture IκBα with other species*

The comparison of the deduced amino acid sequence of vulture IκBα with the sequence of chicken, human, mouse, pig, and rat IκBα indicated that the deduced protein had a higher degree of similarity to chicken (91% of amino acid similarity) than to human (73%), mouse (74%), pig (73%) and rat (73%) sequences (Fig. 7). The analysis of the vulture IκBα sequence using the software NetPhos 2.0 (cita) revealed 14 potential phosphorylation sites: 10 Ser (S35, S39, S89, S160, S251, S263, S282, S287, S288, and S290), 1 Thr (T295) and 3 Tyr (Y16, Y45, and Y301). Although many of these residues were conserved in the aligned sequences from chicken, human, mouse, pig and rat IκBα, two phosphorylation sites (Y16 and S160) were distinctive to the vulture sequence (Fig.8).
