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

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.

Figure 1

Structural organization of vertebrate TLRs. Mangenta is signal peptide sequence. Green is LRRNT (the cysteine clusters on the N-terminal side of LRRs) and LRRCT (the cysteine clusters on the C-terminal side of LRRs). Yellow is LRR domain. Blue is transmembrane region. Light blue is TIR domain.

Figure 2

The multiple sequence alignment of LRRs within mammalian TLR2 from 14 species. bTLR2 [Q95LA9], nTLR2 [Q2V897], dwbTLR2 [Q2PZH4], gTLR2 [ABI31733], pTLR2 [Q59HI8], hoTLR2 [AAR08196], hTLR2 [O60603], cmTLR [Q95M53], dTLR2 [Q689D1], raTLR2 [AAM50059], mTLR2 [Q9QUN7], rTLR2 [Q6YGU2], chTLR2 [Q9R1F8], cTLR2.1 [Q9DD78], cTLR2.2 [Q9DGB6]. Abbreviations: b, Bovine; n, nilgai; dwb, domestic water buffalo; g, goat; p, pig; ho, horse; h, human; cm, Cynomolgus monkey; d, dog ; ra, rabbit; m, mouse; r, rat; ch, Chinese hamster; c, chicken. This panel shows the sequences from the N-termini to LRR10.

Figure 3

The multiple sequence alignment of LRRs within mammalian TLR2 from 14 species. This panel continued from Figure 2 shows the sequences from LRR11 to the C-termini.

Figure 4

The secondary structure prediction of human TLR2 by SSpro4.0 and Proteus. The signal peptide and extracellular domain of hTLR2 [O60603] with 784 residues is shown; residues 1–588. The highly conserved segment of individual LRRs is highlighted by a shadow. Abbreviations: h, helix; c, coil; e, β-strand.

Figure 5

Sequence alignment of LRR domains within the six families of TLRs. (1) hTLR1 [Q15399]; hTLR2 [O60603]; hTLR6 [Q9Y2C9]; hTLR10 [Q9BXR5]; tTLR14 [Q5H726]. (2) hTLR3 [O15455]; jfTLR3 [Q76CT7]. (3) hTLR4 [O00204]; dTLR4 [Q8SQH3]. (4) hTLR5 [O60602]. (5) mTLR11 [Q6R5P0]; mTLR12 [Q6QNU9]; mTLR13[Q6R5N8]: tTLR21 [NP_001027751]; tTLR22 [Q5H723]; tTLR23 [AAW70378]; (6) hTLR7 [Q9NYK1]; hTLR8 [Q9NR97]; hTLR9 [Q9NR96]. (7) jlTLRa [Q33E93]; cTLR15 [ABB71177]. The complete amino acid sequences are shown for hTLR1 with 786 residues (res.), hTLR2 with 784 res, hTLR6 with 796 res., hTLR10 with 811 res., tTLR14 with 871 res., hTLR3 with 904 res., jfTLR3 with 961 res., hTLR4 with 839 res., dTLR4 with 636 res., hTLR5 with 858 res., hTLR7 with 1049 res., hTLR8 with 1041 res., hTLR9 with 1032 res., mTLR11 with 926 res., mTLR12 with 906 res., mTLR13 with 991 res., tTLR21 with 965 res., tTLR22 with 950 res., tTLR23 with 941res., jlTLRa with 813res., and cTLR15 with 868 res., Cysteine is highlighted in magenta. Its boldface indicates cysteines in LRRNT or LRRCT. Residues of missense mutation are highlighted in blue boldface. SIGNAL, signal peptide sequence; LRRNT, the cysteine clusters on the N-terminal side of LRRs; LRRCT, the cysteine clusters on the C-terminal side of LRRs; TRANS, transmembrane region; CYTOP, cytoplasmic region. Abbreviations: h, human; m, mouse; t, Takifugu rubripes; c, chicken; d, dog; jf, Japanese flounder. This panel shows hTLR1, hTLR2, jfTLR2 and TLR6 in the TLR1 family.

Figure 6

Sequence alignment of LRR domains within the six families of TLRs. This panel continued from Figure 5 shows hTLR10 and hTLR14 in the TLR1 family, and hTLR3 and jfTLR3 in the TLR3 family. .

Figure 7

Sequence alignment of LRR domains within the six families of TLRs. This panel continued in Figure 6 shows jfTLR3 (from LRR25 to CYTOP in the TLR3 family, hTLR4 and dTLR4 in the TLR4 family, hTLR5in the TLR5 family, and mtLR11 (from SIGNAL to LRR15) in the TLR11 family.

Figure 8

Sequence alignment of LRR domains within the six families of TLRs. This panel continued in Figure 7 shows mtTLR11 (from LRR16 to CYTOP), mTLR12, mTLR13, and tTLR21 in the TLR11 family.

Figure 9

Sequence alignment of LRR domains within the six families of TLRs. This panel continued in Figure 8 shows tTLR22 and tTLR23 in the TLR11 family, and hTLR7 and hTLR8 (from SIGNAL to LRR12) in the TLR7 family.

Figure 10

Sequence alignment of LRR domains within the six families of TLRs. This panel continued in Figure 9 shows hTLR8 (from LRR13 to CYTOP) and hTLR9 in the TLR7 family, and jlTLRa and cTLR15.

Figure 11

Super-repeat of LRRs in the TLR7 family of TLR7, TLR8 and TLR9. Forty-two superimposed, cross-dot matrices from human TLR7 [Q9NYK1], mouse TLR7 [P58682], human TLR8 [Q9NR97], mouse TLR8 [P58682], human TLR9 [Q9NR96], mouse TLR9 [Q9EQU3], and green puffer TLR [Q4S0D3] with the widow size of 21 residues and the stringency of 10 (upper) and with the widow size of 41 residues and the stringency of 20 (lower). The summed scores for the 21 ((7 × 6)/2) comparisons are represented by color. The order of higher scores is red > purple > blue > light blue. Residue 46–291, 46–291, 44–288, 44–283, 40–285, 40–285, and 23–268 of human TLR7, mouse TLR7, human TLR8, mouse TLR8, human TLR9, and green puffer TLR, respectively, were used for the cross-dot matrices. The abscissa axis and the ordinate axis are residues number.

Figure 12

Sequence alignment of super-repeat of LRRs within TLR7, TLR and TLR9 from human and mouse and TLR from green puffer. human TLR7 [Q9NYK1]; mouse TLR7 [P58682]; human TLR8 [Q9NR97]; mouse TLR8 [P58682]; human TLR9 [Q9NR96]; mouse TLR9 [Q9EQU3]; green puffer TLR [Q4S0D3]. Abbreviations: h, human; m, mouse; gp, green puffer.