Dominant negative mechanism underlies autosomal dominant Stargardt-like macular dystrophy linked to mutations in ELOVL4

ELOVL4 (elongation of very long chain fatty acids 4) is a member of the ELO family of proteins involved in the biosynthesis of very long chain fatty acids. Protein truncation mutations in ELOVL4 have been identified in patients with autosomal dominant Stargardt-like macular degeneration. To determine whether a dominant negative mechanism is responsible for the autosomal dominant inheritance pattern of this disease, we studied the subcellular localization and interaction of wild type and mutant ELOVL4 in COS-7 and HEK 293T cultured cells by immunofluorescence and co-immunoprecipitation. Wild type ELOVL4 containing an endoplasmic reticulum retention sequence was localized to the endoplasmic reticulum as expected. In contrast, disease-associated C-terminal truncation ELOVL4 mutants accumulated as large inclusions exhibiting aggresome-like characteristics in a juxtanuclear position within COS-7 cells. When the wild type and mutant proteins were co-expressed incultured cells, wild type ELOVL4 co-purified with mutant ELOVL4 on an immunoaffinity column and co-localized with the mutant protein in aggresome-like inclusions adjacent to the nucleus. These results indicate that wild type and mutant ELOVL4 form a complex that exhibits an abnormal subcellular localization found for individually expressed mutant ELOVL4. From these studies, we conclude that disease-linked C-terminal truncation mutants of ELOVL4 exert a dominant negative effect on wild type ELOVL4, altering its subcellular localization. This dominant negative mechanism contributes to the autosomal dominant inheritance of Stargardt-like macular dystrophy.     

Endoplasmic reticulum microenvironment and conserved histidines govern ELOVL4 fatty acid elongase activity

Autosomal dominant Stargardt-like macular dystrophy (STGD3) in humans results from mutations in elongation of very long chain FAs-like 4 (ELOVL4), which leads to vision loss in young adults. ELOVL4 is an integral endoplasmic reticulum (ER) protein that mediates the elongation of very long chain (VLC) FAs. Mutations in ELOVL4 lead to truncation and mislocalization of the translated protein from the ER, the site of FA elongation. Little is known about the enzymatic elongation of VLC-FAs by ELOVL4. We over-expressed full-length mouse ELOVL4, an N-glycosylation-deficient mutant, an ER-retention mutant, and mutants of active site histidines to parse their individual roles in VLC-FA elongation. ELOVL4 elongated appropriate precursors to the corresponding VLC-FA species ≥28 carbons. Active site histidine mutants of ELOVL4 did not elongate appropriate precursors, establishing ELOVL4 as the elongase. Displacing ELOVL4 from the ER was sufficient to cause loss of condensation activity, while absence of N-glycosylation was irrelevant for enzyme function. This study shows that ELOVL4 enzymatic activity is governed by individual histidines in its active site and the ER microenvironment, both of which are essential for elongation of VLC-FAs.     

Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice

Very long chain fatty acids (VLCFA), either free or as components of glycerolipids and sphingolipids, are present in many organs. Elongation of very long chain fatty acids-4 (ELOVL4) belongs to a family of 6 members of putative fatty acid elongases that are involved in the formation of VLCFA. Mutations in ELOVL4 were found to be responsible for an autosomal dominant form of Stargardt's-like macular dystrophy (STGD3) in human. We have previously disrupted the mouse Elovl4 gene, and found that Elovl4+/- mice were developmentally normal, suggesting that haploinsufficiency of ELOVL4 is not a cause for the juvenile retinal degeneration in STGD3 patients. However, Elovl4-/- mice died within several hours of birth for unknown reason(s). To study functions of ELOVL4 further, we have explored the causes for the postnatal lethality in Elovl4-/- mice. Our data indicated that the mutant mice exhibited reduced thickness of the dermis, delayed differentiation of keratinocytes, and abnormal structure of the stratum corneum. We showed that all Elovl4-/- mice exhibited defective skin water permeability barrier function, leading to the early postnatal death. We further showed that the absence of ELOVL4 results in depletion in the epidermis of ceramides with omega-hydroxy very long chain fatty acids (> or = C28) and accumulation of ceramides with non omega-hydroxy fatty acids of C26, implicating C26 fatty acids as possible substrates of ELOVL4. These data demonstrate that ELOVL4 is required for VLCFA synthesis that is essential for water permeability barrier function of skin.     

The retention factor p11 confers an endoplasmic reticulum-localization signal to the potassium channel TASK-1

The interaction of the adaptor protein p11, also denoted S100A10, with the C-terminus of the two-pore-domain K+ channel TASK-1 was studied using yeast two-hybrid analysis, glutathione S-transferase pull-down, and co-immunoprecipitation. We found that p11 interacts with a 40 amino-acid region in the proximal C-terminus of the channel. In heterologous expression systems, deletion of the p11-interacting domain enhanced surface expression of TASK-1. Attachment of the p11-interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C-terminal end of p11. Imaging of EGFP-tagged TASK-1 channels in COS cells suggested that wild-type TASK-1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK-1 to the surface membrane. Our results suggest that binding of p11 to TASK-1 retards the surface expression of the channel, most likely by virtue of a di-lysine retention signal at the C-terminus of p11. Thus, the cytosolic protein p11 may represent a 'retention factor' that causes localization of the channel to the ER.     

A Stargardt disease-3 mutation in the mouse Elovl4 gene causes retinal deficiency of C32-C36 acyl phosphatidylcholines

Stargardt disease-3 (STGD3) is a juvenile dominant macular degeneration caused by mutations in elongase of very long chain fatty acid-4. All identified mutations produce a truncated protein which lacks a motif for protein retention in endoplasmic reticulum, the site of fatty acid synthesis. In these studies of Stgd3-knockin mice carrying a human pathogenic mutation, we examined two potential pathogenic mechanisms: truncated protein-induced cellular stress and lipid product deficiency. Analysis of mutant retinas detected no cellular stress but demonstrated selective deficiency of C32-C36 acyl phosphatidylcholines. We conclude that this deficit leads to the human STGD3 pathology.     

Three families with autosomal dominant nephrogenic diabetes insipidus caused by aquaporin-2 mutations in the C-terminus

The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetramerize in the apical membrane of the renal tubular cells and contributes to urine concentration. We identified three novel mutations, each in a single allele of exon 4 of the AQP2 gene, in three families showing autosomal dominant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a deletion of 10 nucleotides starting at nucleotide 763 (763-772del), and a deletion of 7 nucleotides starting at nucleotide 812 (812-818del). The wild-type AQP2 is predicted to be a 271-amino acid protein, whereas these mutant genes are predicted to encode proteins that are 330-333 amino acids in length, because of the frameshift mutations. Interestingly, these three mutant AQP2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes injected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was much smaller than that of oocytes with the AQP2 wild-type (14%-17%). Immunoblot analysis of the lysates of the oocytes expressing the mutant AQP2s detected a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surface expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decreased the oocyte Pf in parallel with the surface expression of the wild-type AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQP2 indicated the formation of mixed oligomers composed of wild-type and mutant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant-negative effect is attributed to oligomerization of the wild-type and mutant AQP2s. Segregation of the mutations in the C-terminus of AQP2 with dominant-type NDI underlies the importance of this domain in the intracellular trafficking of AQP2.     

The C-terminal domain of TRPV4 is essential for plasma membrane localization

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.     

The C-terminal domain of TRPV4 is essential for plasma membrane localization

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.     

Characterization of mouse orthologue of ELOVL4: genomic organization and spatial and temporal expression

Mutations in ELOVL4 are associated with dominant macular degeneration (adMD/STGD3). This gene is highly expressed in the retina and is conserved through evolution. Here we report the genomic organization of the mouse orthologue of ELOVL4 and its temporal and spatial expression. A significant amount of ELOVL4 mRNA expression is detected in the adult retina, brain, skin, testis, and lens. During development, expression is first noted at embryonic day 7 (E7). A significant level of the mRNA is observed both in brain and in eyes at postnatal day 1 (P1), after which levels decrease in the brain and increase in the retina until they stabilize at P30. ELOVL4 protein is evident in the ocular tissues by E10.5 and becomes restricted predominantly to the photoreceptor layer in the mature retina. These observations suggest that ELOVL4 may play an important role in embryonic development and in maintaining normal physiology of retina and brain at later stages of development.     

Dominant-negative mutants are clustered in a domain of the human T-cell leukemia virus type I Rex protein: implications for trans dominance.

The 27-kDa Rex trans-acting protein appears to be essential for replication of human T-cell leukemia virus type I. Mutations introduced outside of the Rex RNA-binding domain-nucleolar localization signal display either wild-type activity or, conversely, yield dominant-negative proteins. We generated missense mutations in a particular domain of the Rex protein (amino acid residues 54 to 69) which is characterized by a cluster of dominant-negative mutants. Our results indicate that amino acids 57 to 67 are critically important for Rex function mediated through the RxRE cis-acting RNA sequence. Within this domain, only amino acids 61 to 63 could be mutated without loss of function. All other missense and deletion mutants yielded dominant-negative proteins. In vitro RNA-binding studies performed with glutathione S-transferase-Rex fusion proteins demonstrated that all of the mutant Rex proteins interacted specifically with RxRE RNA. Analysis of chimeric Rex-Rev proteins suggests that this Rex domain is important for oligomerization.     

Mutation of a tyrosine localization signal in the cytosolic tail of yeast Kex2 protease disrupts Golgi retention and results in default transport to the vacuole.

Kex2 protease processes pro-alpha-factor in a late Golgi compartment in Saccharomyces cerevisiae. The first approximately 30 residues of the 115 amino acid CO2H-terminal cytosolic tail (C-tail) of the Kex2 protein (Kex2p) contain a Golgi retention signal that resembles coated-pit localization signals in mammalian cell surface receptors. Mutation of one (Tyr713) of two tyrosine residues in the C-tail or deletion of sequences adjacent to Tyr713 results in loss of normal Golgi localization. Surprisingly, loss of the Golgi retention signal resulted in transport of C-tail mutant Kex2p to the vacuole (yeast lysosome), as judged by kinetics of degradation and by indirect immunofluorescence. Analysis of the loss of Kex2 function in vivo after shutting off expression of wild-type or mutant forms proved that mutations that cause rapid vacuolar turnover do so by increasing the rate of exit of the enzyme from the pro-alpha-factor processing compartment. The most likely explanation for these results is that mutation of the Golgi retention signal in the C-tail results in transport of Kex2p to the vacuole by default. Wild-type Kex2p also was transported to the vacuole at an increased rate when overproduced, although apparently not due to saturation of a Golgi-retention mechanism. Instead, the wild-type and C-tail mutant forms of Kex2p may follow distinct paths to the vacuole.     

The juxtamembrane sequence of cytochrome P-450 2C1 contains an endoplasmic reticulum retention signal

The N-terminal signal anchor of cytochrome P-450 2C1 mediates retention in the endoplasmic reticulum (ER) membrane of several reporter proteins. The same sequence fused to the C terminus of the extracellular domain of the epidermal growth factor receptor permits transport of the chimeric protein to the plasma membrane. In the N-terminal position, the ER retention function of this signal depends on the polarity of the hydrophobic domain and the sequence KQS in the short hydrophilic linker immediately following the transmembrane domain. To determine what properties are required for the ER retention function of the signal anchor in a position other than the N terminus, the effect of mutations in the linker and hydrophobic domains on subcellular localization in COS1 cells of chimeric proteins with the P-450 signal anchor in an internal or C-terminal position was analyzed. For the C-terminal position, the signal anchor was fused to the end of the luminal domain of epidermal growth factor receptor, and green fluorescent protein was additionally fused at the C terminus of the signal anchor for the internal position. In these chimeras, the ER retention function of the signal anchor was rescued by deletion of three leucines at the C-terminal side of its hydrophobic domain; however, deletion of three valines from the N-terminal side did not affect transport to the cell surface. ER retention of the C-terminal deletion mutants was eliminated by substitution of alanines for glutamine and serine in the linker sequence. These data are consistent with a model in which the position of the linker sequence at the membrane surface, which is critical for ER retention, is dependent on the transmembrane domain.     

ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3

We hypothesized that reduction/loss of very long chain PUFAs (VLC-PUFAs) due to mutations in the ELOngase of very long chain fatty acid-4 (ELOVL4) protein contributes to retinal degeneration in autosomal dominant Stargardt-like macular dystrophy (STGD3) and age-related macular degeneration; hence, increasing VLC-PUFA in the retina of these patients could provide some therapeutic benefits. Thus, we tested the efficiency of elongation of C20-C22 PUFA by the ELOVL4 protein to determine which substrates are the best precursors for biosynthesis of VLC-PUFA. The ELOVL4 protein was expressed in pheochromocytoma cells, while green fluorescent protein-expressing and nontransduced cells served as controls. The cells were treated with 20:5n3, 22:6n3, and 20:4n6, either individually or in equal combinations. Both transduced and control cells internalized and elongated the supplemented FAs to C22-C26 precursors. Only ELOVL4-expressing cells synthesized C28-C38 VLC-PUFA from these precursors. In general, 20:5n3 was more efficiently elongated to VLC-PUFA in the ELOVL4-expressing cells, regardless of whether it was in combination with 22:6n3 or with 20:4n6. In each FA treatment group, C34 and C36 VLC-PUFAs were the predominant VLC-PUFAs in the ELOVL4-expressing cells. In summary, 20:5n3, followed by 20:4n6, seems to be the best precursor for boosting the synthesis of VLC-PUFA by ELOVL4 protein.     

Epidermal expression of an Elovl4 transgene rescues neonatal lethality of homozygous Stargardt disease-3 mice

Elongase of very long chain fatty acids-4 (ELOVL4) is the only mammalian enzyme known to synthesize C28-C36 fatty acids. In humans, ELOVL4 mutations cause Stargardt disease-3 (STGD3), a juvenile dominant macular degeneration. Heterozygous Stgd3 mice that carry a pathogenic mutation in the mouse Elovl4 gene demonstrate reduced levels of retinal C28-C36 acyl phosphatidylcholines (PC) and epidermal C28-C36 acylceramides. Homozygous Stgd3 mice die shortly after birth with signs of disrupted skin barrier function. In this study, we report generation of transgenic (Tg) mice with targeted Elovl4 expression driven by an epidermal-specific involucrin promoter. In homozygous Stgd3 mice, this transgene reinstates both epidermal Elovl4 expression and synthesis of two missing epidermal lipid groups: C28-C36 acylceramides and (O-linoleoyl)-omega-hydroxy C28-C36 fatty acids. Transgene expression also restores skin barrier function and rescues the neonatal lethality of homozygous Stgd3 mice. These studies establish the critical requirement for epidermal C28-C36 fatty acid synthesis for animal viability. In addition to the skin, Elovl4 is also expressed in other tissues, including the retina, brain, and testes. Thus, these mice will facilitate future studies to define the roles of C28-C36 fatty acids in the Elovl4-expressing tissues.     

C-terminal mutations destabilize SIL1/BAP and can cause Marinesco-Sjögren syndrome

Marinesco-Sjögren syndrome (MSS) is an autosomal recessive, neurodegenerative, multisystem disorder characterized by severe phenotypes developing in infancy. Recently, mutations in the endoplasmic reticulum (ER)-associated co-chaperone SIL1/BAP were identified to be the major cause of MSS. SIL1 acts as a nucleotide exchange factor for BiP, the ER Hsp70 orthologue, which plays an essential role in the folding and assembly of nascent polypeptide chains in the ER. SIL1 facilitates the release of BiP from unfolded protein substrates, enabling the subsequent folding and transport of the protein. Although most mutations leading to MSS result in deletion of the majority of the protein, three separate mutations have been identified that disrupt only the last five or six amino acids of the protein, which were assumed to encode a divergent ER retention motif. This study presents an in depth analysis of two of these mutants and reveals that the phenotype in the affected individuals is not likely to be due to depletion of SIL1 from the ER via secretion. Instead, our analyses show that the mutant proteins are particularly unstable and either form large aggregates in the ER or are rapidly degraded via the proteasome. In agreement with our findings, homology modeling suggests that the very C-terminal residues of SIL1 play a role in its structural integrity rather than its localization. These new insights might be a first step toward a possible pharmacological treatment of certain types of MSS by specifically stabilizing the mutant SIL1 protein.     

Overlapping signal sequences control nuclear localization and endoplasmic reticulum retention of GRP58

Glucose-regulated GRP58 has shown clinical applications to endoplasmic reticulum (ER) stress and cancer. GRP58 is localized in the cytosol, endoplasmic reticulum (ER) and nucleus. Twenty-four amino acids at the N-terminal hydrophobic region are known to target GRP58 to ER for synthesis at the ER membrane and translocation into the ER lumen. In addition, GRP58 contains putative nuclear localization (494KPKKKKK500) and ER retention (502QEDL505) signals. However, the role of these signals in nuclear import and ER retention of GRP58 remains unknown. Present studies investigated the signals that control nuclear localization and ER retention of GRP58. Deletion/mutation of nuclear localization signal (NLS) abrogated nuclear import of GRP58. NLS attached to EGFP localized EGFP in the nucleus. However, deletion/mutation of putative ER retention signal alone did not alter ER retention of GRP58. Interestingly, a combined deletion/mutation of NLS and ER retention signals blocked the GRP58 retention in the ER. These results concluded that overlapping NLS and ER retention signal sequences regulate nuclear localization and ER retention of GRP58.     

ER retention may play a role in sorting of the nuclear pore membrane protein POM121

Integral membrane proteins of the nuclear envelope (NE) are synthesized on the rough endoplasmic reticulum (ER) and following free diffusion in the continuous ER/NE membrane system are targeted to their proper destinations due to interactions of specific domains with other components of the NE. By studying the intracellular distribution and dynamics of a deletion mutant of an integral membrane protein of the nuclear pores, POM121, which lacks the pore-targeting domain, we investigated if ER retention plays a role in sorting of integral membrane proteins to the nuclear envelope. A nascent membrane protein lacking sorting determinants is believed to diffuse laterally in the continuous ER/NE lipid bilayer and expected to follow vesicular traffic to the plasma membrane. The GFP-tagged deletion mutant, POM121(1-129)-GFP, specifically distributed within the ER membrane, but was completely absent from the Golgi compartment and the plasma membrane. Experiments using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that despite having very high mobility within the whole ER network (D = 0.41 +/- 0.11 micro m(2)/s) POM121(1-129)-GFP was unable to exit the ER. It was also not detected in post-ER compartments of cells incubated at 15 degrees C. Taken together, these experiments show that amino acids 1-129 of POM121 are able to retain GFP in the ER membrane and suggest that this retention occurs by a direct mechanism rather than by a retrieval mechanism. Our data suggest that ER retention might be important for sorting of POM121 to the nuclear pores.     

Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is suggested to localize to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that acts as a membrane anchor for core protein and as a signal sequence for E1 protein. The signal sequence of core protein is further processed by signal peptide peptidase (SPP). We examined the regions of core protein responsible for ER retention and processing by SPP. Analysis of the intracellular localization of deletion mutants of HCV core protein revealed that not only the C-terminal signal-anchor sequence but also an upstream hydrophobic region from amino acid 128 to 151 is required for ER retention of core protein. Precise mutation analyses indicated that replacement of Leu(139), Val(140), and Leu(144) of core protein by Ala inhibited processing by SPP, but cleavage at the core-E1 junction by signal peptidase was maintained. Additionally, the processed E1 protein was translocated into the ER and glycosylated with high-mannose oligosaccharides. Core protein derived from the mutants was translocated into the nucleus in spite of the presence of the unprocessed C-terminal signal-anchor sequence. Although the direct association of core protein with a wild-type SPP was not observed, expression of a loss-of-function SPP mutant inhibited cleavage of the signal sequence by SPP and coimmunoprecipitation with unprocessed core protein. These results indicate that Leu(139), Val(140), and Leu(144) in core protein play crucial roles in the ER retention and SPP cleavage of HCV core protein.     

Evaluation of the ELOVL4 gene in a Chinese family with autosomal dominant STGD3-like macular dystrophy

Stargardt disease-3 (STGD3) is an autosomal dominant juvenile-onset macular dystrophy characterized by progressive decreasing visual acuity, bilateral atrophic changes in the macula and absence of characteristic dark choroids. We identified a STGD3-like macular dystrophy pedigree by clinical examination. To explore whether the STGD3-like phenotype in the kindred is linked to ELOVL4 gene or associated with any other identified STGD gene, we extracted genomic DNA from leukocytes of peripheral blood from the available family members and 50 normal controls for mutation analysis. Then the exons of ELOVL4, RDS and the three exons of ABCR were amplified by polymerase chain reaction (PCR). All PCR products were screened for mutations by combination of denaturing high-performance liquid chromatography (DHPLC) analysis and DNA sequencing. No mutation was found in the exons of three candidate genes, but we obtained three non-pathogenic polymorphisms, IVS5–2533T A in ELOVL4, 558C T (Val106Val) and 1150G C (Glu304Gln) in RDS. And IVS5–2533T A is never shown in the previous references. These data suggested that there exist other unknown genes responsible for the STGD3-like phenotype in the pedigree.     

Functional analysis of the Notch ligand Jagged1 missense mutant proteins underlying Alagille syndrome

Heterozygous mutations in the JAG1 gene, encoding Notch ligand Jagged1, cause Alagille syndrome (ALGS). As most of the mutations are nonsense or frameshift mutations producing inactive truncated proteins, haplo-insufficiency is considered the major pathogenic mechanism of ALGS. However, the molecular mechanisms by which the missense mutations cause ALGS remain unclear. Here we analyzed the functional properties of four ALGS missense mutant proteins, P163L, R184H, G386R and C714Y, using transfected mammalian cells. P163L and R184H showed Notch-binding activities similar to that of the wild-type when assessed by immunoprecipitation. However, their trans-activation and cis-inhibition activities were almost completely impaired. These mutant proteins localized mainly to the endoplasmic reticulum (ER), suggesting that the mutations induced improper protein folding. Furthermore, the mutant proteins bound more strongly to the ER chaperone proteins calnexin and calreticulin than the wild-type did. C714Y also localized to the ER, but possessed significant trans-activation activity and lacked enhanced binding to the chaperones, indicating a less severe phenotype. The properties of G386R were the same as those of the wild-type. Dominant-negative effects were not detected for any mutant protein. These results indicate that accumulation in the ER and binding to the chaperones correlate with the impaired signal-transduction activities of the missense mutant proteins, which may contribute to the pathogenic mechanism of ALGS. Our findings, which suggest the requirement for cell-surface localization of Jagged1 for cis-inhibition activities, also provide important information for understanding the molecular basis of Notch-signaling pathways.