Human Molecular Genetics, 2001, Vol. 10, No. 4 405-413
© 2001 Oxford University Press
Defective intracellular transport and processing of JAG1 missense mutations in Alagille syndrome
Division of Human Genetics and Molecular Biology, Children’s Hospital of Philadelphia, 1006 Abramson Research Center, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104, USA
Received 6 November 2000; Revised and Accepted 12 December 2000.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Jagged1 (JAG1) is a cell surface ligand in the Notch signaling pathway and mutations in this gene cause Alagille syndrome (AGS). JAG1 mutations have been identified in 60–70% of AGS patients studied, and these include total gene deletions (
6%), protein-truncating mutations (insertions, deletions and nonsense mutations) (82%) and missense mutations (12%). Based on the finding that total JAG1 deletions cause AGS, haploinsufficiency has been hypothesized to be a mechanism for disease causation; however, the mechanism by which missense mutations cause disease is not understood. To date, 25 unique missense mutations have been observed in AGS patients. Missense mutations are non-randomly distributed across the protein with clusters at the 5' end of the protein, in the conserved DSL domain, and two clusters within the EGF repeats. To understand the effect of the missense mutations on protein localization and function, we have studied four missense mutations (R184H, L37S, P163L and P871R). In two assays of JAG1 function, R184H and L37S are associated with loss of Notch signaling activity relative to wild-type JAG1. Neither R184H or L37S is present on the cell surface and both are abnormally glycosylated. Furthermore, these mutations lead to abnormal accumulation of the protein, possibly in the endoplasmic reticulum. Both P163L and P871R are associated with normal levels of Notch signaling activity and are present on the cell surface, consistent with these changes being polymorphisms rather than disease-causing mutations. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The Notch signaling pathway is an evolutionarily conserved signal transduction pathway involved in cell fate determination (1). Evidence from Drosophila, Caenorhabditis elegans, mice and humans has demonstrated that the members of this pathway are expressed in many tissues throughout development, and the consequences of mutations in members of this pathway affect many organ systems with effects ranging from lethality to minor abnormalities (1–7). There are many known members of this pathway including at least four Notch receptors (receptors 1, 2, 3 and 4), five ligands (Jagged1, Jagged2, Delta like-1, Delta-like 3 and Delta-like 4) and numerous other proteins (8). Three congenital human diseases have been associated with defects in this pathway. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a dominantly inherited adult-onset vascular dementia syndrome caused by missense mutations in the Notch3 receptor (9). A variety of types of mutation in the Notch ligand Jagged1 (JAG1) gene cause the dominantly inherited disorder Alagille syndrome (AGS) (6,7), and autosomal recessive spondylocostal dysostosis has been found to be caused by mutations in the Delta-like 3 gene in some consanguineous families (10).
AGS is a rare multisystem developmental disorder, affecting 1 in 50 000 to 1 in 70 000 individuals (11). The clinical findings associated with AGS are paucity of intrahepatic bile ducts, congenital heart defects, skeletal defects, ophthalmological abnormalities and particular facial features; however, the expressivity of the disorder is highly variable (12,13). The JAG1 protein is expressed on the cell surface and is composed of several domains. These include the DSL domain, which is a region conserved among Notch ligands (delta and serrate in Drosophila, and lag2 in C.elegans), 16 epidermal growth factor (EGF)-like repeats, a cysteine-rich (CR) region, a transmembrane (TM) domain and a less conserved small intracellular region (14).
Mutations in the JAG1 gene have been identified in 70% of AGS patients (15–20). Protein truncating, splicing, whole gene deletions and missense mutations have all been identified, with no apparent genotype–phenotype correlation. That there is no phenotypic difference between total gene deletions and protein-truncating mutations suggests that haploinsufficiency is a pathogenic mechanism causing AGS (15,16). However, the mechanism by which missense mutations in AGS lead to disease is unknown and a dominant negative mechanism cannot be ruled out. In Drosophila, mutant delta and serrate (Notch ligands that are highly homologous to JAG1) have been studied and found to be secreted, and act in a dominant negative fashion (21,22). A non-transmembrane (truncated, secreted) form of JAG1 has been shown to be active in a cell culture assay for differentiation of JAG1-transfected NIH-3T3 cells grown on a collagen matrix. These cells form visible, multicellular chords (a component of the endothelial cell differentiation pathway) under the influence of JAG1 (23).
The distribution of missense mutations observed in patients with AGS does not appear to be random (Fig. 1). Twenty-five unique missense mutations have been identified. Thirteen of these occur in the N-terminal region of the protein either within the conserved DSL region (3 mutations) or N-terminal to the DSL (10 mutations). The arginine residue at position 184 is the most mutable amino acid in the protein and has been found to be changed to another amino acid (G, C, H or L) in seven unrelated patients (15–17,20,24). The three mutations within the DSL all lead to the loss of a conserved cysteine residue. Eleven of the mutations were in the EGF repeats and one in the CR region. Two of the mutations found within the EGF repeats (R744Q and P871R) appear to be polymorphisms, as they were seen in unaffected individuals or AGS patients with another mutation (15,16). Eight of the twelve mutations in the EGF repeats or CR region result in loss or gain of a conserved cysteine residue. Loss of conserved cysteine residues within the EGF repeats have been found to be disease causing in other disorders, such as Marfan syndrome (25–27) and CADASIL (9,28,29).
|
Here we present studies on JAG1 missense mutations found in patients with AGS. We have studied these mutations to determine whether these single amino acid changes lead to non-functional proteins. We have hypothesized that studies of missense mutations that impair protein function will provide insight into amino acids that are crucial for normal expression and function of the JAG1 protein.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
R184H and L37S do not activate the Notch signaling pathway
Two assays were utilized to measure the ability of JAG1 to function as a ligand in the Notch signaling pathway. In the first assay, we tested the ability of JAG1-expressing cells to inhibit differentiation of a skeletal muscle-derived cell line (C2C12 cells) in a Notch-dependent manner (14,30). In mitogen-poor medium, C2C12 cells can be induced to undergo terminal differentiation and form multinucleated myotubes, which can be directly observed on microscopic examination. In the presence of functional JAG1–Notch signaling, this differentiation is inhibited and no myotubes are formed. For the experiments reported here, C2C12 cells were co-cultured with NIH-3T3 cells expressing wild-type JAG1, JAG1-R184H, -L37S, -P163L, -P871R or the retroviral expression vector pBABE alone.
In the absence of JAG1 expression, C2C12 cells co-cultured with NIH-3T3 cells undergo differentiation to myotubes as previously reported (30). When C2C12 cells were co-cultured with NIH-3T3 cells expressing wild-type JAG1, differentiation was inhibited (Fig. 2A). In contrast, when C2C12 cells were co-cultured with NIH-3T3 or JAG1-R184H, the differentiation pathway was activated and myotubes were observed, similar to results with C2C12 cells containing L37S (data not shown) and pBABE alone (empty vector). Co-culture of C2C12 cells with NIH-3T3 cells expressing P871R (Fig. 2A) or P163L (data not shown) were capable of inhibition of differentiation, suggesting no loss of signaling function. This is consistent with a lack of Notch signaling activity for JAG1-R184H and -L37S. Similar results were obtained when C2C12 cells constitutively expressing Notch1 were used in this co-culture assay.
|
To determine the level of JAG1–Notch signaling, we used an assay in which NIH-3T3 cells containing a Notch-sensitive luciferase construct were co-cultured with cells expressing either JAG1 or a JAG1 mutant. NIH-3T3 cells were transfected with a luciferase reporter construct containing four upstream binding sites of wild-type CBF1-binding elements (4xwtCBF1Luc) (31). CBF1 (the human homolog of Drosophila Suppressor of Hairless) is a DNA-binding protein which switches from a transcriptional repressor to an activator in a Notch-dependent manner (32,33). Under conditions where Notch is not activated, the promoter is repressed and the luciferase gene is off. When Notch is activated, the promoter complex is activated through loss of histone deacetylase-1 (HDAC1) and recruitment of histone acetylases. Thus, activation of Notch by JAG1 results in derepression of the promoter and the luciferase gene is turned on, with a 6-fold increase in luciferase activity. Following transfection with the reporter plasmid 4xwtCBF1Luc, cells were co-cultivated with cell lines containing pBABE alone, pBABE–wild-type JAG1 or pBABE–JAG1 mutant (R184H, L37S, P163L, P871R) and assayed for luciferase activity. NIH-3T3 cells that expressed wild-type JAG1, as well as P163L and P871R, were able to simulate transcription of luciferase (Fig. 2B). JAG1 mutants L37S and R184H have a 5- to 8-fold lower activity than wild-type JAG1, similar to that of the empty vector pBABE. These results suggest that the missense mutations JAG1-L37S and -R184H have lost Notch signaling function in this assay, consistent with their being disease-causing mutations.
R184H and L37S alter the glycosylation pattern of JAG1
To gain insight into the cause for loss of activity of the mutant JAG1 molecules R184H and L37S, we studied the proteins by western blot analysis. Blots containing cell lysates from NIH-3T3 cell lines expressing wild-type JAG1, JAG1-R184H and -L37S were probed with a JAG1 C-terminal antibody. JAG1 is predicted to have a molecular weight of 134.5 kDa. However, on SDS–PAGE, the apparent molecular weight is higher, 180 kDa. Both R184H and L37S exhibited an altered molecular weight, which was slightly smaller than wild-type JAG1, suggesting a difference in post-translational modification.
To test this hypothesis we treated cell lysates from cells expressing empty vector, wild-type JAG1 or JAG1-R184H with peptide-N-glycosidase F (PNGaseF), an endoglycosidase which removes all N-linked carbohydrates, or calf intestinal alkaline phosphatase (CIP), which removes phosphates from proteins and nucleic acids. In untreated lysates a difference was observed in the migration pattern between wild-type JAG1 and R184H. After treatment with PNGaseF, both JAG1-R184H and wild-type JAG1 migrated through the gel faster and at the same apparent molecular weight (Fig. 3, lanes 4–6). The migration patterns of the R184H or wild-type JAG1 proteins was not affected by treatment with CIP. The C-terminal antibody reacts with the endogenous JAG1 in NIH-3T3 cells, seen in the faint band co-migrating with our expressed wild-type JAG1, and reacting in a similar pattern to wild-type in response to the various treatments. These results are consistent with JAG1 being modified by the addition of N-linked carbohydrates. Similar studies comparing wild-type JAG1 and R184H with L37S demonstrate that L37S also migrates at a lower molecular weight than wild-type, and treatment with PNGaseF results in co-migration of the wild-type and mutant proteins (Fig. 4, lanes 1–6).
|
|
These results demonstrated a difference in N-linked glycosylation between wild-type JAG1 and R184H, with the mutant having fewer moieties attached. This led us to hypothesize that either the mutants completely lack one glycosylation site or there is an alteration in the subsequent modification of the pre-existing carbohydrate structure.
To differentiate between these possibilities, cell lysates were treated with endoglycosidase H (EndoH), an enzyme which will remove only simple carbohydrate structures (high-mannose and hybrid) but will not remove complex carbohydrate structures. Treatment of wild-type JAG1 with EndoH did not alter its migration pattern, indicating the presence of complex carbohydrate linkages (Fig. 4, lane 7). However, treatment of JAG1-R184H and -L37S resulted in complete conversion to the deglycosylated form, as observed by western analysis (Fig. 4, lanes 8 and 9). These results suggest that wild-type JAG1 contains more complex modifications than the R184H and L37S mutants. The differential sensitivity to EndoH observed between the wild-type JAG1 and mutant proteins suggests that the mutations prevented further modification of the core carbohydrate structures. Either this is due to improper recognition by Golgi specific enzymes or the mutant is not properly targeted to the Golgi complex.
We next investigated the timing of EndoH sensitivity in the wild-type and mutant JAG1 proteins. This was done to determine whether the wild-type demonstrated some sensitivity over increased assay time, or whether it was completely resistant. We also hypothesized that there might be multiple glycosylation sites with variable sensitivity and that with incubation times of <60 min we might be able to identify an intermediate form of the protein that was partially degylcosylated. These results demonstrated that deglycosylation was very fast, occurring in <1 minute for the R184H mutant, whereas the majority of wild-type JAG1 protein was resistant to EndoH treatment throughout the experiment (Fig. 5). The low amount (5%) of wild-type JAG1 that converted to the deglycosylated form occurred within 5 min of enzyme addition and was probably due to wild-type JAG1 in transit between the endoplasmic reticulum (ER) and Golgi. These results suggested that JAG1-R184H was very sensitive to EndoH treatment and no glycosylation intermediates were observed, suggesting either that there is a single glycosylation site or that they are equally accessible to the enzyme. Wild-type JAG1 was relatively insensitive to EndoH treatment, with no observable increase in sensitivity over time.
|
Subcellular distribution of wild-type JAG1 and R184H
The loss of activity in the mutant JAG1 molecules (JAG1-R184H and -L37S) could be due to either inability of the mutant ligands to signal the Notch receptor or improper targeting to the cell surface. To determine whether the mutant molecules were expressed on the cell surface, we treated cells with the protease trypsin, such that only JAG1 present on the cell surface would be degraded. The trypsin was inactivated at 1, 5 and 10 min time points. Cells were harvested for protein, which was then analyzed by western blot (Fig. 6). As expected, when cells expressing wild-type JAG1 were treated, the amount of full-length JAG1 decreased due to proteolysis (Fig. 6, lanes 1–4 and 9–12). This was also true of P163L and P871R (Fig. 6, lanes 17–23), the two missense mutations that showed normal JAG1 activity. Conversely, the R184H and L37S proteins did not demonstrate any changes in size with increasing exposure (Fig. 6, lanes 5–8 and 13–16), suggesting that they were not accessible to the trypsin and therefore not present on the cell surface.
|
Immunofluorescence was used to visualize the subcellular localization of the NIH-3T3 cell lines expressing JAG1 and JAG1-R184H proteins, using the JAG1 C-terminal antibody H-114 (Fig. 7). Immunofluorescence demonstrated that the subcellular distribution of wild-type JAG1 and JAG1-R184H and -L37S are different. The R184H and L37S mutants appear localized in the perinuclear region of the cytoplasm, whereas the wild-type JAG1, P163L and P871R were more diffuse and suggestive of their presence on the cell surface and throughout the cell. The subcellular distribution of JAG1 may not be typical due to high levels of expression from the retroviral promoter. However, we can conclude that the distribution of the R184H and L37S mutants is different from wild-type JAG1.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Mutations in the Notch ligand JAG1 cause the autosomal dominant, developmental disorder AGS. Over 200 JAG1 mutations have been identified in AGS patients and the majority of these are predicted to lead to the presence of a premature termination codon (34). A smaller percentage are total gene deletions and splicing or missense mutations. The mechanism of how missense and protein truncating mutations cause disease is unknown. There is no difference in phenotype between these AGS individuals and those with a total gene deletion, suggesting that functional haploinsufficiency is one mechanism for disease causation. This could be due to simple gene dosage effects or a resulting stoichiometric imbalance with (as yet unknown) JAG1-interacting proteins. Alternatively, some missense mutants may act via another mechanism. They may be present on the cell surface, but be incapable of Notch signaling. We have undertaken studies of JAG1 missense mutations to determine whether the mechanism of action of these mutations is consistent with haploinsufficiency or whether they provide evidence for a dominant negative mechanism. Results presented here are consistent with two JAG1 missense mutations (R184H and L37S) acting via haploinsufficiency, as these mutant molecules were not present on the cell surface, and are incapable of interaction with Notch on an adjacent cell. We have further hypothesized that the specific residues mutated in AGS patients will provide clues regarding the amino acid residues required for normal functioning and targeting of JAG1.
We have identified two missense mutations in JAG1 that result in defects in intracellular transport and processing of the JAG1 protein. Based on the results presented here we suggest that the JAG1-R184H and -L37S mutants are improperly targeted through the cell, and fail to appear on the cell surface, resulting in functional inactivation. There are differences in glycosylation between the wild-type and mutant JAG1 proteins. Studies using the endoglycosidase PNGaseF demonstrate that wild-type JAG1, JAG1-R184H and -L37S are sensitive to cleavage by this enzyme, indicating that they contain N-linked glycosylation sites. However, R184H and L37S are sensitive to EndoH whereas the wild-type is not, indicating differences in carbohydrate structure. R184H and L37S do not fall within consensus sequences for N-linked glycosylation [N-X-S/T-X (where X is anything but proline)], leading us to hypothesize that this mutation results in a conformational change inhibiting targeting to the Golgi for proper processing. Consistent with this, we have shown that the mutant protein appears to be retained in the cytoplasm, with a pattern suggestive of the ER. This phenomenon of mutations outside a glycosylation site altering glycosylation and subsequent processing has been identified in a number of disorders. In cystic fibrosis (CF), a number of mutations including 
F508, the most common mutation seen in CF patients, lead to incomplete protein processing and defective intracellular trafficking. These mutants fail to develop complex glycosylation structures preventing export from the ER (35).
We also studied two missense mutations that we found lead to production of phenotypically normal protein (JAG1-P163L and -P871R). The mutation P163L was previously reported in one patient with AGS (16), and we identified this mutation in a patient with pulmonic stenosis, who did not meet the clinical criteria for a diagnosis of AGS (I.D. Krantz, R.P. Colliton and N.B. Spinner, unpublished data). We hypothesize that this mutation may in fact represent a polymorphic variant of JAG1, but further studies will be necessary to determine whether the patient identified by Crosnier et al. (16) has another mutation in JAG1. Alternatively, P163L may cause an abnormality in JAG1 function that is not evident in the specific assays employed in this study. P871R has been reported as a polymorphism in multiple studies based on its presence in unaffected individuals (15,16).
The ER possesses a mechanism which prevents transport of misfolded, mutant and improperly complexed proteins (36). It is unclear what factors in the ER recognize the R184H mutant and prevent transport to the Golgi complex. However, any mutation that prevents proper protein folding also blocks movement of the peptide from the ER into the Golgi. Failure to transport mutant proteins from the ER to the Golgi complex may be the default fate of mutant proteins that are abnormally folded and not properly chaperoned. Protein misfolding secondary to mutation is thought to be a common mechanism which underlies the pathogenesis of a number of human diseases, including CF (35), long QT syndrome (37) and Saethre–Chotzen (38). There are many examples in the literature describing glycosylation changes and disease, which include hemophilia A (39), long QT syndrome (40), retinitis pigmentosa (41) and 
1-antiprotease deficiency. In 1-antiprotease deficiency (due to the missense mutation E342L) the protein is synthesized in the ER, but does not fold properly, forming an aggregate which is not transported from the ER (42).
JAG1 is a cell surface ligand for the Notch transmembrane receptor. Current understanding of Notch signaling is that the mature Notch receptor is present on the cell surface as a heterodimer. The Notch receptors have an extracellular domain which contains 36 EGF-like repeats and a C-terminal fragment which contains the TM domain and cytoplasmic sequences. These two fragments are non-covalently linked in a calcium-dependent manner (43). The cleavage that produces the heterodimer occurs in the secretory pathway and is independent of ligand binding. On binding of ligand to the Notch receptor, there is a second cleavage just N-terminal to the TM domain, followed by a third cleavage just C-terminal to the extracellular domain that releases the intracellular fragment from the membrane. The intracellular domain then translocates into the nucleus where it influences transcription of downstream genes (8). The DSL region of Notch ligands has been shown to be required for receptor binding (44); however, very little else is understood about the ligand–receptor interaction. It is not currently known which of the Notch proteins (1, 2, 3 and 4 in humans) serves as the physiological receptor for JAG1.
We have demonstrated that the R184H and L37S mutations result in functional haploinsufficiency, in that they are not present on the cell surface and thus are unable to signal Notch. To date, 25 JAG1 missense mutations have been identified in AGS patients. The pattern of missense mutations observed in JAG1 to date is intriguing. Ten of the mutations lie at the 5' end of the JAG1 gene, 5' to the conserved DSL region. Three lie within the conserved DSL and the remainder are found in the EGF repeats and one in the CR region. Two of the mutations in the EGF repeats have been considered to be polymorphisms, based on their presence in unaffected individuals or in a patient with another mutation. The mutations in the EGF repeats are clustered in two regions (between EGF repeats 1 and 6, and within repeat 13) and 7/9 lead to gain or loss of a cysteine residue.
Disease-causing cysteine substitutions within EGF-like repeats have been documented in various proteins and the mechanism by which these mutations lead to abnormal protein function is variable. Each EGF-like domain contains six cysteine residues that form stable disulfide bridges creating stable three-dimensional conformations important for calcium binding, correct folding and protein–protein interaction (45). The majority of CADASIL patients have mutations that result in gain or loss of cysteine residues within the EGF-like repeats of the Notch3 protein (28,29,46). These mutations do not affect protein targeting to the cell surface (29). Three-dimensional modeling of the effects of cysteine substitutions in Notch3 demonstrated that loss or gain of the evenly numbered cysteine residues is incompatible with normal folding of the EGF-like repeats (46). These mutations in CADASIL patients lead to decreased turnover of the extracellular domain of Notch3, resulting in protein accumulation of Notch3 extracellularly (29). In this case, the disease mechanism may be similar to other progressive neurologic diseases, such as Alzheimer’s, Parkinson’s and Huntington’s diseases, in which abnormal protein accumulation plays a role (47). Mutations affecting cysteines in the EGF-like repeats of the fibrillin-1 gene most often result in reduced extracellular matrix deposition of fibrillin-1 protein (27). When the protein is misfolded and calcium is no longer able to bind, increased proteolysis is observed (48). Further studies of the missense mutations in the EGF-like repeats of Jagged1 may provide another example of how these mutations result in disease.
We have studied two mutations at the 5' end of the protein and found that neither appears on the cell surface. We therefore hypothesize that this region of the protein is important for proper targeting to the ER and Golgi. Future studies will elucidate the mechanism by which the other mutations disturb JAG1–Notch signaling.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
DNA constructs and cell lines
The N-terminal portion of the Jagged1 cDNA containing the R184H mutation was cloned from total cDNA. A 1771 bp portion of R184H-containing cDNA was amplified by PCR using the following primers: X1AF (5'-CGCGAGCTAGGCTGGGTTT-3') and 8-10R (5'-TAATGACTGCCTTGGCCA-3'). This product was digested with NotI and BglII and cloned into a pCRII-TA vector (Invitrogen) containing wild-type JAG1. The R184H-containing product therefore replaced a portion of the wild-type sequence. Vectors containing the proper-sized insert were sequenced to confirm the presence of the mutation. For expression studies, the full-length JAG1 containing the R184H mutation was subcloned into the BamHI–SalI site of the pBABE-puro vector (45). This retroviral vector contains an endogenous promoter from which JAG1 can be expressed. It also contains an SV40 promoter driving the puromycin resistance gene. This vector can be transfected into packaging cell lines (BOSC 23 in these experiments) which allows formation of infectious viral particles, which we used to infect NIH-3T3 cells.
The L37S, P163L and P871R mutant constructs were made by site-directed mutagenesis (QuikChange kit; Stratagene). In brief, two complementary oligonucleotides were synthesized containing the desired mutation, flanked by unmodified sequences for each mutation. Amplification of the vector plus JAG1, using primers containing the mutation to be introduced, results in a new plasmid containing a mutated JAG1. PfuTurbo DNA polymerase was used with an extension time of 20 min for 16 cycles. The reaction was digested with DpnI to remove the parent (methylated) vector and transformed into bacteria. Several clones of each mutant were sequenced to assay for the presence of the mutation.
Cell lines.
For expression studies, wild-type JAG1 and JAG1 with missense mutations (JAG1-R184H, -L37S, -P163L and -P871R) were created by infection of NIH-3T3 cells using the retroviral expression vector, pBABE-puro (49), into which JAG1 or mutant JAG1 was cloned. Selected clones were transfected into a retroviral producer cell line (BOSC 23, a gift from Dr Warren Pear) (50) using the transfection reagent FuGene 6 (Roche). Virus was harvested and JAG1-expressing cells were created by infection of NIH-3T3 cells (a mouse fibroblast cell line with low endogenous levels of JAG1) with the pBABE–JAG1 constructs in the presence of polybrene. These cell lines were selected in 1.5 µg/ml puromycin-containing medium and used to analyze the effect of missense mutations on JAG1 function. C2C12 cells are a skeletal muscle-derived cell line which can be induced to differentiate in a Notch-dependent manner (14). These cells were a gift from Jon Aster (30). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 2 mM glutamine.
C2C12 myogenesis assay.
The C2C12 myogenesis assay was performed as described by Lindsell et al. (14) and Luo et al. (30). Briefly, C2C12 cells were plated to 60–70% confluence. NIH-3T3 (5 x 106) cells containing wild-type JAG1, JAG1-R184H, -L37S, -P163L or -P871R were seeded onto each dish in DMEM with 10% FBS, resulting in 100% confluence of the combined C2C12 cells and layered NIH-3T3 cells. One day after seeding, the media was changed to DMEM containing 10% horse serum (differentiation medium). Plates were analyzed each day under the microscope and fixed in 10% formaldehyde and stained by methylene blue, 3 days following introduction into differentiation medium.
Western blot analysis.
NIH-3T3 cells expressing wild-type JAG1, JAG1-R184H, -L37S, -P163L and -P871R were counted, and equal numbers of cells were plated and harvested the following day for western analysis (51). For analysis of protein, 10 cm dishes of cells were washed twice in phosphate-buffered saline (PBS) and lysed for 10 min in NP-40 lysis buffer [150 mM NaCl, 1.0% NP-40, 50 mM Tris pH 8.0 containing 0.25 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin and 1 mM dithiothreitol (DTT)]. Supernatant was collected after centrifugation. Protein concentration was determined using the BioRad Protein Assay kit and equal amounts of protein were loaded onto a 7.5% SDS–polyacrylamide gel and subsequently transferred to a PVDF membrane (51). To detect JAG1 protein, a polyclonal antibody to the C-terminal region was used (a gift from Gerry Weinmaster), with a horseradish peroxidase–goat anti-rabbit secondary antibody (Amersham), and visualized using an ECL kit (Amersham).
Glycosylation assays.
Cell lysates were denatured at 100°C for 10 min, then treated either with PNGaseF or EndoH for 60 min according to the manufacturer’s protocol (New England Biolabs). Treated lysates were separated on a 7.5% SDS–polyacrylamide gel and analyzed by western blot.
Luciferase assays.
NIH-3T3 cells were maintained in DMEM with 10% FBS. Cells were transfected with 2 µg of plasmid DNA 4XwtCBF1Luc (31) using Fugene 6 (Roche). The following morning 1 x 106 NIH-3T3 cells containing wild-type JAG1, JAG1-R184H, -L37S, -P163L or -P871R were seeded onto each dish. To quantitate luciferase activity, cells were harvested 48 h after seeding and lysed in 100 µl of a buffer containing 25 mM Tris–HCl pH 7.8, 2 mM EDTA, 2 mM DTT, 10% glycerol and 1% Triton X-100 for 10 min at room temperature and centrifuged at 7000 g for 5 min. Luciferase activity was determined using 20 µl of cleared lysate using a luminometer (Lumat LB 9507; EG&G Berthold), using a luciferase assay system (Promega). All transfections were normalized for the amount of total cellular protein (BioRad). All assays were repeated three times.
Immunofluorescence
Cells were plated on glass coverslips in six-well plates and after 24 h in culture were washed in PBS and fixed in 4% paraformaldehyde for 10 min. Following fixation, coverslips were washed in PBS then blocked in buffer containing FBS. Primary antibody was diluted to 5 µg/ml in the presence of Triton X-100 and incubated for 1 h at room temperature. Coverslips were washed in PBS and then incubated for 30 min at room temperature in biotin-labeled goat anti-rabbit (or mouse depending on primary). Coverslips were washed then incubated for 30 min with fluorescently labeled avidin (either rhodamine or FITC). Slides were stained with Hoechst (diluted 1:500) for 5 min, washed and fixed in methanol at –20°C for 8–10 min. The coverslips were washed with PBS, mounted on slides using Vectashield (Vector) and visualized using a Zeiss fluorescence microscope.
|
ACKNOWLEDGEMENTS |
|---|
We would like to thank Drs Gerry Weinmaster and Carol Hicks for generously providing Jagged1 antibody, Drs Jeff Sklar and Jon Aster for providing C2C12 cells and helpful advice and Dr Warren Pear for the BOSC 23 cells. We thank Tom Kadesch, Susan DeRocco and Prakash Rao for helpful discussion and reagents. We are grateful to Ayala Laufer-Cahana, Lynn Bason, Ian Krantz, Feng Min Lu and Mike Marino for discussion and critical reading of the manuscript. We thank Drs Mary Catherine Glick and Thomas Scanlin for helpful discussions regarding glycosylation studies. Dr Peter Bannerman provided expertise and advice for confocal microscopy. This work is supported in part by NIH training grant no. 5T32NS07413 (J.J.D.M.) and NIH grant no. RO1 DK 53104 from the NIDDK (N.B.S.).
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +1 215 590 3316; Fax: +1 215 590 3850; Email: spinner@mail.med.upenn.edu
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Artavanis-Tsakonas, S., Rand, M.D. and Lake, R.J. (1999) Notch signaling: cell fate control and signal integration in development. Science, 284, 770–776.
2 Lindsell, C.E., Shawber, C.J., Boulter, J. and Weinmaster, G. (1995) Jagged: a mammalian ligand that activates Notch1. Cell, 80, 909–917.[Web of Science][Medline]
3 Krebs, L.T., Xue, Y., Norton, C.R., Shutter, J.R., Maguire, M., Sundberg, J.P., Gallahan, D., Closson, V., Kitajewski, J., Callahan, R. et al. (2000) Notch signaling is essential for vascular morphogenesis in mice. Genes Dev., 14, 1343–1352.
4 Kusumi, K., Sun, E.S., Kerrebrock, A.W., Bronson, R.T., Chi, D.C., Bulotsky, M.S., Spencer, J.B., Birren, B.W., Frankel, W.N. and Lander, E.S. (1998) The mouse pudgy mutation disrupts Delta homologue Dll3 and initiation of early somite boundaries. Nature Genet., 19, 274–278.[Web of Science][Medline]
5 Jiang, R., Lan, Y., Chapman, H.D., Shawber, C., Norton, C.R., Serreze, D.V., Weinmaster, G. and Gridley, T. (1999) Defects in limb, craniofacial, and thymic development in Jagged2 mutant mice. Genes Dev., 12, 1046–1057.
6 Li, L., Krantz, I.D., Den, Y., Genin, A., Banta, A.B., Collins, C.C., Qi, M., Trask, B.J., Kuo, W.L., Cochran, J. et al. (1997) Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nature Genet., 16, 243–251.[Web of Science][Medline]
7 Oda, T., Elkahloun, A.G., Pike, B.L., Okajima, K., Krantz, I.D., Genin, A., Piccoli, D.A., Meltzer, P.S., Spinner, N.B., Collins, F.S. and Chandrasekharappa, S.C. (1997) Mutations in the human Jagged1 gene are responsible for Alagille syndrome. Nature Genet., 16, 235–242.[Web of Science][Medline]
8 Kadesch, T. (2000) Notch signaling: a dance of proteins changing partners. Exp. Cell Res., 260, 1–8.[Web of Science][Medline]
9 Joutel, A., Corpechot, C., Ducros, A., Vahedi, K., Chabriat, H., Mouton, P., Alamowitch, S., Domenga, V., Cecillion, M., Marechal, E. et al. (1996) Notch3 mutations in CADASIL, an hereditary adult onset condition causing stroke and dementia. Nature, 383, 707–710.[Medline]
10 Bulman, M.P., Kusumi, K., Frayling, T.M., McKeown, C., Garrett, C., Lander, E.S., Krumlauf, R., Hattersley, A.T., Ellard, S. and Turnpenny, P.D. (2000) Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis. Nature Genet., 24, 438–441.[Web of Science][Medline]
11 Danks, D.M., Campbell, P.E., Jack, I., Rodgers, J. and Smith, A.L. (1977) Studies of the aetiology of neonatal hepatitis and biliary atresia. Arch. Dis. Child, 52, 360–367.
12 Krantz, I.D., Piccoli, D.A. and Spinner, N.B. (1997) Alagille syndrome (syndrome of the month). J. Med. Genet., 34, 152–157.
13 Krantz, I.D., Smith, R., Colliton, R.P., Tinkel, H., Zackai, E.H., Piccoli, D.A., Goldmuntz, E. and Spinner, N.B. (1999) Jagged1 mutations in patients ascertained with isolated congenital heart defects. Am. J. Med. Genet., 84, 56–60.[Web of Science][Medline]
14 Lindsell, C.E., Shawber, C.J., Boulter, J. and Weinmaster, G. (1995) Jagged: a mammalian ligand that activates Notch1. Cell, 80, 909–917.
15 Krantz, I.D., Colliton, R.P., Genin, A., Rand, E.B., Li, L., Piccoli, D.A. and Spinner, N.B. (1998) Spectrum and frequency of Jagged1 (JAG1) mutations in Alagille syndrome patients and their families. Am. J. Hum. Genet., 62, 1361–1369.[Web of Science][Medline]
16 Crosnier, C., Driancourt, C., Raynaud, N., Dhorne-Pollet, S., Pollet, N., Bernard, O., Hadchouel, M. and Meunier-Rotival, M. (1999) Analysis of mutations of the Jagged1 gene in patients with Alagille syndrome: evidence for most cases being sporadic. Gastroenterology, 116, 1141–1148.[Web of Science][Medline]
17 Pilia, G., Uda, M., Macis, D., Frau, F., Crisponi, L., Balli, F., Barbera, C., Colombo, C., Frediani, T., Gatti, R. et al. (1999) Jagged-1 mutation analysis in Italian Alagille syndrome patients. Hum. Mutat., 14, 394–400.[Web of Science][Medline]
18 Onouchi, Y., Kurahashi, H., Tajiri, H., Ida, S., Okada, S. and Nakamura, Y. (1999) Genetic alterations in the JAG1 gene in Japanese patients with Alagille syndrome. J. Hum. Genet., 44, 235–239.[Web of Science][Medline]
19 Yuan, Z.R., Kohsaka, T., Ikegaya, T., Suzuki, T., Okano, S., Abe, J., Kobayashi, N. and Yamada, M. (1998) Mutational analysis of the Jagged 1 gene in Alagille syndrome families. Hum. Mol. Genet., 7, 1363–1369.
20 Colliton, R.P., Bason, L., Lu, F.M., Piccoli, D.A., Krantz, I.D. and Spinner, N.B. (2001) Jagged1 mutations in Alagille Syndrome (AGS). Hum. Mutat., in press.
21 Rebay, I., Fleming, R.J., Fehon, R.G., Cherbas, L., Cherbas, P. and Artavanis-Tsakonas, S. (1991) Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell, 67, 687–699.[Web of Science][Medline]
22 Sun, X. and Artavanis-Tsakonas, S. (1996) The intracellular deletions of Delta and Serrate define dominant negative forms of the Drosophila Notch ligands. Development, 122, 2465–2474.[Abstract]
23 Wong, M.K.K., Prudovsky, I., Vary, C., Booth, C., Liaw, L., Mousa, S., Small, D. and Maciag, T. (2000) A non transmembrane form of Jagged-1 regulates the formation of matrix dependent chord-like structures. Biochem. Biophys. Res. Commun., 268, 853–859.[Web of Science][Medline]
24 Heritage, M.L., MacMillan, J.C., Colliton, R.P., Genin, A., Spinner, N.B. and Anderson, G.J. (2000) Mutation detection in an Australian Alagille syndrome population. Hum. Mutat., 16, 408–416.[Web of Science][Medline]
25 Kielty, C.M. and Shuttleworth, C.A. (1993) The role of calcium in the organization of fibrillin microfibrils. FEBS Lett., 336, 323–326.[Web of Science][Medline]
26 Dietz, H.C., Saraiva, J.M., Pyeritz, R.E., Cutting, G.R. and Francomano, C.A. (1992) Clustering of fibrillin FBN1 missense mutations in Marfan syndrome patients at cysteine residues in EGF-like domains. Hum. Mutat., 1, 336–374.
27 Schrijver, I., Liu, W., Brenn, T., Furthmayr, H. and Francke, U. (1999) Cysteine substitutions in epidermal growth factor-like domains of fibrilllin-1: distinct effects on biochemical and clinical phenotypes. Am. J. Hum. Genet., 65, 1007–1020.[Web of Science][Medline]
28 Joutel, A., Vahedi, K., Corpechot, C., Troesch, A., Chabriat, H., Vayssiere, C., Cruaud, C., Maciazek, J., Weissenbach, J., Bousser, M.G. et al. (1997) Strong clustering and stereotyped nature of Notch3 mutations in CADASIL patients. Lancet, 350, 1511–1515.[Web of Science][Medline]
29 Joutel, A., Andreux, F., Gaulis, S., Domenga, V., Cecillon, M., Battail, N., Piga, N., Chapon, F., Godfrain, C. and Tournier-Lasserve, E. (2000) The ectodomain of Notch3 receptor accumulates within the cerebrovasculature of CADASIL patient. J. Clin. Invest., 105, 597–605.[Web of Science][Medline]
30 Luo, B., Aster, J.C., Hasserjian, R.P., Kuo, F. and Sklar, J. (1997) Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor. Mol. Cell Biol., 17, 6057–6067.[Abstract]
31 Hsieh, J.J.D., Henkel, T., Salmon, P., Robey, E., Peterson, M.G. and Hayward, S.D. (1996) Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein–Barr virus EBNA2. Mol. Cell. Biol., 16, 952–959.[Abstract]
32 Kao, H.Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, C.R., Evans, R.M. and Kadesch, T. (1998) A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev., 12, 2269–2277.
33 Kurooka, H. and Honjo, T. (2000) Functional interaction between the mouse notch1 intracellular domain region and histone acetyltransferases PCAF and GCN5. J. Biol. Chem., 275, 17211–17220.
34 Spinner, N.B., Colliton, R.P., Crosnier, C., Krantz, I., Hadchouel, M. and Muenier-Rotival, M. (2001) Jagged1 mutations in Alagille syndrome. Hum. Mutat., 17, 18–33.[Web of Science][Medline]
35 Cheng, S.H., Gregory, R.J., Marshall, J., Paul, S., Souza, D.W., White, G.A., O’Riordan, C.R. and Smith, A.E. (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell, 63, 827–834.[Web of Science][Medline]
36 Hammond, C. and Helenius, A. (1995) Quality control in the secretory pathway. Curr. Opin. Cell Biol., 7, 523–529.[Web of Science][Medline]
37 Zhou, Z., Gong, Q., Epstein, M.L. and January, C.T. (1998) HERG channel dysfunction in human long QT syndrome. Intracellular transport and functional defects. J. Biol. Chem., 273, 21061–21066.
38 El Ghouzzi, V., Legeai-Mallet, L., Aresta, S., Benoist, C., Munnich, A., de Gunzburg, J. and Bonaventure, J. (2000) Saethre–Chotzen mutations cause TWIST protein degradation or impaired nuclear location. Hum. Mol. Genet., 9, 813–819.
39 Aly, A.M., Higuchi, M., Kasper, C.K., Kazazian Jr, H.H., Antonarakis, S.E. and Hoyer, L.W. (1992) Hemophilia A due to mutations that create new N-glycosylation sites. Proc. Natl Acad. Sci. USA, 89, 4933–4937.
40 Petrecca, K., Atanasiu, R., Akhavan, A. and Shrier, A. (1999) N-linked glycosylation sites determine HERG channel surface membrane expression. J. Physiol., 515, 41–48.
41 Kaushal, S. and Khorana, H.G. (1994) Structure and function in rhodopsin. 7. Point mutations associated with autosomal dominant retinitis pigmentosa. Biochemistry, 33, 6121–6128.[Medline]
42 Yoshida, A., Lieberman, J., Gaidulis, L. and Ewing, C. (1976) Molecular abnormality of human alpha-1-antitrypsin variant (Pi-SZ) associated with plasma activity deficiency. Proc. Natl Acad. Sci. USA, 73, 1324–1328.
43 Rand, M.D., Grimm, L.M., Artavanis-Tsakonas, S., Patriub, V., Blacklow, S.C., Sklar, J. and Aster, J.C. (2000) Calcium depletion dissociates and activates heterodimeric notch receptors. Mol. Cell Biol., 20, 1825–1835.
44 Shimizu, K., Chiba, S., Kumano, K., Hosoya, N., Takahashi, T., Kanda, Y., Hamada, Y., Yazaki, Y. and Hirai, H. (1999) Mouse jagged1 physically interacts with notch2 and other notch receptors. Assessment by quantitative methods. J. Biol. Chem., 274, 32961–32969.
45 Campbell, I.D. and Bork, P. (1993) Epidermal growth factor-like modules. Curr. Opin. Struct. Biol., 3, 385–392.
46 Dichgans, M., Ludwig, H., Muller-Hocker, J., Messerschmidt, A. and Gasser, T. (2000) Small in frame deletions and missense mutations in CADASIL: 3D models predict misfolding of Notch3 EGF-like domains. Eur. J. Hum. Genet., 8, 280–285.[Web of Science][Medline]
47 Spinner, N.B. (2000) CADASIL: Notch signaling defect or protein accumulation problem? J. Clin. Invest., 105, 561–562.[Web of Science][Medline]
48 Reinhardt, D.P., Ono, R.N. and Sakai, L.Y. (1997) Calcium stabilizes Fibrillin-1 against proteolytic degradation. J. Biol. Chem., 272, 1231–1236.
49 Morgenstern, J.P. and Land, H. (1990) Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res., 18, 3587–3596.
50 Pear, W.S., Nolan, G.P., Scott, M.L. and Baltimore, D. (1993) Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl Acad. Sci. USA, 90, 8392–8396.
51 Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, NY.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. B.-D. Ponio, C. Wright-Crosnier, M.-T. Groyer-Picard, C. Driancourt, I. Beau, M. Hadchouel, and M. Meunier-Rotival Biological function of mutant forms of JAGGED1 proteins in Alagille syndrome: inhibitory effect on Notch signaling Hum. Mol. Genet., November 15, 2007; 16(22): 2683 - 2692. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T Haritunians, T Chow, R P J De Lange, J T Nichols, D Ghavimi, N Dorrani, D M St Clair, G Weinmaster, and C Schanen Functional analysis of a recurrent missense mutation in Notch3 in CADASIL J. Neurol. Neurosurg. Psychiatry, September 1, 2005; 76(9): 1242 - 1248. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Trifonova, D. Small, D. Kacer, D. Kovalenko, V. Kolev, A. Mandinova, R. Soldi, L. Liaw, I. Prudovsky, and T. Maciag The non-transmembrane form of Delta1 but not of jagged1 Induces normal migratory behavior accompanied by FGF receptor 1-dependent transformation J. Biol. Chem., February 9, 2004; (2004) 300564200. [Abstract] [PDF]
|
|
|
|
|
|
T. Gridley Notch signaling and inherited disease syndromes Hum. Mol. Genet., April 2, 2003; 12(90001): R9 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. McCright, J. Lozier, and T. Gridley A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency Development, March 4, 2003; 129(4): 1075 - 1082. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. B. McElhinney, I. D. Krantz, L. Bason, D. A. Piccoli, K. M. Emerick, N. B. Spinner, and E. Goldmuntz Analysis of Cardiovascular Phenotype and Genotype-Phenotype Correlation in Individuals With a JAG1 Mutation and/or Alagille Syndrome Circulation, November 12, 2002; 106(20): 2567 - 2574. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Sasaki, S. Ishida, I. Morimoto, T. Yamashita, T. Kojima, C. Kihara, T. Tanaka, K. Imai, Y. Nakamura, and T. Tokino The p53 Family Member Genes Are Involved in the Notch Signal Pathway J. Biol. Chem., January 4, 2002; 277(1): 719 - 724. [Abstract] [Full Text]
|
|
|
|
|
|
D. Small, D. Kovalenko, D. Kacer, L. Liaw, M. Landriscina, C. Di Serio, I. Prudovsky, and T. Maciag Soluble Jagged 1 Represses the Function of Its Transmembrane Form to Induce the Formation of the Src-dependent Chord-like Phenotype J. Biol. Chem., August 17, 2001; 276(34): 32022 - 32030. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||