|Coordinate||66,841,097 bp (GRCm38)|
|Base Change||A ⇒ T (forward strand)|
|Gene Name||toll-like receptor 4|
|Synonym(s)||Rasl2-8, Lps, lipopolysaccharide response|
|Chromosomal Location||66,827,584-66,930,284 bp (+)|
|MGI Phenotype||Homozygotes for spontaneous or targeted mutations are hyporesponsive to bacterial lipopolysaccharide and more susceptible to infection by gram negative bacteria.|
|Amino Acid Change||Aspartic acid changed to Valine|
|Institutional Source||Beutler Lab|
D709V in Ensembl: ENSMUSP00000045770 (fasta)
|Gene Model||not available|
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Phenotypic Category||immune system, TLR signaling defect: TNF production by macrophages, TLR signaling defect: type I IFN production by macrophages|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice, Embryos, Sperm|
|Last Updated||2017-03-28 1:34 PM by Katherine Timer|
The lps3 phenotype was identified in a G3 screen for mutants with impaired response to Toll-like receptor (TLR) ligands (TLR Signaling Screen). Peritoneal macrophages from lps3 mice produce normal amounts of tumor necrosis factor (TNF)-α in response to all TLR ligands tested, except lipopolysaccharide (LPS). In response to LPS, homozygous lps3 macrophages produce no TNF-α. In addition, they fail to produce type I interferon (IFN) in response to LPS. Heterozygous lps3 macrophages produce TNF-α and type I IFN at wild type levels (Figure 1).
|Nature of Mutation|
The Tlr4 gene on Chromosome 4 of Lps3 mice was sequenced, and an A to T transversion was identified in exon 3 (of 3 total exons) at position 2147 of the Tlr4 transcript.
The mutated nucleotide is indicated in red lettering, and results in an aspartic acid to valine change at amino acid 709 of the TLR4 protein.
TLR4, a type I integral membrane glycoprotein containing 835 amino acids, is the receptor for LPS (1). Like the other TLRs, its cytoplasmic domain (at its C terminus) shares similarity with the interleukin-1 and IL-18 receptors (IL-1R and IL-18R) in a conserved region of approximately 200 amino acids known as the Toll/IL-1R (TIR) domain (2;6), which mediates homo- and heterotypic protein interactions during signal transduction. TIR domains contain six α-helices (αA, αB, αC, αC’, αD and αE) and five β-strands (βA, βB, βC, βD and βE) which are connected by seven loops (named for the α-helix and β-strand they connect; e.g. AA connects βA with αA). The crystal structures of the TLR1 and TLR2 TIR domains reveal that they fold into a structure with the five β-strands forming a central parallel β-sheet which is surrounded by five α-helices (3) (see the record for languid). Many of the α-helices and connecting loops are predicted to participate in binding partner recognition, and their mutation is expected to abrogate specific binding interactions. Indeed, TIR domains in TLRs, IL receptors and the adapters MyD88 and TIRAP contain 3 conserved boxes (boxes 1, 2 and 3) required for signaling, which form part of the βA-strand, BB loop and αE-helix, respectively (4;5). A proline to histidine mutation in the BB loop, identified in Tlr4Lps-d mice abolishes LPS-induced signaling in mice (1), and MyD88 binding to the TLR4 TIR domain (7) (Figure 2).
The extracellular domains of TLRs, unlike those of the interleukin receptors, contain multiple leucine-rich repeats (LRRs), which mediate ligand recognition by TLRs and consist of 24-29 amino acids with two conserved leucine-rich sequences: XLXXLXLXXN (residues 1-10, present in all LRR subtypes) followed by XØXXØX4FXXLX (residues ~11-24, but variable in length, sequence and structure), where X is any amino acid and Øis a hydrophobic amino acid [discussed in (8)]. Crystal structures of several TLRs reveal that each LRR forms a loop such that the juxtaposition of several LRR loops forms a horseshoe structure, with the hydrophobic residues of the LRR consensus sequence pointed inward (9-12). In addition, the XLXXLXLXX sequence folds into a β-strand, with the remaining LRR residues oriented on the convex side of the structure.
TLR4 has 22 predicted LRRs in its ectodomain encoded by the N-terminal half of the protein (2;6;8). The crystal structure of the TLR4 ectodomain reveals a horseshoe structure, but composed of distinct N-terminal, central and C-terminal domains (12). The β-sheet of the central domain has an unusually small radius and large twist angle (12). The concave N-terminal and central domains of the TLR4 ectodomain provide the binding surface for MD-2 (see Background; Figure 3, PDB ID 2Z64) (12).
The lps3 mutation results in an aspartic acid to valine change at position 709 of the TLR4 protein, which lies in the BB loop of the TIR domain (Figure 3).
Tlr4 transcripts of two sizes are detected in multiple human tissues, including heart, brain, placenta, lung, skeletal muscle, kidney, pancreas, thymus, prostate, testis, ovary, small intestine and colon, but not in liver (2;6). Tlr4 expression is highest in spleen and peripheral blood leukocytes (6). RT-PCR analysis of several cell lines indicates Tlr4 expression in monocytes, macrophages, dendritic cells, γ/δ T cells, Th1 and Th2 α/β T cells, a small intestinal epithelial cell line, and a B cell line (6). TLR4 is localized on the cell surface.
Many years before the identification of TLR4 as its receptor, LPS, a glycolipid of the outer membrane of Gram-negative bacteria, was known to cause fever and shock and sometimes death by inducing cytokine production (particularly TNF) by hematopoietic cells (13-15). However, two spontaneously occurring mouse strains, C3H/HeJ and C57BL/10ScCr, were found to be resistant to the lethal effect of LPS (16-18). The existence of a single locus responsible for the response to LPS was strongly supported by analysis of progeny of C3H/HeJ mice backcrossed to LPS-sensitive CWB mice (19), and later confirmed and mapped to mouse Chromosome 4 using classical phenotypic markers (20;21). These mapping studies demonstrated that the locus, designated Lps, lay between the major urinary protein (Mup1) and polysyndactyly (Ps) loci (21). Furthermore, complementation studies showed that the affected loci in C3H/HeJ and C57BL/10ScCr strains were allelic (17;18).
Although the existence of the mammalian TLR4 sequence was known from EST databases, and a full-length sequence was cloned and shown to activate NF-κB in transfected Jurkat cells (2;6), its definitive identity as the LPS receptor was unknown until the positional cloning of the Lps gene. Lps was identified through high-resolution mapping studies using LPS-resistant or –sensitive mice (22-25), and ultimately through sequencing a minimal contig composed of 12 BACs and one YAC, which revealed Tlr4 as a promising candidate gene within the critical region (1). Sequencing of Tlr4 cDNA from both C3H/HeJ and LPS-sensitive strains revealed a point mutation in the Tlr4 coding sequence of C3H/HeJ mice, predicted to substitute histidine for proline at position 712 of TLR4, located in the cytoplasmic domain (1). A small deletion encompassing the entire Tlr4 locus but no other genes was found in the C57BL/10ScCr strain and results in a protein-null animal (1;26). These results were subsequently confirmed by other workers (27), and later, by phenotypic analysis of mice with a targeted deletion of TLR4 (28). Still later, the LPS unresponsive phenotype was corrected by BAC transgenesis in animals lacking the Tlr4 locus (29). This study also showed that the Tlr4 locus is haploinsufficient, and that augmented LPS signaling occurred with increasing copy number over a fairly broad range. Hence, the quantity of TLR4 protein (rather than another component of the LPS sensing apparatus) seems to limit the LPS response.
The MyD88-independent pathway was evidenced by the intact (but slightly delayed) LPS-dependent activation of NF-κB, JNK and MAP kinases in MyD88-deficient macrophages (32). This pathway relies on the adapter Trif, and its hallmark is the production of type I IFN. Trif signals to TRAF3 and TBK1, both of which are required for IRF-3 activation and subsequent IFN induction downstream of TLR4 (37;38).
While TLR4 is absolutely required for LPS sensing, the membrane-associated molecules CD14 (see heedless) and MD-2 have also been implicated in the TLR4-dependent LPS response (39-41). In MD-2-deficient fibroblasts, TLR4 is not properly localized at the plasma membrane, and MD-2-null mice do not respond to LPS (41). Cd14-/- mice are resistant to LPS-induced shock, demonstrating that CD14 is required for the LPS response (39). CD14 allows the TLR4-MD-2 complex to distinguish between smooth and rough LPS chemotypes (40). Evidence indicates that CD14 may serve to bind to and transfer LPS to the TLR4-MD-2 (42;43).
In a study comparing the coding sequences of TLR4 in healthy patients or patients with meningococcal sepsis, rare heterozygous missense mutations of TLR4 were found in excess frequency among patients with disease compared to ethnically matched healthy patients (44). Interestingly, no one mutation was predominant in the diseased group (44), and in particular, the commonest variant allele, exhibiting a double amino acid substitution (D299G; T399I), was not represented at an excess frequency in the diseased group. These findings suggest that rare TLR4 mutations can affect LPS responsiveness to certain infections, and can contribute to increased risk of disease in humans. In another report, the single nucleotide polymorphisms D299G and T399I were reportedly associated with an impaired response to inhaled LPS in humans (45). However, this finding was challenged by the observation that the same polymorphic allele has no effect on TNF production by human mononuclear cells (46).
Residues of the BB loop are highly conserved between TLRs, and mutation of prolines corresponding to the Lps-d mutation in TLR2 (3;47), MyD88 (48), Mal (49) or IL-1 receptor accessory protein (IL1RAcP) (50) abrogates signaling by these proteins. The crystal structure of the TLR2 TIR domain indicates that the BB loop forms part of a large conserved surface patch, with the BB loop extending away from the rest of the TIR domain in a protruding hydrophobic knob (3). Interestingly, studies of the structure and binding functions of the P681H mutant of TLR2 (corresponding to TLR4 P712H) demonstrate that P681 is not likely to contribute to the structural integrity of TLR2, since mutation to histidine had no significant effects on overall TIR domain structure (3). The effect on signaling is therefore likely to be caused by impaired binding of TLR2 P681H to its signaling partners. Consistent with this, the P681H-mutated TLR2 TIR domain fails to bind MyD88 in vitro (3), and the MyD88 TIR domain with P200H mutation fails to act as a dominant negative inhibitor of IL-1β signaling in HEK293 cells (5).
The lps3 mutation substitutes valine for aspartic acid at position 709 of TLR4. With the exception of TLRs 1 and 6, an aspartic acid at this position is conserved in all TLRs, as well as in MyD88, Mal and IL1RAcP. This residue, only 3 amino acids from the canonical P712H Tlr4Lps-d mutation, lies within the critical BB loop. In contrast to the conserved proline residue of the BB loop, the nearby aspartic acid residue which is mutated in lps3, in addition to other adjacent amino acids (arginine, glycine), are predicted to form ion-pair interactions that may stabilize the conformation of the BB loop (3). Mutation of the arginine, aspartic acid or glycine residue in TLR4 prevents activation of an NF-κB reporter (3), and mutation of arginine and aspartic acid in MyD88 results in a dominant negative protein that inhibits signaling stimulated by IL-1β (5). Thus, the effect of such mutations is ultimately to block signal transduction by TIR domains, whether primarily by destabilizing the BB loop, or by otherwise preventing interactions between signaling partners. The lps3 phenotype further supports this hypothesis. Interestingly, lps3 is a purely recessive phenotype, suggesting that residual signaling from the Tlr4lps3 allele can support a normal response to LPS stimulation in heterozygous lps3 mice. TLR4 functions as a dimer, and codominance of the Lps-d mutation suggests that the presence of one P712H-mutant TLR4 molecule blocks recruitment of signaling partners to the receptor complex. In contrast, the recessive nature of the lps3 mutation suggests that both subunits of the complex must contain the mutation to abrogate signaling.
The P681H mutation in TLR2 only partially abrogates signaling, in a ligand-dependent manner, from the TLR2/6 complex (48). Although it may be predicted that engrafting the lps3 mutation onto TLR2 would result in signaling similar to that supported by TLR2 P681H, examination of the effect of the lps3 mutation on signaling by the TLR2/6 complex may provide further insight into the mechanism of this receptor:adapter interaction.
|Primers||Primers cannot be located by automatic search.|
Lps3 genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.
Primers for PCR amplification
Lps3(F): 5’- AGCACCTGGATTTTCAGCACTCTAC -3’
Lps3(R): 5’- AAGGCCCAGTGAAGACTGGTTCTC -3’
1) 94°C 2:00
2) 94°C 0:15
3) 60°C 0:30
4) 68°C 1:00
5) repeat steps (2-4) 35X
6) 68°C 10:00
7) 4°C ∞
Primers for sequencing
Lps3_seq(F): 5’- GGGGTATTTGACACCCTCCATAG -3’
Lps3_seq(R): 5’- TAGGTAATTCATACCCCTGGAAAGG -3’
The following sequence of 1528 nucleotides (from Genbank genomic region NC_000070 for linear DNA sequence of Tlr4) is amplified:
12429 ag cacctggatt ttcagcactc tactttaaaa agggtcacag aattctcagc
12481 gttcttatcc cttgaaaagc tactttacct tgacatctct tatactaaca ccaaaattga
12541 cttcgatggt atatttcttg gcttgaccag tctcaacaca ttaaaaatgg ctggcaattc
12601 tttcaaagac aacacccttt caaatgtctt tgcaaacaca acaaacttga cattcctgga
12661 tctttctaaa tgtcaattgg aacaaatatc ttggggggta tttgacaccc tccatagact
12721 tcaattatta aatatgagtc acaacaatct attgtttttg gattcatccc attataacca
12781 gctgtattcc ctcagcactc ttgattgcag tttcaatcgc atagagacat ctaaaggaat
12841 actgcaacat tttccaaaga gtctagcctt cttcaatctt actaacaatt ctgttgcttg
12901 tatatgtgaa catcagaaat tcctgcagtg ggtcaaggaa cagaagcagt tcttggtgaa
12961 tgttgaacaa atgacatgtg caacacctgt agagatgaat acctccttag tgttggattt
13021 taataattct acctgttata tgtacaagac aatcatcagt gtgtcagtgg tcagtgtgat
13081 tgtggtatcc actgtagcat ttctgatata ccacttctat tttcacctga tacttattgc
13141 tggctgtaaa aagtacagca gaggagaaag catctatgat gcatttgtga tctactcgag
13201 tcagaatgag gactgggtga gaaatgagct ggtaaagaat ttagaagaag gagtgccccg
13261 ctttcacctc tgccttcact acagagactt tattcctggt gtagccattg ctgccaacat
13321 catccaggaa ggcttccaca agagccggaa ggttattgtg gtagtgtcta gacactttat
13381 tcagagccgt tggtgtatct ttgaatatga gattgctcaa acatggcagt ttctgagcag
13441 ccgctctggc atcatcttca ttgtccttga gaaggttgag aagtccctgc tgaggcagca
13501 ggtggaattg tatcgccttc ttagcagaaa cacctacctg gaatgggagg acaatcctct
13561 ggggaggcac atcttctgga gaagacttaa aaatgcccta ttggatggaa aagcctcgaa
13621 tcctgagcaa acagcagagg aagaacaaga aacggcaact tggacctgag gagaacaaaa
13681 ctctggggcc taaacccagt ctgtttgcaa ttaataaatg ctacagctca cctggggctc
13741 tgctatggac cgagagccca tggaacacat ggctgctaag ctatagcatg gaccttaccg
13801 ggcagaagga agtagcactg acaccttcct ttccaggggt atgaattacc taactcggga
13861 aaagaaacat aatccagaat ctttaccttt aatctgaagg agaagaggct aaggcctagt
13921 gagaacagaa aggagaacca gtcttcactg ggcctt
PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated A is shown in red text.
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|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine|
|Authors||Owen M. Siggs, Bruce Beutler|