Phenotypic Mutation 'Tlr13rev1' (pdf version)
AlleleTlr13rev1
Mutation Type missense
ChromosomeX
Coordinate105,200,983 bp (GRCm39)
Base Change A ⇒ G (forward strand)
Gene Tlr13
Gene Name toll-like receptor 13
Synonym(s) LOC279572
Chromosomal Location 105,186,881-105,204,099 bp (+) (GRCm39)
MGI Phenotype PHENOTYPE: Macrophages isolated from mice carrying an ENU-induced mutation respond normally to known TLR ligands and can contain various viral infections; however, mice carrying this allele have not been tested for immune responses in vivo. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_205820; MGI:3045213

MappedYes 
Amino Acid Change Aspartic acid changed to Glycine
Institutional SourceBeutler Lab
Gene Model not available
AlphaFold Q6R5N8
SMART Domains Protein: ENSMUSP00000043101
Gene: ENSMUSG00000033777
AA Change: D240G

DomainStartEndE-ValueType
LRR 106 125 5.12e1 SMART
LRR_TYP 126 149 8.81e-2 SMART
LRR 150 172 1.45e1 SMART
LRR_TYP 173 196 5.67e-5 SMART
LRR 197 219 1.49e1 SMART
LRR 223 245 3.76e1 SMART
LRR 246 269 1.33e1 SMART
LRR 395 418 4.7e0 SMART
LRR_TYP 419 442 8.47e-4 SMART
LRR 443 466 1.16e-1 SMART
LRR 467 490 6.06e1 SMART
LRR_TYP 491 514 2.09e-3 SMART
LRR_TYP 515 538 3.39e-3 SMART
LRR_TYP 539 562 6.23e-2 SMART
LRR 563 586 1.97e2 SMART
LRR 592 617 3.36e2 SMART
LRR_TYP 670 693 3.58e-2 SMART
LRR 694 717 8.26e1 SMART
TIR 833 978 1.8e-29 SMART
Predicted Effect probably benign

PolyPhen 2 Score 0.000 (Sensitivity: 1.00; Specificity: 0.00)
(Using ENSMUST00000040065)
Meta Mutation Damage Score 0.0898 question?
Is this an essential gene? Not available question?
Phenotypic Category Unknown
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance unknown 
Alleles Listed at MGI

All alleles(3) : Targeted(2) Chemically induced(1)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL02044:Tlr13 APN X 105200703 missense probably damaging 1.00
IGL02644:Tlr13 APN X 105200503 missense probably benign 0.12
IGL02809:Tlr13 APN X 105200297 splice site probably benign
X0024:Tlr13 UTSW X 105200334 missense probably benign
X0028:Tlr13 UTSW X 105200928 missense probably benign 0.04
X0067:Tlr13 UTSW X 105200839 missense probably benign 0.00
Mode of Inheritance Unknown
Local Stock Sperm, gDNA
Repository

none

Last Updated 2016-05-13 3:09 PM by Anne Murray
Record Created unknown
Record Posted 2014-02-17
Phenotypic Description

The Tlr13rev1 allele was discovered in a sequencing screen of G1 mice born to N-ethyl-N-nitrosourea (ENU)-mutagenized sires. DNA collected from animals was sequenced using primers directed against the Tlr13 gene. 

Peritoneal macrophages from Tlr13rev1 hemizygous males were tested in several ex vivo assays. Tumor necrosis factor (TNF)-α production by these macrophages was normal in response to Toll-like receptor (TLR) stimulation with the TLR2/6 ligands MALP2 (macrophage-activating lipopeptide 2) and peptidoglycan (PGN), the TLR9 ligand CpG DNA, the TLR4 ligand lipopolysaccharide (LPS), the TLR1/2 ligand Pam3CSK4 (a triacyl lipopeptide), the TLR7 ligand resiquimod (a ssRNA mimetic), and the TLR3 ligand poly I:C (a dsRNA mimetic) (TLR Signaling Screen). Tlr13rev1 macrophages produced normal amounts of double-stranded DNA-induced type I interferon (IFN) (Double-stranded DNA Macrophage Screen), LPS/nigericin-induced interleukin (IL)-1β (NALP3 Inflammasome Screen), and were resistant to infection with mouse cytomegalovirus (MCMV), influenza virus, Rift Valley Fever virus (RVFV), and an adenoviral vector (Ex Vivo Macrophage Screen for Control of Viral Infection).

Nature of Mutation
The Tlr13rev1 mutation is an A to G transition identified at position 898 of the Tlr13 transcript NM_205820 within exon 3 out of 3 total exons.
 
882 AGCATCATGTACTTGGACCACAGCCCTAGGTCA
235 -S--I--M--Y--L--D--H--S--P--R--S-
 
The mutated nucleotide is indicated in red lettering, and results in a conversion of aspartic acid to glycine at residue 240 of the TLR13 protein.
Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 1. Protein and domain structure of TLR13. A) Schematic representation of TLR13 based on crystalized structures of mouse TLR3 LRR (PBD 3CIG) and human TLR2 TIR (1FYW) domains. The residue affected by the Tlr13rev1 mutation is shown. 3D image was created using UCSF Chimera. B) TLR13 is a 991 amino acid protein with an extracellur domain (pink) of leucine rich repeats (LRR), a short transmembrane domain and an cytoplasmic Toll/Interleukin-1 receptor (TIR) domain. The CpG11 mutation (red asterisk) results in an aspartic acid to glycine substitution at amino acid 240 of the TLR13 protein in the predicted sixth leucine rich repeat (LRR).

TLR13 is a type I integral membrane glycoprotein containing 1348 amino acids and belongs to the TLR11/12 subfamily based on phylogenetic analysis. This subfamily is represented in humans by only one pseudogene (1). Like the other TLRs, its C-terminal 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 (2-4), which mediates homo- and heterotypic protein interactions during signal transduction. TIR domains in TLRs and in IL receptors contain 3 conserved boxes (boxes 1, 2 and 3), which are required for signaling (5). 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 (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 a central five-stranded parallel β-sheet surrounded by five helices (6) (see the record for languid for a picture of the TLR2 TIR domain). 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 (see the record for lps3), which has been reported to abolish MyD88 binding to a constitutively active TLR4 mutant (7) and LPS-induced signaling in mice (8).

The extracellular domains of TLRs, unlike those of the interleukin receptors, contain multiple leucine-rich repeats (LRRs) (Figure 1), 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 Ø is a hydrophobic amino acid [discussed in (9)]. TLR13 has 27 such LRRs in its ectodomain encoded by the N-terminal half of the protein (10;11). Crystal structures of TLR1, TLR2 (12) and other LRR-containing proteins revealed that the XLXXLXLXXN sequence folds into a β-strand. 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).

The Tlr13rev1 mutation results in substitution of a glycine for a phenylalanine at residue 240 of TLR13 located in the sixth LRR of the TLR13 extracellular region.

Expression/Localization

According to SymAtlas, mouse Tlr13 mRNA is most highly expressed in cells of the myeloid lineage such as macrophages, myeloid dendritic cells, osteoclasts and microglia. Interestingly, macrophage expression is inhibited by TLR4 stimulation. This observation is supported by Shi et al. who found that NF-κB activation, including activation by both TLR2 and TLR 4 ligands, inhibited Tlr13 transcription in a macrophage cell line (13).

TLR13 protein is strongly expressed in the spleen, in which it is abundant in macrophages and conventional dendritic cells (cDCs) but not plasmacytoid dendritic cells (pDCs) (14).  It is also expressed at low levels in mouse Sertoli cells in the testes (15), and in the brain in neurons, ependymal cells, endothelial cells and astrocytes (16).

TLR13 is localized intracellularly and colocalizes with Unc93b1 when transiently transfected into fibroblast cell lines (14).

Background

A sequence within gram-negative and gram-positive bacterial 23S ribosomal RNA was identified as a TLR13 ligand (17;18).  The minimal activating ribonucleotide sequence was CGGAAAGACC, representing a portion of S. aureus 23S rRNA surrounding adenosine 2085 (bold).  The ligand is recognized in the endosome, as bafilomycin treatment or the 3d mutation of Unc93b1 abrogated activation of Tlr23479-/- macrophages, as measured by TNF or IL-6 production after challenge with heat inactivated S. aureus in vitro.  Single stranded (ss)RNA-specific RNase A treatment prevented heat inactivated S. aureus from inducing TNF production by Tlr23479-/- macrophages, indicating that the ligand is single stranded.   Interestingly, the stimulatory rRNA segment binds to MLS (macrolide, lincosamide, streptogramin) group antibiotics, and adenosine 2085 is N6 methylated in antibiotic-resistant S. aureus strains grown in erythromycin.  The methylation of adenosine 2085 modulates the binding of antibiotic to the rRNA.  Antibiotic-resistant strains of S. aureus grown in the presence of erythromycin, as well as S. aureus strains with 23S rRNA mutations that confer resistance to MLS antibiotics, failed to activate Tlr23479-/- macrophages.  Thus, the acquisition of MLS antibiotic resistance by S. aureus was also accompanied by the ability to evade host innate immune detection through TLR13.

To demonstrate that TLR13 was the relevant receptor, siRNA was used to knockdown TLR13 expression in Tlr23479-/- macrophages, and the stimulatory oligoribonucleotide (ORN) GGUUACCCGCGACAGGACGGAAAGACCCCGUGGAGCUUUACUGUAGCC was applied (17).  Whereas macrophages treated with a scrambled control siRNA produced IL-6 in response to the ORN, those treated with TLR13 siRNA produced a greatly reduced amount.  In addition, ectopic expression in HEK293 cells of TLR13 but not CD14, TLR3, TLR7, TLR8, TLR9, or TLR12 conferred NF-κB responsiveness to cells treated with heat inactivated S. aureus or stimulatory ORNs.  No role for TLR8 in ligand recognition was found.  Tlr2-/-Tlr4-/-Unc93b13d/3d mice, which lack TLR13 function, were resistant to i.v. injection of a TLR13 stimulatory ORN together with IFN-γ, and D-galactosamine, while Tlr23479-/- mice died within 16 hours of treatment.   

Constitutively active TLR13 (the TLR13 intracellular domain fused to the CD4 extracellular domain) has been reported to activate NF-κB and interferon (IFN)-β in a MyD88- and TAK1-dependent, TRIF-independent manner (14).  In agreement, MyD88 and MAPK1 were required for TLR13-dependent responses to heat inactivated S. aureus (17).  TLR13 activated NF-κB in response to bacterial RNA or Streptococcus pyogenes in Chinese Hamster Ovary (CHO) cells (19). In dendritic cells, siRNA-mediated reduction in Tlr13 expression resulted in less IL-12p40 induction in response to bacterial DNA than control siRNA-transfected cells. In addition Il6 (interleukin-6) and Il12 (interleukin-12) mRNA levels and secretion were reduced in TLR13 siRNA-transfected cells. The complete signaling pathway and signaling compartments utilized by TLR13 remain to be elucidated.

TLR13 was upregulated in response to various stimuli including parasite infection in the brain (16), fungal infection in the eye (20), and infection with paramyxovirus followed by secondary Streptococcus pneumonia infection in the lung (21).  In order to determine the regulatory mechanisms underlying Tlr13 expression, the Tlr13 promoter was analyzed and was found to bind to the Ets2, Sp1, and PU.1 transcription factors. These transcription factors, as well as IFN-β, were all able to upregulate the expression of a Tlr13 reporter in vitro.  Sp1 is a ubiquitous factor that regulates the constitutive expression of many genes, but both Ets2 and PU.1 have been shown to play important roles in the development and function of various immune cells (13).

Putative Mechanism

Macrophages from Tlr13rev1 mice responded normally to TLR2/6, TLR9, TLR4, TLR1/2, TLR7, and TLR3 ligands and contained various viral infections. Macrophages from Tlr13rev1 mice have not been tested for responsiveness to bacterial 23S rRNA.

Primers Primers cannot be located by automatic search.
Genotyping
Tlr13rev1 genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transition. 
 
Primers
Tlr13rev1 (F): 5’- CAGAGAATACTCTGGAAGTGGGTTTTGAAAGCTGG -3’
Tlr13rev1 (R): 5’- TGGGTAGGTTTCCGAGCAGGTGACAAACAG -3’
 
PCR program
1) 95°C             2:00
2) 95°C             0:30
3) 56°C             0:30
4) 72°C             1:00
5) repeat steps (2-4) 29X
6) 72°C             7:00
7) 4°C               ∞
 
Primers for sequencing
Tlr13rev1_seq(F): 5'- TGGAACTCAATTTGGAAGATCGAC -3'
Tlr13rev1_seq(R): 5’- TAAGTGACCATGTCTAAGCTCC -3’
 
The following sequence of 1333 nucleotides (NCBI Mouse Genome Build 37.1, Chromosome X, bases 103,351,707 to 103,353,039) is amplified: 
 
                              cagagaa tactctggaa gtgggttttg aaagctggtg ttggactgag
catagtggtt catgccttta atcccagcac tcaggaagca aacagatctc tctgtgtttg
agggcagcct gctctatgga gtgagttcca tgatagtcag ggttacacag agaaaccctg
caggaggtgg aggtggggac aaaaccaaac caagctaaaa caaaacaaaa aacaacccag
gttgatgttg agattgaact tttttgtttc cctgaagcag ctataaaaca cttattggga
tagggaaatt ttaactaaaa acctttgttt ttctttgcag caaagtccat atgtgaagac
cgtgcctttg aacatgagaa gggatttttt tttcctggtt gtaacctgga tgcctaagac
agtcaagatg aatgggagca gctttgtgcc atctctacag ctcctgctca tgttagtagg
attttctctg ccgcctgtgg cagagacata tgggttcaac aagtgcacac agtatgaatt
tgatattcac catgtgctct gcattaggaa gaagatcacc aacttgacag aggccattag
tgacatacct agatatacta ctcaccttaa cctcacacac aacgaaattc aagtcctccc
tccctggagt tttaccaatc tgtctgctct ggtggacttg agactagagt ggaactcaat
ttggaagatc gacgaaggtg cctttagggg acttgaaaat ttgactctgc tgaatttagt
ggaaaataag attcaaagtg tgaataactc ctttgagggc ctgtccagcc tgaagaccct
gctcctgagc cataatcaga ttacccatat tcacaaagac gccttcactc ctctaatcaa
attgaaatat ttgagcctat ctcgaaacaa cattagcgat ttttctggta ttcttgaagc
agtccagcat cttccatgcc tggagcgcct tgatctaact aacaacagca tcatgtactt
ggaccacagc cctaggtcac tggtttctct gacccacctg agttttgagg ggaacaaact
aagggagtta aacttctctg ctttgtcatt acctaactta accaatctaa gtgcttcccg
gaatggcaat aaagtcattc agaatgtgta tcttaaaact ctgccccaac ttaaaagctt
gaatctgagt ggaacggtga taaaattgga aaatctttcg gccaaacacc tgcagaatct
aagagctatg gatctcagta attgggagct tagacatggt cacttagata tgaaaactgt
ttgtcacctg ctcggaaacc taccca
 
Primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is indicated in red.
References
Science Writers Nora G. Smart
Illustrators Diantha La Vine
AuthorsMicha Berger, Owen Siggs, Yu Xia, Kevin Khovananth & Bruce Beutler
Edit History
2011-08-23 9:49 AM (current)
2011-08-23 9:49 AM
2011-08-10 10:47 AM
2011-01-07 9:55 AM
2010-02-25 3:13 PM