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. 2010 Sep 24:11:39.
doi: 10.1186/1471-2091-11-39.

Similarity of molecular phenotype between known epilepsy gene LGI1 and disease candidate gene LGI2

Vachiranee Limviphuvadh  1 , Ling Ling Chua, Rabi Atul Adawiyah Bte Rahim, Frank Eisenhaber, Sebastian Maurer-Stroh, Sharmila Adhikari
Affiliations

Similarity of molecular phenotype between known epilepsy gene LGI1 and disease candidate gene LGI2

Vachiranee Limviphuvadh et al. BMC Biochem. .

Abstract

Background: The LGI2 (leucine-rich, glioma inactivated 2) gene, a prime candidate for partial epilepsy with pericentral spikes, belongs to a family encoding secreted, beta-propeller domain proteins with EPTP/EAR epilepsy-associated repeats. In another family member, LGI1 (leucine-rich, glioma inactivated 1) mutations are responsible for autosomal dominant lateral temporal epilepsy (ADLTE). Because a few LGI1 disease mutations described in the literature cause secretion failure, we experimentally analyzed the secretion efficiency and subcellular localization of several LGI1 and LGI2 mutant proteins corresponding to observed non-synonymous single nucleotide polymorphisms (nsSNPs) affecting the signal peptide, the leucine-rich repeats and the EAR propeller.

Results: Mapping of disease-causing mutations in the EAR domain region onto a 3D-structure model shows that many of these mutations co-localize at an evolutionary conserved surface region of the propeller. We find that wild-type LGI2 is secreted to the extracellular medium in glycosylated form similarly to LGI1, whereas several mutant proteins tested in this study are secretion-deficient and accumulate in the endoplasmic reticulum. Interestingly, mutations at structurally homologous positions in the EAR domain have the same effect on secretion in LGI1 and LGI2.

Conclusions: This similarity of experimental mislocalization phenotypes for mutations at homologous positions of LGI2 and the established epilepsy gene LGI1 suggests that both genes share a potentially common molecular pathogenesis mechanism that might be the reason for genotypically distinct but phenotypically related forms of epilepsy.

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Figures

Figure 1
Figure 1
Domain architectures of LGI2 and LGI1. We annotated four SNPs of LGI2, 16 ADLTE missense mutations found in LGI1 (and the artificial LGI1 L26R) and two additional mutations (LGI1 K353E and LGI2 V420E) constructed in this study. Mutations are indicated as lollipops (red for mutations experimentally tested in this study and grey otherwise).
Figure 2
Figure 2
Phylogenetic analysis of the EAR repeats containing proteins and structural model analysis of the EAR domain. A) Phylogenetic tree of the representative EAR repeats containing proteins. The EAR regions of LGI family, C21orf29, GPR98 of representative species were aligned with MAFFT v6.240 using the L-INS-I algorithm and then the Neighbor-Joining method was used to construct the phylogenetic tree (see Method section for details). WDR5 is used as out-group. Phylogenetic analyses were conducted in MEGA4. NCBI accession numbers of all used sequences are shown. Species are abbreviated as follows: Hs ... Homo sapiens, Rn ... Rattus norvegicus, Dr ... Danio rerio and Cf ... Canis familiaris. B) Structural model and conservation mapping of the EAR domain. A) Shows the propeller domains of LGI2 and LGI1 with residues involved in SNPs of LGI2 (red colored) and mutations of ADLTE found in LGI1 (blue colored). B) Shows the conservation within the whole LGI family mapped to the surface of the propeller domain (see Method section for details) in the same orientation as in a), while c) is rotated by 180 degree to show the other side of the domain. Grey color means no conservation, while the other colors signify conservation of physical properties, i.e., yellow: hydrophobic, green: uncharged polar, blue: positive charge, red: negative charge. Color intensity is proportional to strength of conservation see Method section for more details).
Figure 3
Figure 3
Analysis of the effect on the secretion of A) LGI1 mutants B) LGI2 mutants. As detailed in the Methods section, HEK293 cells were transfected with the indicated constructs and cell lysates and concentrated culture media were analysed by western blotting with an anti-GFP antibody 17-18 hours post-transfection. Simplyblue™safe staining is shown to demonstrate that all conditioned media samples were loaded equally and that all cell lysate samples were loaded similar to each other.
Figure 4
Figure 4
Glycosylation of LGI1 and LGI2 mutations. HEK 293 cells were transfected with LGI1WT and LGI2 WT constructs respectively, harvested 17-18 hours post transfection, lysed and subjected to treatment in the absence (-) or presence (+) of PNGFase. The gel shifts indicate glycosylation events.
Figure 5
Figure 5
Subcellular localization of A) LGI1 and B) LGI2 GFP-tagged wild-type and mutant proteins. COS7 cells were transfected with GFP-fused protein (green) as indicated and treated with either an anti-PDI followed by alexa 546 (red) to stain the endoplasmic reticulum or TR Ceramide to detect the Golgi apparatus and DAPI (blue) to stain the nuclei and then examined by laser fluorescence confocal microscopy. The fields shown were visualized independently at the appropriate wavelength for GFP (488 nm) and anti-PDI or TR Ceramide (546 nm), and then the two images were merged. Magnification: 63×. Scale bar is 10 μm.
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