Phylogenetic variation and polymorphism at the Toll-like receptor 4 locus (TLR4)

Abstract

4), the product of which is thought to bind LPS and mediate LPS signal transduction in immune system cells. and, in both humans and mice, flanking sequences and introns are rich in repeats of retroviral origin. Interstrain analyses reveal that Tlr4 is a polymorphic protein and that the extracellular domain is far more variable than the cytoplasmic domain, both among strains and among species. The cytoplasmic domain of the Tlr4 protein is highly variable at the carboxy-terminal end. We suggest that selective evolutionary pressure exerted by microbes expressing structurally distinguishable LPS molecules has produced the high level of variability in the Tlr4 extracellular domain. The highly variable carboxy-terminal region of the cytoplasmic domain is likely to determine the magnitude of the response to LPS within a species.

Background:

4), the product of which is thought to bind LPS and mediate LPS signal transduction in immune system cells.

Results:

and, in both humans and mice, flanking sequences and introns are rich in repeats of retroviral origin. Interstrain analyses reveal that Tlr4 is a polymorphic protein and that the extracellular domain is far more variable than the cytoplasmic domain, both among strains and among species. The cytoplasmic domain of the Tlr4 protein is highly variable at the carboxy-terminal end.

Conclusions:

We suggest that selective evolutionary pressure exerted by microbes expressing structurally distinguishable LPS molecules has produced the high level of variability in the Tlr4 extracellular domain. The highly variable carboxy-terminal region of the cytoplasmic domain is likely to determine the magnitude of the response to LPS within a species.

Background

].

], baboons and most other primates are highly resistant. It is likely that part of the difference in sensitivity may be explained at a very proximal level, although differences in responses to cytokines (for example TNF) may also have a role. Neither interspecific differences nor interindividual differences in LPS responses have, until recently, been accessible to systematic genetic analysis. Advances in understanding LPS signal transduction now permit these issues to be examined.

].

].

sequence from two species of subhuman primates that have dramatically different responses to LPS.

) that includes a fourth exon, positioned between the 'normal' first and second introns. When included in the processed transcript, however, this exon specifies early termination of the polypeptide chain. Although it is possible that translation is initiated distal to the added stop codon, and that a shorter product results in the human than in the mouse, this would be unusual, given the length of the 5' untranslated region (UTR) that would then exist and the presence of multiple upstream initiation codons. Moreover, there is no murine sequence homologous to the alternative second exon of the human gene. The biological significance of this exon is therefore unclear and, in all likelihood, its inclusion in the mRNA leads to the formation of a nonfunctional protein.

, the grayscale images of the human and mouse genes call attention to the repetitive elements in the region and illustrate the relationship between exons and spacing in the two species.

], in which early truncation of the protein is predicted. The grayscale image was generated using X-GRAIL, version 1.3c, and depicts GC content as well as repetitive elements (both complex and simple) identified by RepeatMasker (which appear as unbroken stretches of white). GC-rich areas appear darker than AT-rich areas. Grail exons are shown in green (highest quality) and blue (intermediate quality) above each grayscale image. Restriction sites indicate enzymes that cut at single sites within the interval.

locus

gene exhibits 11 mutations the distinguish it from the most common haplotype, six of them specifying changes in the Tlr4 amino-acid sequence; the SEA/GnJ strain differs by nine mutations, and the strains NZW/J and VM/Dk, which are identical to one another, differ from the most common haplotype at six sites. Shared mutations suggest that introgression took place after mutational separation had occurred, leading to the introduction of groups of mutations by genetic recombination. Hence, mice of the P/J, NZW/J, and VM/Dk strains have several mutations that are observed in the A/J and BALB/c strains, but also lack some of the mutations of the latter strains and have unique mutations of their own.

in mice

*Strains are as follows: I, NZO/HILt; 2, SI/Col; 3, DBA/IJ; 4, A/J; 5, EL/Suz; 6, CBA/J; 7, AKR/J; 8, BALB/cJ; 9, DDY/JcI; 10, P/J; I I, MRL/MPJ; 12, SJL/J; 13, NOD/LtJ; 14, 129/J; 15, FL/IRe; 16, MA/MyJ; 17, SWR/J; 18, LP/J; 19, PRO/IReJ; 20, SODI/Ei; 21, SEA/GnJ; 22, SM/J; 23, KK/HIJ; 24, ST/bJ; 25, WB/Re; 26, YBR/Ei; 27, FVB/NJ; 28, PL/J; 29, LT/ChReSv; 30, RILLS/J; 31, RF/J; 32, NZB/BINJ; 33, AU/SsJ; 34, NZW/LacJ; 35, VM/Dk.

Exon I, 26041-26424; Exon 2, 32397-32563; Exon 3, 37732-41297. ATG at 26335; TGA at 39982. Ecto, extracellular domain; Cyto, cytoplasmic domain;

TM, transmembrane domain.

Yes implies one or two forms; No implies three to five forms among the six mammalian species examined.

strains. All coding mutations reside within exon 3. Most occupy portions of the gene corresponding to the extracellular domain. The transmembrane domain is denoted by a blue-green bar. Mutations occurring at sites that are relatively conserved among species (only one or two forms among six species) are shown in blue; those occurring at sites that are less conserved (three to five forms among six species) are shown in black.

. Arrows point in the direction of descent, and their lengths are proportional to distance. Dotted lines suggest introgression, given the similarity of the haplotypes observed. The symbol ? denotes the likelihood of an intermediate form before interbreeding of strains.

.) Of these, however, one mutation (R761H) is fairly common among the strains surveyed, and the corresponding residue has been reported as an H in the hamster. A single conservative substitution (V637I) was noted within the transmembrane domain of the P/J strain.

sequences, and their relationship to the human and rodent sequences

).

Conservation of Tlr4 among six mammalian species, calculated according to region

With respect to the human sequence, the extracellular domain is amino acids 1-624; transmembrane domain, amino acids 625-658; proximal cytoplasmic domain, amino acids 659-618; distal cytoplasmic domain, amino acids 619-638. Homology estimates were generated by multiplex FASTA comparison; ns, not significant.

Spline curve illustrating interspecific sequence variation across the Tlr4 protein. A multiple alignment of Tlr4 sequences from three rodent species (mouse, rat and hamster) and three primate species (human, chimpanzee and baboon) was generated using the GCG program Pileup. The number of amino acids observed at each residue was plotted using the program Prism 3.0 (a value of 1 was assigned if a single amino acid was observed in the six species; a value of 5 was assigned if five different amino acids were observed among the six species, and so on). The points were then connected using a cubic spline curve. TM, transmembrane domain. Numbering refers to the human sequence. Where a deletion was introduced by Pileup, a single mismatch was assumed. Where the sequence was truncated, each missing residue was tabulated as a separate mismatch.

Discussion

that are absent in closely related LPS-sensitive strains.

].

. The first step in determining whether different isoforms of Tlr4 confer added risk of, or protection against, sepsis is the assessment of genetic variability at this locus in the normal population.

locus implicated in the defect.

].

], and have particular 'set points' for responses to toxic LPS molecules. An assessment of variability may be made through comparison of different species, but is complemented by the study of a large number of individuals within species. Both approaches reveal that the extracellular domain of Tlr4 is highly variable compared with the transmembrane domain and proximal cytoplasmic domain of the protein. Pooling the number of mutable sites in the extracellular domain and transmembrane domain of humans and mice, 17 coding changes are observed, compared with two in the proximal cytoplasmic domain. Moreover, variability does not seem confined to any particular region of the extracellular domain, but is spread uniformly across its length.

], and the relatively high frequency of polymorphism observed in Tlr4 may be viewed as a consequence of the protective effect rendered by LPS recognition and the variability of LPS structure.

]. The highly variable carboxy-terminal end of the Tlr4 cytoplasmic domain may be seen as the embodiment of interspecific differences in LPS sensitivity, although the poor conservation of this portion of the molecule might alternatively be taken to indicate a neutral effect of mutation. It is also possible that this region of the molecule is subject to a higher rate of mutation than that applying to the rest of the protein. Although most such mutations might be removed by selection, some might be discovered in populations defined by the occurrence of Gram-negative sepsis.

) genomic sequences

for the human sequence). All data related to mutations are presented with reference to these sequences.

Sequencing DNA from mouse, chimpanzee and baboon samples

strains, was ordered from the Jackson Laboratories. Chimpanzee and baboon DNA were obtained from Kurt Benirschke (University of California, San Diego) and Gregory Delzoppo (Scripps Research Institute), respectively.

were amplified independently from mouse genomic DNA samples, leaving a margin of approximately 50 bp to each side of the exons so as to indentify intronic mutations that might alter splicing. All exons of the chimpanzee were amplified and sequenced using the same primers used to amplify and sequence the human exons. For the baboon, the first two exons were also amplified using these same primers; however, the third exon of the baboon was amplified with a substituted primer at the 5' end.

; separate sets were used to amplify and sequence mouse and the primate samples.

genes

↑ Primer matches + strand; ↓primer matches - strand.

alpha (obtained from David Gordon, University of Washington Genome Center) was used to visualize reads and mutations.

sequences were also used.

Genetic computation

A 500 MHz DEC-alpha system equipped with 256 Mbytes of memory was used for direct analysis of sequence data as described above. In addition to the programs already mentioned, the GCG software (version 9.0) was used for alignment analysis, with the program Pileup used in multiple alignments of protein sequences. The windows-based program Generunner 3.0 (Hastings Software) was used for the design of oligonucleotide primers. A spline curve describing heterogeneity of the Tlr4 polypeptide sequence from different species was produced using the program Prism 3.0 (Graphpad Software). Sequences were prepared for submission with the use of the program Sequin 2.90 (obtained from the National Center for Biotechnological Information).

Acknowledgments

We thank the Zoologic Society of San Diego, which was the source of the chimpanzee genomic DNA used in these studies.