Phenotypic Mutation 'languid' (pdf version)
Allelelanguid
Mutation Type missense
Chromosome3
Coordinate83,837,315 bp (GRCm38)
Base Change T ⇒ A (forward strand)
Gene Tlr2
Gene Name toll-like receptor 2
Synonym(s) Ly105
Chromosomal Location 83,836,272-83,841,767 bp (-)
MGI Phenotype Homozygous null mice demonstrate abnormal responses to bacterial and viral infections.
Accession Number
NCBI RefSeq: NM_011905; MGI: 1346060
Mapped Yes 
Amino Acid Change Asparagine changed to Isoleucine
Institutional SourceBeutler Lab
Ref Sequences
N487I in Ensembl: ENSMUSP00000029623 (fasta)
Gene Model not available
SMART Domains

DomainStartEndE-ValueType
signal peptide 1 24 N/A INTRINSIC
LRR 51 74 1.45e2 SMART
LRR 75 98 2.33e2 SMART
LRR_TYP 99 122 3.69e-4 SMART
low complexity region 268 281 N/A INTRINSIC
LRR 359 384 6.78e1 SMART
LRR 386 409 2.54e2 SMART
LRR 412 435 8.49e1 SMART
LRR_TYP 476 499 3.34e-2 SMART
LRRCT 533 586 5.04e-7 SMART
transmembrane domain 588 610 N/A INTRINSIC
TIR 640 784 5.08e-38 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using Ensembl: ENSMUSP00000029623)
Phenotypic Category immune system, TLR signaling defect: TNF production by macrophages
Penetrance 100% 
Alleles Listed at MGI

Tlr2tm1Aki, Tlr2tm1Dgen, Tlr2tm1Kir (Allele List at MGI)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01762:Tlr2 APN 3 83836994 missense probably benign 0.00
IGL02160:Tlr2 APN 3 83837371 missense possibly damaging 0.47
IGL02405:Tlr2 APN 3 83836674 missense probably damaging 1.00
IGL02940:Tlr2 APN 3 83836474 missense probably benign 0.08
IGL03165:Tlr2 APN 3 83837948 missense probably benign 0.00
R1177:Tlr2 UTSW 3 83838734 missense probably benign 0.02
R1251:Tlr2 UTSW 3 83838269 missense possibly damaging 0.64
R1346:Tlr2 UTSW 3 83836593 missense probably damaging 0.99
R1553:Tlr2 UTSW 3 83837463 missense probably benign 0.00
R1613:Tlr2 UTSW 3 83837353 missense probably damaging 1.00
R1816:Tlr2 UTSW 3 83838209 missense probably damaging 1.00
R2312:Tlr2 UTSW 3 83837540 missense probably damaging 1.00
R3023:Tlr2 UTSW 3 83837871 missense probably benign
R4724:Tlr2 UTSW 3 83838185 missense probably damaging 1.00
R4950:Tlr2 UTSW 3 83837332 missense probably damaging 1.00
R5109:Tlr2 UTSW 3 83837723 missense probably damaging 1.00
R5764:Tlr2 UTSW 3 83838512 missense probably damaging 1.00
R5859:Tlr2 UTSW 3 83836503 missense possibly damaging 0.94
Mode of Inheritance Autosomal Recessive
Local Stock Live Mice, Embryos, gDNA
MMRRC Submission 012838-UCD
Last Updated 03/28/2017 1:24 PM by Katherine Timer
Record Created unknown
Record Posted 10/30/2007
Phenotypic Description
The languid phenotype was identified in a G3 screen for mutants with altered response to Toll-like receptor (TLR) ligands (TLR Signaling Screen).  Peritoneal macrophages from homozygous languid mice fail to produce tumor necrosis factor (TNF)-α in response to the lipopeptides Pam3CSK4 (triacyl lipopeptide), Pam2CSK4 (diacyl lipopeptide), MALP-2 (macrophage-activating lipopeptide-2; diacyl lipopeptide) and peptidoglycan (all TLR2 ligands).  The deficit in TNF-α production by languid cells mirrors that of Tlr2-null cells. Languid cells produce normal levels of TNF-α in response to lipopolysaccharide (LPS; TLR4 ligand).  Heterozygous languid macrophages behave as wild type.

 

Nature of Mutation
The languid mutation was mapped to Chromosome 3, and corresponds to an A to T transversion at position 1971 of the Tlr2 transcript, in exon 3 of 3 total exons.
 
1955 CTCTATATTTCCAGAAATAAGCTGAAAACACTC
482  -L--Y--I--S--R--N--K--L--K--T--L-
 
The mutated nucleotide is indicated in red lettering, and results in an asparagine to isoleucine change at position 487 of the TLR2 protein.
Protein Prediction
TLR2 is a 784-amino acid type 1 transmembrane glycoprotein receptor, with regions of hydrophobicity corresponding to a signal peptide at its N-terminus and a transmembrane domain near the middle of the protein (1;2).  Like the other TLRs, its cytoplasmic domain 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 (1;2), which mediates homo- and heterotypic protein interactions in signal transduction.  TIR domains in TLRs and in IL receptors contain 3 conserved boxes (boxes 1, 2 and 3), which are required for signaling (3).  In addition, 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 (2;4).  The crystal structures of the TLR1 and TLR2 TIR domains revealed that they fold into a structure with a central five-stranded parallel β-sheet surrounded by five helices (4) (Figure 1; PDB ID 1FYW).  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.  This is true of a proline to histidine mutation in the BB loop of TLR4, which abolishes MyD88 binding (5) and LPS-induced signaling in mice (6).
 
The extracellular domains of TLRs, unlike those of the interleukin receptors, contain multiple leucine-rich repeats (LRRs), each of which consists of 24-29 amino acids with two conserved leucine-rich sequences: XLXXLXLXXN (residues 1-10, present in all LRR subtypes) and XØXXØX4FXXLX (variable in length, sequence and structure), where X is any amino acid and Ø is a hydrophobic amino acid [discussed in (7)].  The XLXXLXLXX sequence folds into a β-strand.  TLR2 has 20 such LRRs (2;8).  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 (7).  While “typical” LRR family proteins normally bind ligands on the concave surfaces of the horseshoe, the crystal structure of a TLR1-TLR2-Pam3CSK4 complex (ectodomains of the TLRs fused to the hagfish Variable Lymphocyte Receptor VLRB.61) revealed that ligand binding to TLR2 occurs in a crevice on the convex side of the horseshoe (8) (Figure 2; PDB ID 2Z7X).  The surface of this crevice is completely lined with hydrophobic resides from LRR modules 9-12 (8).  Crystallization of the complex also revealed that the Pam3CSK4 ligand is indispensable for heterodimerization of TLR1 and TLR2, because the ligand bridges the two proteins with its lipid chains (8).  In addition, Pam2CSK4, a diacylated peptide, could not induce heterodimerization of TLR1 and TLR2 (8).
 
The conserved asparagine residues at the end of XLXXLXLXXN consensus sequences of the LRRs create an “asparagine ladder” in which a continuous network of hydrogen bonds forms between the asparagines and neighboring backbone oxygens (8).  Since the β strands form the inner core of the ectodomain, disruption of the ladder is predicted to significantly affect the overall protein structure (9).  The languid mutation results in an asparagine to isoleucine change at position 487 of the TLR2 protein, in the conserved asparagine residue of LRR module 18.  An asparagine residue at this position is conserved in TLR2 orthologues from 14 mammalian species (10).  Mutant protein expression has not been examined in languid cells.

 

Expression/Localization
Northern blot analysis of human tissues revealed strong Tlr2 expression in peripheral blood leukocytes (1).  Tlr2 transcript is also detected in spleen, thymus, prostate, testis, ovary, small intestine, colon, lung, heart, brain and muscle (1;2).  TLR2 is localized on the cell surface, but may be recruited to macrophage phagosomes where it can be activated by phagocytosed ligands (11).
Background
Figure 3. Overview of Toll-like receptor (TLR) signaling pathways. Shown are the signaling events downstream of TLR activation that ultimately lead to the induction of thousands of genes including TNF and type I IFN, which are critical in activating innate and adaptive immune responses. TLR1,2,4,5 and 6 are located at the cell surface, while TLR3,7, and 9 are localized in the endosome. Once TLR complexes recognize their ligands, they recruit combinations of adaptor proteins (MyD88, TICAM, TRAM, TIRAP) via homophilic TIR domain interactions.

In the MyD88-dependent pathway utilized by all TLRs except TLR3, MyD88 (lime green) recruits IRAK kinases through their death domains (DD). TRAF6 and IRF5 are also recruited to this complex. Phosphorylation of IRAK1 by IRAK4 allows dissociation of IRAK1 and TRAF6. K63 ubiquitination (small light blue circles) of TRAF6 recruits TAK1 and the TAK1 binding proteins, TAB1 and TAB2. Activation of TAK1 leads to activation of MAP kinase cascades and the IKK complex. NEMO polyubiquitination by TRAF6 is necessary for IKK complex function. The IKK complex phosphorylates IκB, p105, and TPL2 (or MAP3K8), resulting in IκB and p105 ubiquitination and degradation (small pink circles), releasing NF-κB into the nucleus and permitting TPL2 to become activated, respectively. Activation of the p38, JNK and ERK1/2 kinases leads to the activation of both CREB and AP1, which in turn induce many target genes. In pDCs, activation of TLR7 and 9 in endosomes recruits MyD88 and IRAK4, which then interact with TRAF6, TRAF3, IRAK1, IKKα, osteopontin (OPN), and IRF7. IRAK-1 and IKKα phosphorylate and activate IRF7, leading to transcription of interferon-inducible genes and production of large amounts of type I IFN.
 
In the TICAM-dependent pathway stimulated by TLR3 or 4 activation, TICAM (bright yellow) recruits polyubiquitinated RIP1, which interacts with the TRAF6/TAK1 complex and leads to NF-κB activation and proinflammatory cytokine induction. TICAM signaling also leads to type I IFN production through phosphorylation and activation of IRF3 by a complex containing TRAF3, TBK1 and IKKe; RIP1 is not required for TICAM-dependent activation of IRF3.

Note that TLR4 signals through the MyD88-dependent pathway from the cell membrane and is subsequently internalized into late endosomes to signal through the TICAM-dependent pathway. When bound to vesicular stomatitis virus glycoprotein G (VSV-G) (far left), TLR4 can signal through TRAM to induce IRF7 activation, a process that is partially dependent on TICAM. Upon viral stimulation, TLR2 may also be internalized into endosomes to activate both IRF3 and IRF7 by an unknown mechanism. LTA = lipoteichoic acid; LP2 = lipopeptide 2. PAM3CSK4 is a triacyl lipopeptide. Phosphorylation events are represented by small yellow circles labeled with a “P”. This image is interactive. Click on the image to view mutations found within the pathway (red) and the genes affected by these mutations (black). Click on the mutations for more specific information.
Figure 4. Toll-like receptor 2 (TLR2) signaling pathways. TLR2 associates with TLR1 and TLR6 to initiate a MyD88-dependent response that produces proinflammatory cytokines. Upon viral stimulation, TLR2 may also be internalized into endosomes to activate both IRF3 and IRF7 by an unknown mechanism.

TLRs are type I transmembrane proteins that sense molecules of microbial origin and trigger host cell responses.  There are 12 TLRs in mice, and 10 in humans, and each receptor recognizes a distinct microbial ligand. TLR2 recognizes microbial lipopeptides (12;13), lipoteichoic acid (LTA) (14) and zymosan (11).  TLR2 forms heterodimers with TLR1 or TLR6, to detect triacyl lipopeptides or diacyl lipopeptides, respectively.  The synthetic peptides Pam3CSK4 (tripalmitoyl-cysteinylseryl-(lysyl)3-lysine) and Pam2CSK4 (dipalmitoyl- cysteinylseryl-(lysyl)3-lysine) are commonly used as experimental ligands for TLR2.  Interestingly, some diacyl lipopeptides, such as Pam2CSK4, are recognized by TLR6-deficient cells (in a TLR2-dependent manner), suggesting that a TLR2 homodimer or a new heterodimer may recognize some lipopeptide species (15).

Upon ligand binding, the activated TLR2 heterodimer transduces the signal through the adapter proteins MyD88 and TIRAP (TIR-domain-containing adaptor protein), which recruit and activate several IRAKs (IL-1-receptor-associated kinases) and TRAF6 (tumor necrosis factor receptor-associated factor 6) [reviewed in (16;17)] (Figure 3)  Their functions lead to the activation of the IKK (IκB kinase) complex, which phosphorylates IκB, leading to the liberation of NF-κB for translocation to the nucleus and transcriptional activation. MAP kinase may also be activated through TRAF6; it controls AP-1 regulated gene expression.  Transcriptional activation of cytokines, including TNF and IL-6, results in the initiation of a local inflammatory response.
 
Study of the Cd36obl/obl (oblivious) mouse mutant demonstrated that the Cd36 receptor is required for TLR2-dependent detection of certain lipopeptides (18).  Oblivious macrophages exhibit reduced TNF-α production in response to MALP-2 and LTA, but normal TNF-α production stimulated by peptidoglycan, zymosan and Pam3CSK4 (18). Cd36obl/obl mice, like TLR2-deficient mice, are also highly susceptible to infection by Staphylococcus aureus (18;19).  Thus, while the nature of their interaction is unknown, Cd36 is a co-receptor for TLR2 recognition of certain ligands.
 
The Cd14 receptor also enhances signaling through TLR2 upon stimulation by lipopeptides (23-25).  Cd14 is a glycophosphatidylinositol-anchored LRR-containing protein found in proximity to TLR2 at the cell membrane, and proposed to facilitate the transfer of lipopeptide ligands to TLR2 complexes (23;24).  This nature of this transient interaction is also unknown.
 
In humans, a TLR2 polymorphism resulting in substitution of arginine for tryptophan at position 677 of TLR2 is associated with lepromatous leprosy (20), a disease caused by Mycobacterium leprae invasion of the peripheral nerves that primarily causes skin lesions (OMIM #246300).  TLR2 mediates innate immune recognition of M. leprae, and the Arg677Trp mutant fails to activate NF-κB in response to M. leprae when expressed in HEK293 cells (21).
Putative Mechanism
The languid mutation replaces the conserved asparagine residue of LRR module 18 of TLR2 with an isoleucine.  As mentioned above (Protein prediction), these conserved asparagines form an “asparagine ladder,” in which a continuous network of hydrogen bonds forms between the asparagines and neighboring backbone oxygens (8).  The β strands form the inner core of the ectodomain, and disruption of the ladder is predicted to significantly affect the overall protein structure (9).  Furthermore, LRR module 18 is near the end of the LRR series in TLR2, and would be positioned close to the C-terminal LRR (LRR-CT), whose sequence (and probably structure) is highly conserved both in Drosophila Toll and mammalian TLRs (7).  The LRR-CT of TLRs lies only two to ten residues from the transmembrane domain, and is believed to constrain the position of the LRR-CT at the membrane, and in turn the entire TLR ectodomain relative to the cell surface (7).  Mutations in the LRR-CT of Drosophila Toll can result in a constitutively active receptor (22).  The proximity of the languid mutation to the LRR-CT suggests that it may disrupt the position and structure of the LRR-CT, and thereby the entire ectodomain.  Consistent with this hypothesis, the languid phenotype closely resembles the phenotype of Tlr2-/- mice.
Primers Primers cannot be located by automatic search.
Genotyping
The languid mutation introduces an Mse I restriction enzyme site in the Tlr2 genomic DNA sequence.  Languid genotyping is performed by amplifying the region containing the mutation using PCR, followed by Mse I restriction enzyme digestion.
 
Primers
languid_F: 5’- CTCAGACGCTGGAGGTGTTGGATGTTAG -3’
languid_R: 5’- GCAGCCTGGTGACATTCCAAGACG -3’
 
PCR program
1) 94°C             5:00
2) 94°C             0:30
3) 55°C             0:30
4) 68°C             1:00
5) repeat steps (2-4) 39X
6) 68°C             10:00
7) 4°C                ∞
 
The following sequence of 400 nucleotides (from Genbank genomic region NC_000069 for Tlr2 linear genomic sequence) is amplified:
 
4360                                           c tcagacgctg gaggtgttgg
4381 atgttagtaa caacaatctt gactcatttt ctttgttctt gcctcggctg caagagctct
4441 atatttccag aaataagctg aaaacactcc cagatgcttc gttgttccct gtgttgctgg
4501 tcatgaaaat cagagagaat gcagtaagta ctttctctaa agaccaactt ggttcttttc
4561 ccaaactgga gactctggaa gcaggcgaca accactttgt ttgctcctgc gaactcctat
4621 cctttactat ggagacgcca gctctggctc aaatcctggt tgactggcca gacagctacc
4681 tgtgtgactc tccgcctcgc ctgcacggcc acaggcttca ggatgcccgg ccctccgtct
4741 tggaatgtca ccaggctgc
 
Primer binding sites are underlined; the novel Mse I site is highlighted in gray; the mutated A is indicated in red.
 
Restriction Digest
Digest PCR reactions with Mse I. Run on 2% agarose gel with heterozygous and C57BL/6J controls.
Products: languid allele- 94 bp, 306 bp.  Wild type allele- 400 bp.
References
 22.  Schneider, D. S., Hudson, K. L., Lin, T. Y., and Anderson, K. V. (1991) Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal-ventral polarity in the Drosophila embryo., Genes Dev. 5, 797-807.
Science Writers Eva Marie Y. Moresco
Illustrators Diantha La Vine
AuthorsMichael Berger, Bruce Beutler
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