|Coordinate||98,909,263 bp (GRCm38)|
|Base Change||G ⇒ T (forward strand)|
|Gene Name||limb region 1 like|
|Chromosomal Location||98,903,917-98,918,231 bp (-)|
|MGI Phenotype||PHENOTYPE: Mice homozygous for a gene disruption display normal morphology, clinical chemistry, hematology, and behavior. [provided by MGI curators]|
|Limits of the Critical Region||98903921 - 98918231 bp|
|Amino Acid Change||Cysteine changed to Stop codon|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000023736] [ENSMUSP00000104755]|
AA Change: C212*
|Predicted Effect||probably null|
|Predicted Effect||noncoding transcript|
|Meta Mutation Damage Score||0.6256|
|Is this an essential gene?||Non Essential (E-score: 0.000)|
|Candidate Explorer Status||CE: excellent candidate; human score: 1; ML prob: 0.998|
Linkage Analysis Data
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Last Updated||2019-05-14 3:15 PM by Anne Murray|
|Record Created||2016-05-04 11:29 PM by Jin Huk Choi|
The strawberry phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4379, some of which showed reduced frequencies of CD11b+ dendritic cells (DCs) (Figure 1), T cells (Figure 2) CD4+ T cells (Figure 3), CD4+ T cells in CD3+ T cells (Figure 4), naïve CD4 T cells in CD4 T cells (Figure 5), CD8+ T cells (Figure 6), naïve CD8 T cells in CD8 T cells (Figure 7), natural killer (NK) cells (Figure 8), and NK T cells (Figure 9) with concomitant increased frequencies of B1 cells (Figure 10), central memory CD8 T cells in CD8 T cells (Figure 11), effector memory CD4 T cells in CD4 T cells (Figure 12), macrophages (Figure 13), and neutrophils (Figure 14), all in the peripheral blood (1). The B to T cell ratio was increased (Figure 15). CD44 expression was increased on T cells (Figure 16), including both CD4 (Figure 17) and CD8 (Figure 18) T cells. The T-dependent antibody responses to ovalbumin administered with aluminum hydroxide (Figure 19) and to recombinant Semliki Forest virus (rSFV)-encoded β-galactosidase (rSFV-β-gal) were diminished (Figure 20) (1). The T-independent antibody response to 4-hydroxy-3-nitrophenylacetyl-Ficoll (NP-Ficoll) was also diminished (Figure 21). The level of IgE was reduced after administration of OVA/alum (Figure 22). Some mice also showed decreased cytotoxic T lymphocyte (Figure 23) and NK (Figure 24) cell target killing. T cells in the Lmbr1l-deficient mice show exaggerated apoptosis.
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 57 mutations. All of the above anomalies were linked by continuous variable mapping to two mutations on chromosome 15: Lmbr1l and Cers5. The mutation in Lmbr1l was presumed causative, and is a C to A transversion at base pair 98,909,263 (v38) on chromosome 15, or base pair 8,986 in the GenBank genomic region NC_000081 encoding Lmbr1l. The strongest association was found with a recessive model of inheritance to the B to T cell ratio, wherein six variant homozygotes departed phenotypically from 15 homozygous reference mice and 25 heterozygous mice with a P value of 7.155 x 10-17 (Figure 25).
The mutation corresponds to residue 813 in the mRNA sequence NM_029098 within exon 8 of 17 total exons.
The mutated nucleotide is indicated in red. The mutation results in substitution of cysteine 212 to a premature stop codon (C212*) in the LMBR1L protein.
The causative mutation for several of the phenotypes was validated to be in Lmbr1l by CRISPR-mediated knockout of Lmbr1l (Table 1).
LMBR1L (alternatively, lipocalin-interacting membrane receptor; LIMR) is one of four lipocalin receptors. The lipocalin receptors do not share sequence homology with each other or with other receptor families; however, the receptors may share similarities in their topography in the lipid bilayer (2).
LMBR1L has an extracellular N-terminus, nine putative transmembrane domains, a large intracellular loop between the fifth and sixth transmembrane domains, and an intracellular C-terminus (Figure 26) (3). The LMBR1L extracellular N-terminus contains a putative lipocalin (LCN1) binding site and binding sites for other putative ligands (see the Background section for more information about LMBR1L ligands) [(3-5); reviewed in (2)]. LMBR1L is predicted to form dimers and higher order oligomers (2).
The strawberry mutation results in substitution of cysteine 212 to a premature stop codon (C212*); residue 212 is within the fifth transmembrane domain.
LMBR1L is highly expressed in testis, pituitary gland, adrenal gland, trachea, placenta, thymus, cerebellum, stomach, mammary gland, spinal cord, fetal kidney, and fetal lung, and weaker expression in colon, pancreas and prostate (3). LMBR1L is localized to the plasma membrane and on the endoplasmic reticulum membrane.
Lipocalins are secreted proteins (e.g., a-1-microglobulin, a-1-acid glycoprotein, and C8-g) that transport hydrophobic molecules (e.g., steroids, bilins, retinoids, and lipids) to function in several processes, including animal behavior, immunity, iron metabolism, signal transduction, development, and vision [reviewed in (2)]. The lipocalins bind to the lipocalin receptors LMBR1L, megalin, 24p3R, and STRA6. The function of LMBR1L will be discussed in further detail, below. Megalin is an endocytic receptor (6). 24p3R regulates intracellular iron levels and apoptosis upon binding and internalization of its ligand, 24p3 (7). STRA6 is a receptor for retinol binding protein, and mediates uptake of retinol from holo-retinol binding protein (8).
LMBR1L is a receptor for several ligands, including LCN1, bovine lipocalin β-lactoglobulin [BLG] (4), the LCN1 porcine homolog von Ebner's gland protein [pVEG], and the secretoglobin uteroglobin [UG]) (5)) [(3); reviewed in (2)]. Further studies using single cycle kinetics experiments showed that while LMBR1L is a specific receptor for LCN1, it does not show high affinity for pVEG, UG, or BLG (2). These findings indicate that either LMBR1L is not a receptor for pVEG, UG, or BLG, or that these putative ligands bind the receptor at sites distinct from that of LCN1 that are unexposed, inaccessible, or inactive. LCN1 is mainly expressed at epithelial surfaces, and presumably has a scavenging function in which it removes potentially harmful xenobiotics (9). LCN1 comprises 20 to 30 percent of the protein fraction in human tears whereby it reduces surface tension and regulates tear viscosity by binding lipids and interacting with other proteins (10-12). UG putatively suppresses cell motility and invasion upon binding to LMBR1L (5). BLG is the major milk whey protein of most mammals except for primates (13); the biological function of BLG is unknown. Two of the ligands (e.g., LCN1 and BLG) were internalized in the NT2 human teratocarcinoma cell line in a LMBR1L-dependent manner, leading to their degradation (3;4;14). LCN1 internalization was not unique to NT2 cells as LCN1 internalization was also observed in the human breast cancer T-47D cell line (14).
LMBR1L is a putative negative regulator of the Wnt/β-catenin and ERAD signaling pathways independent of its function in lipocalin binding (Figure 27) (1). LMBR1L regulates the expression of mature forms of Wnt co-receptors and phosphorylated GSK-3β as well as the expression of multiple destruction complex proteins. Within the ER, LMBR1L is a component of a destruction complex along with GP78 and UBAC2, which functions to regulate Wnt receptor availability and subsequent Wnt signaling activity (1). LMBR1L also regulates the expression and/or stabilization of the β-catenin destruction complex through its participation in the GP78-UBAC2 complex (1). Co-immunoprecipitation (co-IP) combined with mass spectrometry identified 25 proteins that were more than 50-fold more abundant in the LMBR1L co-IP relative to the control (1). Four of these putative LMBR1L interacting proteins were components of the ERAD pathway (e.g., UBAC2, TERA, UBXD8, and GP78). Several components of the Wnt/β-catenin signaling pathway were also identified in the LMBR1L co-IP, including zinc and ring finger 3 (ZNRF3), low-density lipoprotein receptor-related protein 6 (LRP6), β-catenin, glycogen synthase kinase-3α (GSK3α), and GSK3β (1). Microarray analysis identified additional putative LMBR1L-interacting proteins, including β-catenin and casein kinase 1 (CK1) isoforms CK1α, γ, δ, and ε (1). Co-transfection of HA-tagged LMBR1L with FLAG-tagged Wnt pathway components verified interactions with GSK-3β, β-catenin, ZNRF3, FZD6, LRP6, DVL2, UBAC2, UBXD8, VCP, and GP78.
Mice homozygous for a Lmbr1l gene disruption display normal morphology, clinical chemistry, hematology, and behavior (MGI:3604480).
LMBR1L functions in T and B cell differentiation (Figure 27) (1). Strawberry T cells showed increased T cell apoptosis due to aberrant Wnt/β-catenin signaling activation (1). Loss of LMBR1L expression resulted in upregulation of the expression of mature forms of the Wnt co-receptors and phosphorylated GSK3β with concomitant reduced expression of several destruction complex proteins. As a result, β-catenin accumulated, entered the nucleus, and promoted the transcription of target genes (e.g., Myc, Trp53, and Cd44).
strawberry(F):5'- TGTGTTCTGACCAACTCCAG -3'
strawberry(R):5'- GCAAGACAAGTTGACATGGC -3'
strawberry_seq(F):5'- GTGTTCTGACCAACTCCAGATGATG -3'
strawberry_seq(R):5'- AGACTTCTGGGAGTACTACCTC -3'
Genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the mutation (A>G @ 99751253 bp in the Cers5 gene).
R43790047_PCR_F: 5’- CCATGTGCCGCACCTATTAGTG-3’
R43790047_PCR_R: 5’- AAATAACGGGTTTCTGGTTCCTAG-3’
R43790047_SEQ_F: 5’- AGTGCTACTCTCCATTAGTCCATTAG-3’
R43790047_SEQ_R: 5’- AACGGGTTTCTGGTTCCTAGTCTTG-3’
1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40X
6) 72°C 10:00
7) 4°C hold
The following sequence of 402 nucleotides is amplified (NCBI RefSeq: NC_000081, chromosome 15:99751066-99751467):
ccatgtgccg cacctattag tgctactctc cattagtcca ttagccaagc attcaaacac
atgagtctat gggggccata cctattcaaa cctccacact tacctttctt acatatgctg
tctccccacc tatggagcac ccttctgtgg ccttcatccg gctcacttac cttagtaaca
gatacgaaca ccttctcaag ggtgtcattg ggttccacct tattgacagg actgtcttta
atgccaacac ggagtgcaca gggcttggca ataaatctgt aacaaagaaa atccaaggac
aattctaaat atagtttgct aaatatgacg acggggtcct gattggaata ttaatggagg
gaaaatggaa acccaagact aggaaccaga aacccgttat tt
Primer binding sites are underlined and the sequencing primer is highlighted; the mutated nucleotide is shown in red text (Chr. (+) = A>G; sense strand = T>C).
Genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the mutation (G>T @ 98,909,263 (assembly) in the Lmbr1l gene [see here for the Incidental page]).
R43790046_PCR_F: 5’- TGTGTTCTGACCAACTCCAG-3’
R43790046_PCR_R: 5’- GCAAGACAAGTTGACATGGC-3’
R43790046_SEQ_F: 5’- GTGTTCTGACCAACTCCAGATGATG-3’
R43790046_SEQ_R: 5’- AGACTTCTGGGAGTACTACCTC-3’
1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40X
6) 72°C 10:00
7) 4°C hold
The following sequence of 460 nucleotides is amplified (NCBI RefSeq: NC_000081, chromosome 15):
tgtgttctga ccaactccag atgatgggaa ggggtgctgg ggtgggggtg gggggcaaca
acacagaagc caagcaaggg tgtggccttc aaatgtagga aattggccct gagcaagagg
cagggagcac tacatactac atactacata ctacatacta catactacat acccggggct
tgaccagcaa cttcccagtg acagagaaca tgcgggcgag acctagtgga gtgcacactg
taggcacaag agctggtcag tcacatgcca gacctttcac tccctcccca cacctcctcc
ttccaccctg actgatgccc acgatgccct cttcctctcc cagattctga cactcaccca
gaagcagcag aactccgagg aaggagatac aggagtagag ataggggagg tagtactccc
agaagtctag gacaataaaa gccatgtcaa cttgtcttgc
Primer binding sites are underlined and the sequencing primer is highlighted; the mutated nucleotide is shown in red text (Chr. (+) = G>T; sense strand = C>A)
1. Choi, J. H., Zhong, X., McAlpine, W., Liao, T. C., Zhang, D., Fang, B., Russell, J., Ludwig, S., Nair-Gill, E., Zhang, Z., Wang, K. W., Misawa, T., Zhan, X., Choi, M., Wang, T., Li, X., Tang, M., Sun, Q., Yu, L., Murray, A. R., Moresco, E. M. Y., and Beutler, B. (2019) LMBR1L Regulates Lymphopoiesis through Wnt/beta-Catenin Signaling. Science. 364, 10.1126/science.aau0812.
2. Hesselink, R. W., and Findlay, J. B. (2013) Expression, Characterization and Ligand Specificity of Lipocalin-1 Interacting Membrane Receptor (LIMR). Mol Membr Biol. 30, 327-337.
3. Wojnar, P., Lechner, M., Merschak, P., and Redl, B. (2001) Molecular Cloning of a Novel Lipocalin-1 Interacting Human Cell Membrane Receptor using Phage Display. J Biol Chem. 276, 20206-20212.
4. Fluckinger, M., Merschak, P., Hermann, M., Haertle, T., and Redl, B. (2008) Lipocalin-Interacting-Membrane-Receptor (LIMR) Mediates Cellular Internalization of Beta-Lactoglobulin. Biochim Biophys Acta. 1778, 342-347.
5. Zhang, Z., Kim, S. J., Chowdhury, B., Wang, J., Lee, Y. C., Tsai, P. C., Choi, M., and Mukherjee, A. B. (2006) Interaction of Uteroglobin with Lipocalin-1 Receptor Suppresses Cancer Cell Motility and Invasion. Gene. 369, 66-71.
6. Christensen, E. I., and Birn, H. (2002) Megalin and Cubilin: Multifunctional Endocytic Receptors. Nat Rev Mol Cell Biol. 3, 256-266.
7. Devireddy, L. R., Gazin, C., Zhu, X., and Green, M. R. (2005) A Cell-Surface Receptor for Lipocalin 24p3 Selectively Mediates Apoptosis and Iron Uptake. Cell. 123, 1293-1305.
8. Kawaguchi, R., Yu, J., Honda, J., Hu, J., Whitelegge, J., Ping, P., Wiita, P., Bok, D., and Sun, H. (2007) A Membrane Receptor for Retinol Binding Protein Mediates Cellular Uptake of Vitamin A. Science. 315, 820-825.
9. Gasymov, O. K., Abduragimov, A. R., Prasher, P., Yusifov, T. N., and Glasgow, B. J. (2005) Tear Lipocalin: Evidence for a Scavenging Function to Remove Lipids from the Human Corneal Surface. Invest Ophthalmol Vis Sci. 46, 3589-3596.
10. Fullard, R. J., and Tucker, D. L. (1991) Changes in Human Tear Protein Levels with Progressively Increasing Stimulus. Invest Ophthalmol Vis Sci. 32, 2290-2301.
11. Gouveia, S. M., and Tiffany, J. M. (2005) Human Tear Viscosity: An Interactive Role for Proteins and Lipids. Biochim Biophys Acta. 1753, 155-163.
12. Millar, T. J., Mudgil, P., Butovich, I. A., and Palaniappan, C. K. (2009) Adsorption of Human Tear Lipocalin to Human Meibomian Lipid Films. Invest Ophthalmol Vis Sci. 50, 140-151.
13. Kontopidis, G., Holt, C., and Sawyer, L. (2004) Invited Review: Beta-Lactoglobulin: Binding Properties, Structure, and Function. J Dairy Sci. 87, 785-796.
|Science Writers||Anne Murray|
|Illustrators||Diantha La Vine|
|Authors||Malcolm MacConmara, Evan Nair-Gill, Jin Huk Choi, James Butler, Takuma Misawa, Bruce Beutler|