Phenotypic Mutation 'mousebird' (pdf version)
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Allelemousebird
Mutation Type critical splice donor site (2 bp from exon)
Chromosome18
Coordinate65,475,260 bp (GRCm38)
Base Change T ⇒ A (forward strand)
Gene Malt1
Gene Name MALT1 paracaspase
Synonym(s) paracaspase, D430033E09Rik
Chromosomal Location 65,430,962-65,478,823 bp (+)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene has been found to be recurrently rearranged in chromosomal translocation with two other genes - baculoviral IAP repeat-containing protein 3 (also known as apoptosis inhibitor 2) and immunoglobulin heavy chain locus - in mucosa-associated lymphoid tissue lymphomas. The protein encoded by this gene may play a role in NF-kappaB activation. Two alternatively spliced transcript variants encoding different isoforms have been described for this gene. [provided by RefSeq, Jul 2008]
PHENOTYPE: Homozygous inactivation of this gene disrupts normal B cell development and leads to impaired cytokine production and T cell and B cell proliferative responses after antigen receptor engagement due to failure of NF-kappaB activation. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_172833; MGI:2445027

Mapped Yes 
Amino Acid Change
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000048376] [ENSMUSP00000153585]
SMART Domains Protein: ENSMUSP00000048376
Gene: ENSMUSG00000032688

DomainStartEndE-ValueType
low complexity region 19 35 N/A INTRINSIC
low complexity region 38 51 N/A INTRINSIC
PDB:2G7R|B 52 132 3e-29 PDB
IGc2 145 203 8.19e-9 SMART
IGc2 248 306 2.88e-4 SMART
Pfam:Peptidase_C14 340 557 1.4e-19 PFAM
Predicted Effect probably null
Predicted Effect probably null
Phenotypic Category
Phenotypequestion? Literature verified References
post-MCMV FACS B1b cells - increased
post-MCMV FACS B2 cells - decreased
post-MCMV FACS CD11b+ DCs (gated in CD11c+ cells) - increased
T-dependent humoral response defect- decreased antibody response to OVA+ alum immunization
T-dependent humoral response defect- decreased antibody response to rSFV
T-independent B cell response defect- decreased TNP-specific IgM to TNP-Ficoll immunization
Penetrance  
Alleles Listed at MGI

All mutations/alleles(8) : Gene trapped(3) Targeted(5)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00323:Malt1 APN 18 65448963 nonsense probably null
IGL01354:Malt1 APN 18 65475191 missense probably damaging 1.00
IGL01514:Malt1 APN 18 65476400 missense possibly damaging 0.74
IGL01968:Malt1 APN 18 65449016 missense probably benign 0.08
bryce_canyon UTSW 18 65462915 critical splice donor site probably null
yellowstone UTSW 18 65458200 missense probably damaging 1.00
H8930:Malt1 UTSW 18 65462815 nonsense probably null
R0319:Malt1 UTSW 18 65462915 critical splice donor site probably null
R0512:Malt1 UTSW 18 65458200 missense probably damaging 1.00
R0748:Malt1 UTSW 18 65475260 critical splice donor site probably null
R2085:Malt1 UTSW 18 65473147 missense probably damaging 1.00
R2962:Malt1 UTSW 18 65448335 missense probably benign 0.01
R4193:Malt1 UTSW 18 65447675 missense probably benign 0.00
R4359:Malt1 UTSW 18 65476229 missense probably benign 0.00
R4913:Malt1 UTSW 18 65476280 missense probably damaging 1.00
R5201:Malt1 UTSW 18 65476055 missense probably benign
R5925:Malt1 UTSW 18 65431368 missense possibly damaging 0.86
R6944:Malt1 UTSW 18 65437920 missense probably benign 0.08
Mode of Inheritance Autosomal Recessive
Local Stock Live Mice
MMRRC Submission 038188-MU
Last Updated 2018-04-18 3:09 PM by Diantha La Vine
Record Created 2014-08-28 7:36 PM by Kuan-Wen Wang
Record Posted 2015-04-07
Phenotypic Description

Figure 1. Homozygous mousebird mice exhibit diminished T-dependent IgG responses to ovalbumin administered with aluminum hydroxide. IgG levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

Figure 2. Mousebird mice exhibit diminished T-dependent IgG responses to Semliki Forest virus (rSFV)-encoded β-galactosidase (rSFV-β-gal). IgG levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

Figure 3. Mousebird mice exhibit diminished T-independent antibody response to 4-hydroxy-3-nitrophenylacetyl-Ficoll (NP-Ficoll). IgM levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The mousebird phenotype was identified among N-Nitroso-N-ethylurea (ENU)-mutagenized G3 mice of the pedigree R0748, some of which showed a diminished T-dependent antibody response to ovalbumin administered with aluminum hydroxide (Figure 1), a diminished T-dependent antibody response to Semliki Forest virus (rSFV)-encoded β-galactosidase (rSFV-β-gal; Figure 2), and a diminished T-independent antibody response to 4-hydroxy-3-nitrophenylacetyl-Ficoll (NP-Ficoll) (Figure 3).  

Nature of Mutation
 

Figure 4. Linkage mapping of the reduced T-dependent IgG response to rSFV-β-gal. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 30 mutations (X-axis) identified in the G1 male of pedigree R0748.  Normalized phenotype data are shown for single locus linkage analysis without consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 30 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Malt1:  a T to A transversion at base pair 65,475,260 (v38) on chromosome 18, or base pair 44,325 in the GenBank genomic region NC_000084.  The strongest association was found with an additive model of linkage (P = 2.683 x 10-6) to the normalized T-independent antibody response to rSFV-β-gal, wherein 3 variant homozygotes departed phenotypically from 17 homozygous reference mice and 25 heterozygous mice (Figure 4).  A substantial semidominant effect was observed in most of the assays but the mutation is preponderantly recessive, and in no assay was a purely dominant effect observed.  The mutation corresponds to nucleotide two within the donor splice site of intron 15 of Malt1. The effect of the mutation at the cDNA and protein level has not been tested, but the mutation is predicted to result in skipping of the 126-base pair exon 15 (out of 16 total exons). Skipping of exon 15 would cause an in-frame deletion of amino acids 635-676.

 

             <--exon 14       <--exon 15 intron 15-->     exon 16-->

42208 ……ACTGATTTCCCTCTT……CTACAAAAATTAAAG gtcactgcttttcc…… GAACATCTGATC……TCTGAAAACTGA
630   ……-T--D--F--P--L-……-L--Q--K--L--K-                  -E--H--L--I-……-S--E--N--*-
            correct         deleted                                 correct

 

Genomic numbering corresponds to NC_000084. The donor splice site of intron 15, which is destroyed by the mutation, is indicated in blue; the mutated nucleotide is indicated in red.

Protein Prediction

Figure 5. Domain structure of MALT1. The location of the mousebird mutation within intron 15 is indicated. Abbreviations: DD, death domain, Ig, immunoglobulin-like.

Malt1 encodes mucosa-associated lymphoid tissue translocation gene 1 (MALT1; alternatively paracaspase; Figure 5). MALT1 has several domains including a death domain (DD; amino acids 45-132), a paracaspase domain (amino acids 340-535), and three immunoglobulin (Ig)-like domains [amino acids 145-203 (Ig1), 248-306 (Ig2), and 581-715 (Ig3)] (1).

 

Figure 6. Crystal structure of human MALT1 (amino acids 334-719). Figure was generated by Chimera and is based on PDB:3V55 and Weismann et al. 2012.

The paracaspase domain of MALT1 is essential for its catalytic function (2). Cys464 is a predicted active site of MALT1 and mutation of Cys464 to alanine (Cys465Ala) resulted in reduced activation of NF-κB (1;2). The paracaspase domain of MALT1 may directly associate with CARMA1 (alternatively, CARD11; see the record for king) within the CARMA1/BCL10/MALT (CBM) complex (see the Background section for more details on the CBM complex) (3). The enzyme/protease activity of MALT1 is dependent on the formation of MALT1 dimers, which are stabilized by monoubiquitination of MALT1 on Lys644 (2;4;5). Upon substrate binding, the MALT1 dimer undergoes conformation changes that affect the conformation of both the caspase and Ig3 domains (2). The catalytic domain of MALT1 adopts a general caspase fold [Figure 6; PDB:3V55; (2)]. The paracaspase domain has a six-stranded (β2, β1, β3, β4, β7, and β8) β-sheet that is flanked by three helices (αA, αD, αE) on one side and by two helices (αB and αC) on the other side (2). The MALT1 dimer is formed by interactions between β8 and αF from their respective monomers (2). The MALT1 dimer forms a contiguous 12-stranded β-sheet with the active sties positioned on opposite faces of the central β-sheet.

 

The Ig-like domains of MALT1 are protein-protein interaction domains that facilitate the interaction of MALT1 with the Ser/Thr-rich C-terminus of BCL10 (6). BCL10 binds MALT1 in a region that contains the DD and the Ig1 domain (7). Interaction between MALT1 and BCL10 induces MALT1 oligomerization (8;9). The Ig3 domain is required for the stabilization of the MALT1 protein structure (2). The structure of the DD, Ig1, and Ig2 domains have been solved (10). The DD is a five-helix bundle (10). Ig-like domains typically are comprised of two β-sheets, one formed by strands βA, βB, βE, and βD, and the other of strands βC, βF, and βG (2). The Ig1 and Ig2 domains have a classical sandwich-like Ig fold formed by two antiparallel β-sheets. Ig1 and Ig2 naturally form oligomers and are arranged in head-to-tail fashion (10). Helix α1 of the Ig3 domain packs closely against helix αC and strand β2 of the paracaspase domain. An additional strand, βA0, precedes strand βA in the Ig3 domain of MALT1. In contrast to other Ig-like domains that have one of two β-sheets that contain strands βA, βB, βE, and βD (i.e., a “ABED” topology, the Ig3 domain of MALT1 has an additional strand (βA0), lacks the βD strand, and has a very short βE strand subsequently having a “A0ABE” topology (2).

 

MALT1 has candidate self-cleavage site at Arg149. B- and T-cell stimulation induced MALT1 cleavage resulting in the generation of a p19 and a p76 fragment (11). Baens et al. propose that following self-cleavage of MALT1 at R149, the p76 MALT1 fragment activated NF-κB in a TRAF6-dependent, BCL10-independent manner (11). MALT1 cleavage did not affect the proteolytic activity of MALT1 and was not necessary for IKK activation or nuclear translocation of NF-κB in activated T cells; however, it was required for the expression of NF-κB targets including IL-2 and granulocyte-macrophage-colony stimulating factor (GM-CSF) (11).

 

The mousebird mutation within intron 15 is predicted to result in deletion of amino acids 635-676 which constitutes a portion of the Ig3 domain.

Expression/Localization

Malt1 is ubiquitously expressed (12). Malt1 is highly expressed in peripheral blood mononuclear cells; weaker expression was observed in the bone marrow, thymus, lymph node, colon, and lung (13). MALT1 protein expression has been noted in heart, liver, kidney, spleen, and thymus (12).

Background

Figure 7. Canonical and non-canonical NF-kB signaling pathways. In the canonical pathway, several membrane receptors, including TNFR (tumor-necrosis factor receptor), IL-1R (interleukin-1 receptor) and TLRs (toll-like receptors), signal through kinases and adaptors (TRAFs), resulting in IKK activation. This activation occurs after the K63 ubiquitination of TRAFs and RIP. TAK1 and its adaptor proteins TAB1 and TAB2 bind ubiquitin chains to TRAF and NEMO (IKKγ) resulting in the activation of the IKK complex (NEMO, IKKα and IKKβ). Ubiquitination can be inhibited by deubiquitinating enzymes (DUB). Stimulation of the T-cell receptor (TCR) and B-cell receptor (BCR) results in the recruitment of Src and Syk family kinases. These kinases activate a phosphorylation cascade which leads to the activation of protein kinase C (PKC). The phosphorylation of CARMA1 (CARD (caspase-recruitment domain)-MAGUK (membrane-associated guanylate kinase) protein 1) recruits BCL 10 (B-cell lymphoma 10) and MALT1 (mucosa-associated lymphoid tissue lymphoma translocation gene 1), forming the CBM complex and activating the IKK complex. The IKK complex phosphorylates both IκB, p105 and TPL2 (or MAP3K8), resulting in IκB and p105 ubiquitination and degradation (small pink circles) by 26S proteasome. Degradation of IκB releases activated NF-κB dimers for translocation to the nucleus. A subset of TNFRs such as the lymphotoxin-β receptor (LT-bR),CD40, B-cell-activating factor receptor (BAFFR) and receptor activator of Nf-κB (RANK) can activate the canonical or non-canonical NF-κB signaling pathways. In the non-canonical pathway, the receptors bind to TRAFs to regulate NIK activity. TRAF3 and TRAF2 are recruited to the receptor along with cIAP1/2. TRAF2 undergoes K63 self-ubiquitination and is responsible for the K63 ubiquitination of cIAP1/2. TRAF3 is degraded by K48 ubiquitination, enhanced by the K63 ubiquitination of TRAF2 and cIAP1/2. (Gray arrows represent ubiquitination dependence.) As TRAF levels decrease, NIK is released and phosphorylates IKKa which phosphorylates p100. Phosphorylation and ubiquitination of p100 leads to the 26S proteasomal degradation of p100 and the processing of p52. P52 and RelB are released for translocation to the nucleus. This image is interactive. Click on the image to view mutations in the pathway (red) and the genes affected by these mutations (black). Click on the mutations for more specific information.

B-cell receptor (BCR) or T-cell receptor (TCR) engagement results in the formation of a lipid raft-associated multiprotein complex (called the “immunological synapse” in T cells) at the site of cell-cell contact that controls the events leading to activation of NF-κB [Figure 7; (14)]. Upon T cell activation by the TCR and costimulatory molecule engagement, CARMA1 associates with a complex containing Bcl10 and MALT1 and recruits these proteins to lipid rafts of the immunological synapse and to form a scaffold for the assembly of several signaling complexes including those that involve TNF receptor-associated factor 6 (TRAF6), TGF (transforming growth factor)-β-activated kinase 1 (TAK1), and NF-κB essential modulator (NEMO; see the record for panr2) to facilitate NF-κB activation and lymphocyte stimulation. The CBM complex at the immunological synapse activates the inhibitor of kappa B kinase (IKK) complex. MALT1 is proposed to mediate the activation of the IKK complex by binding to TRAF6-containing ubiquitin ligase complexes to facilitate the addition of K63-linked ubiquitin chains to several proteins and the subsequent proteasome-mediated degradation of BCL10, MALT1, TRAF6, and NEMO (15). Subsequently, the IKK complex is recruited and TRAF6-dependent activation of the Ser/Thr kinase TAK1 promotes the activation of IKKβ by its phosphorylation (16). IKKβ is required for the recruitment of Bcl10/MALT1 to CARMA1 after TCR engagement. IKKβ then catalyzes the phosphorylation of Bcl10 and disengagement of Bcl10 from MALT1, leading to reduced Bcl10-induced IKKγ ubiquitination (17). In effector T cells, the active IKK complex is a component of the TCR-dependent Bcl10-MALT1 signalosome containing the adaptor protein p62 (18). IKK activation occurs after signalosome assembly. Activation of the IKK complex leads to degradation of inhibitor of kappa-Bα (IκBα) and subsequent activation and translocation of NF-κB dimers comprised of p65 (alternatively, RelA) and p50 (see the record for Finlay) to the nucleus (3;19-21). The CBM complex functions similarly in B cells to activate NF-κB in response to BCR engagement (22). In addition to the CBM complex, protein kinase C (PKC) is a critical component of the immunological synapse required for the activation of NF-κB. The PKC isoform PKCβ (see the record for Untied) operates specifically in BCR-dependent NF-κB signaling (23), while PKCθ functions specifically in TCR-dependent NF-κB signaling (24;25). Upon lymphocyte activation, the linker domain of CARMA1 is phosphorylated at several sites by PKCβ or PKCθ. These phosphorylation events are required for release of the inhibitory intramolecular CARD domain-linker domain interactions within CARMA1, allowing CARMA1 to assemble the CBM complex required to activate NF-κB (22;26). CARMA1, BCL10, and MALT1 are all essential for adaptive immunity and for NF-κB activation after B- and T-cell receptor stimulation (27-30).  Deficiency in expression of any of the three proteins leads to defective NF-κB activation and subsequent defects in adaptive immunity (28;31).

 

In addition to T and B cells, MALT1 functions in mast cells, dendritic cells, macrophages and natural killer (NK) cells (32-34). In marginal zone B cells, MALT1 mediates BAFF (see the record for Frozen)-dependent anti-apoptotic gene induction through the noncanonical NF-κB signaling pathway (see the record for xander) (35). In mast cells, MALT1 mediates IgE FcεRI receptor-mediated secretion of TNFα (see the record for Panr1) and IL-6 (33). Malt1-/- mice have normal skin mast cells and exhibit regular IgE-mediated immediate phase anaphylactic reactions; however, late phase anaphylactic reactions are reduced (33). Bone marrow-derived mast cells from Malt1-/- mice did not produce TNF-α or IL-6 in response to FcεRI stimulation due to loss of NF-κB activation, but the production of leukotrienes and degranulation was normal (33). In dendritic cells, MALT1 is necessary for the differentiation of TH17 subset of helper T cells. In macrophages, MALT1 mediates NF-κB activation downstream of toll-like receptor 4 (TLR4; see the record for lps3) and the C-type lectin receptors Dectin-1 and -2 (36-38). In dendritic cells and macrophages, CARD9 can stimulate NF-κB activation during innate immune responses as well as through the C-type lectin receptor pathway. In NK cells, MALT1 functions downstream of several immune receptor tyrosine-based activation motif (ITAM)-coupled lectin-like receptors including NK1.1 (see the record for Unnatural), Ly49D, Ly49H, and NKG2D to activate the canonical NF-κB pathway as well as JNK and p38 MAP kinases that induce NK-cell cytotoxicity and cytokine release (34). NK cells from Malt1-deficient (Malt1-/-) mice exhibit impaired TNF-α, IFN-γ, GM-CSF, and MIP-1β production upon NK cell activation using phorbol myristate acetate (PMA) and the calcium ionophore ionomycin (Iono) (34).

 

BCL10 and MALT1 can interact with other CARD family members including CARMA3 (alternatively, CARD10) and CARD9 to induce NF-κB activation. CARMA3 stimulates NF-κB activation through G protein-coupled receptors (GPCRs) and receptor tyrosine kinases that recognize lysophosphatidic acid (LPA), the proinflammatory growth factor angiotensin II (Ang II), CXCL8/IL-8, CXCL12/SDF-1α, and thrombin (12;39-43). For example, in vascular smooth muscle cells and endothelial cells, MALT1 regulates CXCL8-dependent upregulation of VEGF as well as thrombin-dependent adhesion of monocytes indicating a MALT1 function in neovascularization, endothelial dysfunction, and atherosclerosis (39;41;42). MALT1 has a role in the pathophysiology of some carcinomas and inflammatory diseases through the CARMA3/Bcl10/MALT1 pathway. For example, in ovarian cancer pathogenesis, MALT1 promotes LPA-mediated urokinase plasminogen activator (uPA) upregulation (44). In oral squamous cell carcinoma, MALT1 mediates CXCL12/SDF-1α-induced cellular invasion (43).

 

In addition to its function within the CBM complex, MALT1 is a paracaspase that cleaves substrates after arginine residues in the P1 position (i.e., N-terminal to the cleavage site) [reviewed in (45)]. The known substrates of MALT1 include BCL10, A20, cylindromatosis (CYLD), NF-κB-inducing kinase (NIK), RelB, and Regnase-1 [Table 1; (46-51)].  Cleavage of the MALT1 substrates serves to inactivate negative regulators of both canonical and non-canonical NF-κB signaling as well as JNK signaling.

 

Table 1. Known substrates of MALT1 [reviewed in (45)].

Substrate

Summary of function

Effect of MALT1-induced cleavage

References

BCL10

Adaptor for the CBM signaling complex

Regulates MALT lymphoma cell adhesion to fibronectin; regulates integrin-mediated T cell adhesion

(46)

A20

A deubiquitinating enzyme that removes K63-linked chains from NF-κB activators (e.g., TRAF6, NEMO, and MALT1); see the record for lasvegas

Inactivates A20 and results in amplification and persistent NF-κB signaling

((47)

CYLD

A deubiquitinating enzyme; persistent NF-κB signaling

Positively regulates JNK signaling; results in persistent NF-κB signaling

(48)

NIK

Phosphorylates IKKα (inhibitory κB kinase) to regulate non-canonical NF-κB signaling (see the record for xander for information on the non-canonical NF-κB signaling pathway)

Persistent noncanonical NF-κB signaling; contributes to MALT lymphomagenesis

(52)

RelB

NF-κB subunit in the non-canonical NF-κB signaling pathway

Enforces canonical NF-κB signaling

(49)

Regnase-1 (alternatively, MCPIP-1)

RNA-binding protein that destabilizes mRNAs of T cell effector genes

Alleviates Regnase-1-mediated suppression that regulates mRNA stability of effector genes in T cells

(50)

Roquin-1

Post-transcriptional gene regulation

Alleviates roquin-1-mediated suppression that regulates mRNA stability of genes (e.g., IL-6, ICOS, c-Rel, IRF4, IκBNS and IκBζ) that regulate TH17 cell differentiation

(53)

MALT1

See text for description of function

Necessary for antigen receptor-induced TRAF6-dependent NF-κB-dependent gene (e.g., IL6, c-Rel, Ox40, and Il2) transcription

(11)

 

Malt1-/- mice exhibit defective antigen receptor-induced lymphocyte activation (27;28). In addition, Malt1-/- mice exhibit defects in B1 and marginal zone B cell development as well as TCR-mediated cellular proliferation, activation, and cytokine production (27;28). The role of MALT1 in B2 cell development is unclear (27;28). The numbers and differentiation of B cell precursors in the bone marrow of the Malt1-/- mice was normal (27). In addition, the expression of surface IgM and IgD on mature splenic B cells, maturation of CD3IL-2Rβ+ NK cells and CD3+IL-2Rβ+ NKT cells, the number of thymocytes and maturation of CD4+CD8+ double-positive thymocytes into CD4+ and CD8+ single-positive T cells, and the number and distribution of single positive T cells in the spleen and lymph nodes were normal in the Malt1-/- mice (27). However, there was a reduction in the proportion of DN3 cells (CD25+CD44lo) and an increase in the DN4 cells (CD25CD44lo) in the Malt1-/- mice due to premature maturation of the double negative thymocytes (28). The frequency of peripheral activated T cells was reduced in the Malt1-/- mice. All of the Ig isotypes were reduced in the Malt1-/- mice compared to those in wild-type mice (27;28). Stimulation of T cells from Malt1-/- mice with anti-CD3ϵ antibodies (with or without anti-CD28 costimulation) or with PMA did not result in T cell expansion or IL-2 synthesis (27;28;54). Stimulation of T cells from Malt1-/- mice with PMA led to impaired JNK activation compared to wild-type mice (27). The role of MALT1 in LPS-stimulated B cell proliferation is unclear; studies have reported that MALT1 was or was not necessary for LPS-induced NF-κB signaling (27;28). However, LPS-induced NF-κB activation in macrophages requires MALT1 expression (36). In B cells, MALT1 selectively regulates activation of the NF-κB subunit c-REL to control B-cell survival and Malt1-/- B cells die more rapidly than wild-type cells (55). While wild-type B cells induce the expression of antiapoptotic factor Bcl-xL in response to BCR ligation, Malt1-/- B cells do not (55).

 

Several human pathologies are linked to MALT1. B-cell lymphomas affecting mucosa-associated lymphoid tissue (i.e., MALT lymphomas) are often caused by the t(11;18)(q21;q21) chromosomal translocation, which results in a gain-of-function fusion protein, inhibitor of apoptosis 2 (cIAP2)—MALT1 (13;56-59). The fusion protein consists of the C-terminus of MALT1 linked to the N-terminus of cIAP2 and causes constitutive activation of the canonical NF-κB pathway, promoting cell growth and survival (8;9;57). The cIAP2—MALT1 fusion protein also induces the proteasome-dependent processing of p100 to p52 in the the noncanonical NF-κB pathway (52). The t(14;18)(q32;q21) chromosomal translocation also affects the MALT1 gene, putting it under the control of the immunoglobulin heavy locus (IgH) enhancer and subsequently causing dysregulation of MALT1 expression (58). Mutations in MALT1 are linked to immunodeficiency-12 [IMD12; alternatively, combined immunodeficiency (CID); OMIM: #615468; (60;61)). IMD12 is characterized by early-onset recurring bacterial and candida infections leading to bronchiectasis and growth delay as well as inflammatory gastrointestinal disease, periodontal disease, and dermatitis (60;61). Patients with IMD12 have normal levels of circulating T and B cells as well as serum immunoglobulins, antibody titers were low after immunization, T cells showed impaired proliferative responses to antigens , increased numbers of naïve B cells, loss of marginal zone B cells, and reduced switched memory B cells (60;61). Patients with IMD12 die between ages 7 and 13.5 years (61). Dysregulation of MALT1 expression has also been observed in B-cell non-Hodgkin lymphoma (62).

Putative Mechanism

Malt1-/- mice exhibited reduced T-dependent antibody responses to keyhole limpet haemocyanin (KLH) in complete Freund's adjuvant (CFA) and 2,4-dinitrophenol–conjugated ovalbumin (DNP-OVA) as well as reduced T-independent antibody responses to tri-nitrophenol-(TNP)-Ficoll (27;28;35). The mousebird mice also exhibit defects in both T-independent and T-dependent antibody responses indicating that the MALT1mousebird protein exhibits loss-of-function.

Primers PCR Primer
mousebird(F):5'- TGAATCTTCCTGCAACTTCCCCAAATG -3'
mousebird(R):5'- GGCCTAACTTCTCAACAGCAGAGC -3'

Sequencing Primer
mousebird_seq(F):5'- GGGATTACCTATTCAAGCCAGTACTC -3'
mousebird_seq(R):5'- TTCTCAACAGCAGAGCAGACAG -3'
Genotyping

Mousebird genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transversion.
 

PCR Primers

Mousebird(F): 5’- TGAATCTTCCTGCAACTTCCCCAAATG-3’

Mousebird(R): 5’- GGCCTAACTTCTCAACAGCAGAGC-3’

 

Sequencing Primer

Mousebird_seq(F): 5’- GGGATTACCTATTCAAGCCAGTACTC-3’
 

Mousebird_seq(R): 5’- TTCTCAACAGCAGAGCAGACAG-3’
 

 

PCR program

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               ∞

 

The following sequence of 689 nucleotides is amplified (Chr.18: 65474770-65475458, GRCm38; NC_000083):

43835                                      tgaatc ttcctgcaac ttccccaaat    

43861 gataataaag acagagactc caaaactgaa aactggaaca ggttacaagt gattcaaaga    

43921 atagctgtaa aggcaactat ccttttagaa ataacttgaa gtgtttttaa tgtcgctact    

43981 ttaaaaatgt gtagaatcta gaatacctat ggcaatttat ggccctagaa aatgattttc    

44041 ctgtttcttt agtcctctct aagggattac ctattcaagc cagtactcat gagagaacag    

44101 tatctgtgaa gatattttat gttatacaca atgatataac ttccattgat tgcatcatag    

44161 acatttactg tcttgggttt tgttttgttt tttataggac ctagatattg atccaaaaca    

44221 tgcaaacaag gggactcctg aagaaactgg cagctactta gtatcaaaag accttcccaa    

44281 gcactgcctc tacactagac tcagctcact acaaaaatta aaggtcactg cttttcctgt    

44341 ttccagcagc taacaaaaaa gctgtggggc taagaaggtt gattgtggca tcgtccaggg    

44401 atgagttgct tcttttggga actaacctgg agcagcagta actacagtgg tctttgacat    

44461 taaattctgc ctctgttact ggcctaggga cgtctgtctg ctctgctgtt gagaagttag

44521 gcc

 

FASTA sequence

 

Primer binding sites are underlined and the sequencing primer is highlighted; the mutated T is shown in red text.

References
Science Writers Anne Murray
Illustrators Peter Jurek
AuthorsKuan-Wen Wang, Jin Huk Choi, Apiruck Watthanasurorot, Bruce Beutler
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