Full-length cDNA cloning of Toll-like receptor 4 in dogs and cats

Asahina, Yuka; Yoshioka, Noriyuki; Kano, Rui; Moritomo, Tadaaki; Hasegawa, Atsuhiko

doi:10.1016/s0165-2427(03)00159-4

Cited by 36 publications

(28 citation statements)

References 12 publications

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“…The TLR1 family consists of TLR1, TLR2, TLR6 and TLR10. This family contains 19–21 LRRs and has fewer numbers than do the other families except for Dog TLR4 [Q8SQH3] [56] and human TLR4 variant [Q5VZ17] in the TLR4 family. Mammalian TLR1 contains 20 LRRs (Table 2).…”

Section: Resultsmentioning

confidence: 99%

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Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors

et al. 2007

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Abstract

Background

Toll-like receptors (TLRs) play a central role in innate immunity. TLRs are membrane glycoproteins and contain leucine rich repeat (LRR) motif in the ectodomain. TLRs recognize and respond to molecules such as lipopolysaccharide, peptidoglycan, flagellin, and RNA from bacteria or viruses. The LRR domains in TLRs have been inferred to be responsible for molecular recognition. All LRRs include the highly conserved segment, LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. There are seven classes of LRRs including "typical" ("T") and "bacterial" ("S"). All known domain structures adopt an arc or horseshoe shape. Vertebrate TLRs form six major families. The repeat numbers of LRRs and their "phasing" in TLRs differ with isoforms and species; they are aligned differently in various databases. We identified and aligned LRRs in TLRs by a new method described here.

Results

The new method utilizes known LRR structures to recognize and align new LRR motifs in TLRs and incorporates multiple sequence alignments and secondary structure predictions. TLRs from thirty-four vertebrate were analyzed. The repeat numbers of the LRRs ranges from 16 to 28. The LRRs found in TLRs frequently consists of LxxLxLxxNxLxxLxxxxF/LxxLxx ("T") and sometimes short motifs including LxxLxLxxNxLxxLPx(x)LPxx ("S"). The TLR7 family (TLR7, TLR8, and TLR9) contain 27 LRRs. The LRRs at the N-terminal part have a super-motif of STT with about 80 residues. The super-repeat is represented by STTSTTSTT or _TTSTTSTT. The LRRs in TLRs form one or two horseshoe domains and are mostly flanked by two cysteine clusters including two or four cysteine residue.

Conclusion

Each of the six major TLR families is characterized by their constituent LRR motifs, their repeat numbers, and their patterns of cysteine clusters. The central parts of the TLR1 and TLR7 families and of TLR4 have more irregular or longer LRR motifs. These central parts are inferred to play a key role in the structure and/or function of their TLRs. Furthermore, the super-repeat in the TLR7 family suggests strongly that "bacterial" and "typical" LRRs evolved from a common precursor.

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Section: Resultsmentioning

confidence: 99%

“…As seen in the TLR1 family, 5 of the 7 irregular LRRs are in the central part of the LRR domain. Dog TLR4 [Q8SQH3] [56] and human TLR4 variant [Q5VZ17] are shorter by about 200 amino acids at the N-terminus. These two TLRs contain only 16 LRRs.…”

Section: Resultsmentioning

confidence: 99%

Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors

et al. 2007

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“…Complete cDNA sequences are available only for TLR2, 4, and 9 (Asahina et al 2003;Hashimoto et al 2005;Ishii et al 2006). Compared with human TLR genes, the nucleotide homology is between 65 and 77% (TLR4) or 88% (TLR2, 9).…”

Section: Dogmentioning

confidence: 99%

Toll-like receptors in domestic animals

et al. 2010

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Toll-like receptors are pattern recognition receptors with which hosts recognize pathogen-associated molecular patterns (PAMP). This recognition process is translated rapidly into a meaningful defense reaction. This form of innate host defense is preserved in the animal kingdom: invertebrates heavily depend on it; higher vertebrates also have an adaptive immune system. Both adaptive and innate immune systems are intertwined in that the former also depends on an intact innate recognition and response system. Members of the TLR system cover recognition of parasitic, bacterial or viral germs. Due to the constraints imposed by the necessity to recognize PAMP and to interact with downstream signaling molecules, the TLR system is relatively conserved in evolution. Nevertheless, subtle species differences have been reported for several mammalian TLR members. Examples of this will be given. In all mammalian species investigated, part of the coding sequence is available for the most important TLR members, thus allowing study of expression of these TLR members in various tissues by reverse-transcription polymerase chain reaction in its classical (RT-PCR) and quantitative real time RT-PCR (qRT-PCR) form. In some species, the whole coding sequences of the most important or even all TLR members are known. This allows construction of cDNA and transfection of common host cells, thus permitting functional studies. Extensive investigations were devoted to the study of non-synonymous single nucleotide polymorphisms. In a few cases, expression of a given amino acid in the extracellular (ligand-binding) portion of TLR members could be associated with infectious diseases. This will be discussed below.

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“…TLRs are an ancient pattern recognition system central for signaling in innate immunity [6] . TLR2 expression has been reported in canine blood phagocytes [9] , and TLR4 and TLR9 have been identified in selected dog tissues [8,10,26] . To date, no study has reported on TLR expression in the dog heart.…”

Section: Tlr4mentioning

confidence: 99%

“…Information on TLRs in small animal species is still quite sparse. Recently, the full-length cDNA for TLR4 was cloned in dogs and cats [8] , and TLR2 expression has been identified in canine blood phagocytes [9] . Furthermore, the expression of TLR9 has been investigated in canine tissues [10] , and TLR expression has been reported in feline lymphoid tissues [11] .…”

Section: Introductionmentioning

confidence: 99%

Toll-Like Receptor (TLR) 2 and TLR4 Gene Expression in Canine Heart

Linde¹,

Blecha²,

Melgarejo³

2007

American J. of Animal and Veterinary Sciences

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Toll-like receptors (TLRs) are archetypal pattern recognition receptors of immediate importance for an efficacious innate immune response. TLRs exhibit marked differential tissue activity and their levels within a discrete cell type can be highly dynamic. Of 13 known mammalian paralogues, three TLRs have been identified in the dog. Although cardiac TLR expression has been reported in other species, this study is the first to present evidence that these innate immune receptors are expressed in the canine heart. Heart tissue samples from all four chambers were collected from healthy dogs immediately after euthanasia and stored at -80ºC until analysis. Total RNA was extracted with TRI Regent. Specific primers were designed for amplification of canine TLR2 and TLR4 based on previously reported sequences for these genes. Reverse transcription was performed with M-MLV reverse transcriptase. PCR amplification was performed and PCR products analyzed by agarose gel electrophoresis. Bands were excised from the gel and the DNA isolated and cloned using the TA Cloning ® Kit. The correct sequence for each product was verified by nucleotide sequencing. TLR4 expression was detected in the left ventricle and right atrium; TLR2 was detectable at low levels in the right atrium only. Identity of the RT-PCR products was confirmed by sequencing. Our findings show that at least two TLR paralogues -namely TLR2 and TLR4 -are expressed in the canine heart. Additional studies are warranted to determine these immune receptors' potential implication in the development of naturally occurring heart disease in the dog.

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Full-length cDNA cloning of Toll-like receptor 4 in dogs and cats

Cited by 36 publications

References 12 publications

Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors

Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors

Toll-like receptors in domestic animals

Toll-Like Receptor (TLR) 2 and TLR4 Gene Expression in Canine Heart

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