EGF-like Repeats Research Articles

Diseases related to Notch glycosylation

The Notch receptors are a family of transmembrane proteins that mediate direct cell-cell interactions and control numerous cell-fate specifications in humans. The extracellular domains of mammalian Notch proteins contain 29-36 tandem epidermal growth factor-like (EGF) repeats, most of which have O-linked glycan modifications: O-glucose added by POGLUT1, O-fucose added by POFUT1 and elongated by Fringe enzymes, and O-GlcNAc added by EOGT. The extracellular domain is also N-glycosylated. Mutations in the glycosyltransferases modifying Notch have been identified in several diseases, including Dowling-Degos Disease (haploinsufficiency of POFUT1 or POGLUT1), a form of limb-girdle muscular dystrophy (autosomal recessive mutations in POGLUT1), Spondylocostal Dysostosis 3 (autosomal recessive mutations in LFNG), Adams-Oliver syndrome (autosomal recessive mutations in EOGT), and some cancers (amplification, gain or loss-of-function of POFUT1, Fringe enzymes, POGLUT1, MGAT3). Here we review the characteristics of these diseases and potential molecular mechanisms.

Evaluation of the Role of ITGBL1 in Ovarian Cancer.

Simple SummaryA poorly characterized protein called Integrin beta-like 1 (ITGBL1) may play an important role in ovarian cancer progression. Our previous studies have indicated that increased expression of the gene coding for ITGBL1 is related to poor prognosis for ovarian cancer patients. In the present study we investigated the role of ITGBL1 in ovarian cancer cells using several in-vitro assays and global gene expression analysis. We found that ITGBL1 overexpression affected cellular adhesion, motility and invasiveness. In addition, ITGBL1 caused cells to be more resistant to cisplatin and paclitaxel, major drugs used in ovarian cancer treatment. Our results indicate that higher expression of ITGBL1 in ovarian cancer is associated with the features that may worsen the clinical course of the disease. Further studies will show if ITGBL1 can be exploited as a cancer biomarker and/or molecular target for experimental biological therapy.In our previous microarray study we identified two subgroups of high-grade serous ovarian cancers with distinct gene expression and survival. Among differentially expressed genes was an Integrin beta-like 1 (ITGBL1), coding for a poorly characterized protein comprised of ten EGF-like repeats. Here, we have analyzed the influence of ITGBL1 on the phenotype of ovarian cancer (OC) cells. We analyzed expression of four putative ITGBL1 mRNA isoforms in five OC cell lines. OAW42 and SKOV3, having the lowest level of any ITGBL1 mRNA, were chosen to produce ITGBL1-overexpressing variants. In these cells, abundant ITGBL1 mRNA expression could be detected by RT-PCR. Immunodetection was successful only in the culture media, suggesting that ITGBL1 is efficiently secreted. We found that ITGBL1 overexpression affected cellular adhesion, migration and invasiveness, while it had no effect on proliferation rate and the cell cycle. ITGBL1-overexpressing cells were significantly more resistant to cisplatin and paclitaxel, major drugs used in OC treatment. Global gene expression analysis revealed that signaling pathways affected by ITGBL1 overexpression were mostly those related to extracellular matrix organization and function, integrin signaling, focal adhesion, cellular communication and motility; these results were consistent with the findings of our functional studies. Overall, our results indicate that higher expression of ITGBL1 in OC is associated with features that may worsen clinical course of the disease.

Effect of Site‐Specific O‐Glycosylation on the Structural Behavior of NOTCH1 Receptor Extracellular EGF‐like Domains 11 and 10

Human NOTCH1 receptor contains 36 epidermal growth factor (EGF)-like repeating domains, in which O-glycosylation status of EGF12 domain regulates the interaction with Notch ligands. Our interest is focused on the effect of specific O-glycosylation states on the structural behavior of EGF11 and EGF10, because they appeared to affect molecular mechanism in receptor-ligand interactions by inducing some conformational alterations in these domains and/or the regions connecting two domains. To understand the structural impact of various O-glycosylation patterns on the pivotal EGF-like repeats 10, 11, and 12, we performed chemical synthesis and NMR studies of site-specifically O-glycosylated EGF11 and EGF10. Our strategy enabled us to synthesize four EGF11 and five EGF10 modules. The specific O-glycosylation states affected in vitro folding of EGF10 more than EGF11, while calcium ion had a larger effect on EGF11 folding. Comprehensive NMR studies shed light on the new type "sugar bridges" crosslinking Thr-O-GlcNAc in the consensus sequence C5-X-X-G-X-(T/S)-G-X-X-C6 and an amino acid in the hinge region between the domains, 445Thr-O-GlcNAc-IIe451 in domain 11 and 405Thr-O-GlcNAc-Gln411 in domain 10, respectively.

Psoriasis-associated angiogenesis is mediated by EDIL3

The dermal mesenchymal stem cells (DMSCs) from psoriasis display higher expression level of epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3), while EDIL3 can bind integrins, including αvβ3 and αvβ5, to regulate angiogenesis. To assess the role of EDIL3 derived from DMSCs of psoriasis (P-DMSCs) in angiogenesis, in vitro, EDIL3 of DMSCs from psoriasis was silenced by interfering EDIL3. Then the efficacy of silencing EDIL3 was tested by fluorescent flag, qRT-PCR and western blotting. And, in vitro, the relationship of EDIL3 in DMSCs with the angiogenesis of HUVECs were investigated through co-culture system. In vivo, EDIL3 recombinant protein was injected into IMQ cream-induced psoriasis-like skin lesions of mouse and EDIL3-associated tube formation were determined using Image J software. Our results showed the capacity of the adhesion, migration and tube formation of HUVECs in all psoriatic DMSCs groups were significantly higher compared with the control and si-EDIL3 groups (all P<0.05) in vitro. Moreover, under stimulated by EDIL3 recombinant protein, EDIL3-associated tube formation was dramatically elevated in vivo (P<0.01). In this study, EDIL3 could promote the adhesion, migration and tube formation of ECs and participant in the angiogenesis pathogenesis of psoriasis through affecting biological function on ECs both in vitro and in vivo. The results suggest a potential role of the critical pro-angiogenic factor EDIL3 in psoriasis therapy.

PCSK9: A Key Target for the Treatment of Cardiovascular Disease (CVD)

Proprotein convertase subtilisin/kexin type 9 (PCSK9), as a vital modulator of low-density lipoprotein cholesterol (LDL-C) , is raised in hepatocytes and released into plasma where it binds to LDL receptors (LDLR), leading to their cleavage. PCSK9 adheres to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR which is confirmed by crystallography. LDLR expression is adjusted at the transcriptional level through sterol regulatory element binding protein 2 (SREBP-2) and at the post translational stages, specifically through PCSK9, and the inducible degrader of the LDLR PCSK9 inhibition is an appealing new method for reducing the concentration of LDL-C. In this review the role of PCSK9 in lipid homeostasis was elucidated, the effect of PCSK9 on atherosclerosis was highlighted, and contemporary therapeutic techniques that focused on PCSK9 were summarized. Several restoration methods to inhibit PCSK9 have been proposed which concentrate on both extracellular and intracellular PCSK9, and they include blockage of PCSK9 production by using gene silencing agents and blockage of it’s binding to LDLR through antibodies and inhibition of PCSK9 autocatalytic processes by tiny molecule inhibitors.

Expression, purification, and glycosylation of epidermal growth factor-like repeat 27 from mouse NOTCH1

Notch receptors have large extracellular domains containing up to 36 tandem epidermal growth factor-like (EGF) repeats, which facilitate cell signaling by binding ligands on neighboring cells. Notch receptors play major roles in a variety of developmental processes by controlling cell fate decisions. Each EGF repeat consists of about 40 amino acids with 3 conserved disulfide bonds. Many of the EGF repeats are modified by O-linked fucose glycans, and more than half have calcium-binding sites, but the sequences of the EGF repeats vary giving distinct roles to each repeat. EGF repeat 27 (EGF27) from mouse NOTCH1 is modified with O-fucose and is 1 of 7 repeats that is differentially modified by specific Fringe enzymes, which are known to regulate NOTCH1 activation and ligand binding. To better understand the role of EGF27 in NOTCH1 function and regulation, the 3-dimensional structures of EGF27 and its glycoforms are being pursued. E. coli cells were used to produce EGF27 in sufficient quantities for nuclear magnetic resonance analysis. Previous attempts to express the repeat alone and refold the repeat under a steady redox environment were unsuccessful due to low yields and extensive mixed-disulfide bond cross-linking. A new strategy using a cleavable maltose binding protein fusion tag increased the solubility and yield of EGF27. With the fusion tag, EGF27 was refolded to produce the correct disulfide bond arrangement, which was verified enzymatically with the glycosyltransferases, Protein O-fucosyltransferase 1 (POFUT1) and Lunatic Fringe (LFNG).

Identification of key genes for guiding chemotherapeutic management in ovarian cancer using translational bioinformatics

The emergence of resistance to chemotherapy drugs in patients with ovarian cancer is still the main cause of low survival rates. The present study aimed to identify key genes that may provide treatment guidance to reduce the incidence of drug resistance in patients with ovarian cancer. Original data of chemotherapy sensitivity and chemoresistance of ovarian cancer were obtained from the Gene Expression Omnibus dataset GSE73935. Differentially expressed genes (DEGs) between sensitive and resistant ovarian cancer cell lines were screened by Empirical Bayes methods. Overlapping DEGs between four chemoresistant groups were identified by Venn map analysis. Protein-protein interaction networks were also constructed, and hub genes were identified. The hub genes were verified by in vitro experiments as well as The Cancer Genome Atlas data. Results from the present study identified eight important genes that may guide treatment decisions regarding chemotherapy regimens for ovarian cancer, including epidermal growth factor-like repeats and discoidin I-like domains 3, NRAS proto-oncogene, hyaluronan and proteoglycan link protein 1, activated protein C receptor, CD53, cyclin-dependent kinase inhibitor 2A, insulin-like growth factor 1 receptor and roundabout guidance receptor 2 genes. Their expressions were found to have an impact on the prognosis of different treatment groups (cisplatin, paclitaxel, cisplatin + paclitaxel, cisplatin + doxorubicin and cisplatin + topotecan). The results indicated that these genes may minimise the occurrence of ovarian cancer drug resistance and may provide effective treatment options for patients with ovarian cancer.

Xylosyl Extension of O-Glucose Glycans on the Extracellular Domain of NOTCH1 and NOTCH2 Regulates Notch Cell Surface Trafficking.

Biochemical and genetic studies have indicated that O-linked glycosylation such as O-glucose (Glc), fucose (Fuc), and N-acetylglucosamine (GlcNAc) is critical for Notch signaling; however, it is not fully understood how O-glycans regulate the Notch receptor function. Notch receptors are type-I transmembrane proteins with large extracellular domains (ECD), containing 29–36 epidermal growth factor-like (EGF) repeats. Here, we analyzed O-Glc glycans on NOTCH1 and NOTCH2 expressed in HEK293T cells using an Orbitrap Fusion mass spectrometer and successfully revealed the structures and stoichiometries of all 17 EGF repeats of NOTCH1 with the O-Glc consensus sequence (C1-X-S-X-(P/A)-C2), and 16 out of 17 EGF repeats of NOTCH2 with the same consensus sequence. High levels of O-Glc attachment and xylosyl elongation were detected on most NOTCH1 and NOTCH2 EGF repeats. When both glucoside xylosyltransferases, GXYLT1 and GXYLT2, responsible for the xylosyl elongation of O-glucose, were genetically deleted, the expression of endogenous NOTCH1 and NOTCH2 on the surface of HEK293T cells did not change, but the cell surface expression of overexpressed NOTCH1 and NOTCH2 decreased compared with that in the wild type cells. In vitro secretion assays consistently showed a reduced secretion of both the NOTCH1 and NOTCH2 ECDs in GXYLT1 and GXYLT2 double knockout cells compared with the wild type cells, suggesting a significant role of the elongation of O-Glc glycans on the Notch ECDs in the quality control of Notch receptors.

Prevalence and Atypical Clinical Characteristics of NOTCH3 Mutations Among Patients Admitted for Acute Lacunar Infarctions.

Objectives: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary small vessel disease, with reported frequencies of 2-5/100,000 individuals. Recently, it has been reported that some patients with NOTCH3 gene mutations show atypical clinical symptoms of CADASIL. Assuming that CADASIL is underdiagnosed in some cases of lacunar infarction, this study was designed to examine the prevalence of NOTCH3 gene mutations in the patients at highest risk who were admitted for lacunar infarctions.Methods: From January 2011 to April 2018, 1,094 patients with lacunar infarctions were admitted to our hospital, of whom 31 patients without hypertension but with white matter disease (Fazekas scale 2 or 3) were selected and genetically analyzed for NOTCH3 gene mutations (Phase 1). Furthermore, 54 patients, who were 60 years or younger, were analyzed for NOTCH3 mutations (Phase 2). NOTCH3 exons 2–24, which encode the epidermal growth factor-like repeat domain of the NOTCH3 receptor, were analyzed for mutations by direct sequencing of genomic DNA.Results: Three patients presented NOTCH3 p.R75P mutations: two in the Phase 1 and one in the Phase 2 cohort. Among patients aged 60 years or younger and those without hypertension but with moderate-to-severe white matter lesions, the carrier frequency of p.R75P was 3.5% (3/85), which was significantly higher than that in the Japanese general population (4.7KJPN) (odds ratio [95% CI] = 58.2 [11.6–292.5]). All three patients with NOTCH3 mutations had family histories of stroke, and the average patient age was 51.3 years. All three patients also showed white matter lesions in the external capsule but not in the temporal pole. The CADASIL and CADASIL scale-J scores of the three patients were 6, 17, 7 (mean, 10.0) and 13, 20, 10 (mean, 14.3), respectively.Conclusion: Among patients hospitalized for lacunar infarctions, the p.R75P prevalence may be higher than previously estimated. The NOTCH3 p.R75P mutation may be underdiagnosed in patients with early-onset lacunar infarctions due to the atypical clinical and neuroimaging features of CADASIL. Early-onset, presence of family history of stroke, external capsule lesions, and absence of hypertension may help predict underlying NOTCH3 mutations despite no temporal white matter lesions.

Genotype-phenotype correlations and effect of mutation location in Japanese CADASIL patients.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary cerebral small vessel disease caused by NOTCH3, and characterized by recurrent cerebral ischemic events without vascular risk factors, mood disturbance, and dementia. MRI testing shows cerebral white matter hyperintensities, especially in the external capsule and temporal pole. Typical mutations are cysteine-related missense ones located in one of 34 EGF-like repeats (EGFr) in the NOTCH3 receptor. To identify genotype-phenotype correlations, 179 Japanese CADASIL probands were recruited. Of the 68 mutations identified, p.Cys388Arg, p.Cys435Phe, p.Gly481Cys, p.Cys743Tyr, and p.Cys1009Phe were novel ones. The genotype-phenotype correlation was analyzed based on the three most common mutations: p.Arg75Pro, p.Arg141Cys, and p.Arg182Cys. p.Arg141Cys showed typical CADASIL phenotypes, whereas p.Arg75Pro showed mild and atypical phenotypes, a low frequency of stroke/TIA, high frequency of hypertension, and low frequency of temporal pole lesions. p.Arg182Cys showed various initial symptoms other than stroke/TIA. Subsequently, we analyzed the effect of the mutation location on the age at onset of stroke/TIA. We found that mutations in EGFr 1-6 excluding the cysteine-sparing mutation p.Arg75Pro were significantly correlated with a younger age at onset of stroke/TIA compared with those in EGFr 7-34. This was in agreement with a recent European report, suggesting that the effect of the mutation location is a consensus finding in CADASIL worldwide.

Roles of Notch Glycoslation in Signaling

Notch receptors are single pass transmembrane glycoproteins with an extracellular domain (ECD) comprised of many epidermal growth factor‐like (EGF) repeats. Specific Ser and Thr residues in numerous EGF repeats have an attached O‐glycan initiated by fucose, glucose, or GlcNAc, and elongated with 1–3 more sugars. Notch receptors signal in response to the binding of Notch ligands to Notch ECD. Notch signaling regulates cell fate decisions in many organs during development and differentiation, and its dysregulation leads to developmental disorders and cancer. A point mutation that precludes the addition of a single O‐fucose glycan to EGF12 in the NOTCH1 ligand binding domain alters Notch signaling differently depending on genetic background. In a mixed genetic background a mild effect on T and B cell development is observed, whereas in the C57BL/6J background, lack of the EGF12 O‐fucose glycan is embryonic lethal. Lunatic (LFNG), Manic (MFNG) and Radical (RFNG) Fringe are GlcNAc transferases that modify Notch EGF repeats which already carry a fucose. We are investigating roles for each Fringe in T and B cell development using mice lacking two or three Fng genes. We are also identifying individual roles for Fng genes using a co‐culture assay containing OP9 stromal cells expressing one of the four Notch ligands, DLL1, DLL4, JAG1 or JAG2 and bone marrow hemopoietic cells from mice expressing all Fng genes, no Fng genes (tKO) or a single Fng gene. The combined results show that the O‐fucose glycans on NOTCH1 are key regulators of the strength of NOTCH1 signaling induced by canonical Notch ligands.Support or Funding InformationNational Institutes of Health grant RO1 GM106417National Institutes of Health grant PO1 CA13330

How does GDP‐fucose get into the Endoplasmic Reticulum for O‐fucosylation of EGF Repeats and TSRs?

Fucose is a common monosaccharide found in glycan chains of all mammalian cells. Fucose is transferred from GDP‐fucose to glycoproteins by fucosyltransferases, resulting in terminal fucosylation of N‐linked glycans or mucin‐type O‐glycans, or direct modification of serine or threonine residues by Protein O‐fucosyltransferases (POFUT) 1 or 2. Defects in fucosylation can lead to developmental disorders or cancer. In particular, POFUT1 adds O‐fucose to Epidermal Growth Factor‐like (EGF) repeats of Notch receptors, while POFUT2 adds O‐fucose to Thrombospondin type 1 Repeats (TSRs) found in multiple cell‐surface and secreted proteins, including the A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) family of extracellular proteases. Elimination of Pofut1 or Pofut2 results in early embryonic lethality in mice, indicating these two fucosylation pathways play essential roles in mouse development. Both POFUT1 and POFUT2 are located in the endoplasmic reticulum (ER), while fucosyltransferases involved in terminal fucosyaltion are localized in the trans‐Golgi. GDP‐fucose is synthesized in the cytoplasm either through a de novo or salvage pathway, and the GDP‐fucose is then transported into the lumen of the ER or Golgi for use by the fucosyltransferases. Currently two GDP‐fucose transporters, SLC35C1 and SLC35C2, have been identified, both of which are localized to the Golgi. It is unknown how GDP‐fucose is transported into the ER where Notch receptors and ADAMTS proteins are fucosylated. In order to uncover where the GDP‐fucose for POFUT1 and POFUT2 comes from, we have generated CRISPR‐Cas9 mutant cell lines in which fucosylation is impaired in two ways: 1. Inactivating GDP‐fucose biosynthetic enzymes from the salvage or de novo pathways; 2. Inactivating the GDP‐fucose transporters. HEK293T cells have proven to be an excellent model for fucosylation studies. All the fucose biosynthesis genes are normally expressed, and impaired fucosylation can be expected in knockout mutant cell lines. Using CRISPR‐Cas9 technology, the following genes have been knocked out: GMDS and TSTA3 of the de novo pathway, FUK and GFPP of the salvage pathway, and the GDP‐fucose transporters SLC35C1 and SLC35C2. O‐Fucosylation of EGF repeats and TSRs and terminal fucosylation of N‐linked glycans is being analyzed using Mass Spectrometry and lectin binding. Notch function is also being evaluated. These results should reveal whether a preferred biosynthetic pathway or transporter for different forms of fucosylation exists.Support or Funding InformationSupported by NIH grant GM061126.

Contribution of extracellular O-GlcNAc to the stability of folded epidermal growth factor-like domains and Notch1 trafficking

The Notch signaling pathway is highly conserved and essential in animal development and tissue homeostasis. Regulation of Notch signaling is a crucial process for human health. Ligands initiate a signal cascade by binding to Notch receptors expressed on the neighboring cell. Notch receptors interact with ligands through their epidermal growth factor-like repeats (EGF repeats). Most EGF repeats are modified by O-glycosylation with residues, such as O-linked N-acetylglucosamine (O-GlcNAc), O-fucose, and O-glucose. A recent study revealed the distinct roles of these O-glycans in ligand binding, processing, and trafficking of Notch receptors. In particular, O-GlcNAc glycans are essential for Delta-like (DLL) ligand-mediated Notch signaling. In this study, we showed that O-GlcNAc promotes Notch1 trafficking to the cell surfaces under the condition that O-fucose and O-glucose are removed from consecutive EGF repeats of Notch1. Through in vitro experiments, we showed that O-GlcNAc mediates the stability of EGF domains in the same manner as O-fucose and O-glucose. Thus, O-GlcNAc on EGF domains possesses a shared function in the stability of EGF domains and Notch1 trafficking.

NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease

The small-vessel disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) arises from mutations in the human gene encoding NOTCH3 and results in vascular smooth muscle cell degeneration, stroke, and dementia. However, the structural changes in NOTCH3 involved in CADASIL etiology are unclear. Here, we discovered site-specific fragmentation of NOTCH3 protein in pathologically affected vessels of human CADASIL-affected brains. EM-based experiments to pinpoint NOTCH3 localization in these brains indicated accumulation of NOTCH3 fragmentation products in the basement membrane, collagen fibers, and granular osmiophilic material within the cerebrovasculature. Using antibodies generated against a disease-linked neo-epitope found in degenerating vascular medium of CADASIL brains, we mapped the site of fragmentation to the NOTCH3 N terminus at the peptide bond joining Asp80 and Pro81 Cleavage at this site was predicted to separate the first epidermal growth factor (EGF)-like domain from the remainder of the protein. We found that the cleavage product from this fragmentation event is released into the conditioned medium of cells expressing recombinant NOTCH3 fragments. Mutagenesis of Pro81 abolished the fragmentation, and low pH and reducing conditions enhanced NOTCH3 proteolysis. Furthermore, substitution of multiple cysteine residues of the NOTCH3 N terminus activated proteolytic release of the first EGF-like repeat, suggesting that the elimination of multiple disulfide bonds in NOTCH3 accelerates its fragmentation. These characteristics link the signature molecular genetic alterations present in individuals with CADASIL to a post-translational protein alteration in degenerating brain arteries. The cellular consequences of these pathological NOTCH3 fragments are an important area for future investigation.

Effects of Notch glycosylation on health and diseases.

Notch signaling is an evolutionarily conserved signaling pathway and is essential for cell-fate specification in metazoans. Dysregulation of Notch signaling results in various human diseases, including cardiovascular defects and cancer. In 2000, Fringe, a known regulator of Notch signaling, was discovered as a Notch-modifying glycosyltransferase. Since then, glycosylation-a post-translational modification involving literal sugars-on the Notch extracellular domain has been noted as a critical mechanism for the regulation of Notch signaling. Additionally, the presence of diverse O-glycans decorating Notch receptors has been revealed in the extracellular domain epidermal growth factor-like (EGF) repeats. Here, we concisely summarize the recent studies in the human diseases associated with aberrant Notch glycosylation.

Research advances of EDIL3 in tumor

Epidermal growth factor (EGF)-like repeat and discoidin Ⅰ-like domain 3 (EDIL3) is a extracellular matrix protein that produced and secreted by endothelial cells and plays important roles in embryonic development, angiogenesis, and limit inflammation. Several recent studies indicate that EDIL3 is aberrantly expressed in pancreatic cancer, liver cancer, colorectal cancer, lung cancer and breast cancer, which is associated with cell proliferation, apoptosis, invasion, metastasis, and angiogenesis. EDIL3 is widely participated in the regulating tumor initiation, progression and metastasis, and its abnormal expression is expected to provide a new method for diagnosis and therapy of cancers. Key words: Neoplasms; Angiogenesis; Apoptosis; EGF-like repeat and discoidin Ⅰ-like domain 3

A novel cysteine-sparing G73A mutation of NOTCH3 in a Chinese CADASIL family.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic disease leading to stroke and vascular dementia. CADASIL is an inherited small blood vessel disease caused by mutations in the gene encoding the neurogenic locus notch homolog protein 3 (NOTCH3). NOTCH3 is large type I membrane receptor mainly expressed in vascular smooth muscle cells and pericytes. Most identified mutations result in insert or deletion of a cysteine residue within the EGF-like repeats. To date, some cases with a cysteine-sparing mutant have been described. Genetic analysis revealed a novel mutation in NOTCH3 in a CADASIL family. Molecular analysis revealed its potential pathogenic mechanism in causing CADASIL. In this paper, we present a Chinese family with a novel cysteine-sparing mutation in exon 3 (c.218G>C, p.G73A) of the NOTCH3 gene. Family carriers of the same mutation presented with symptoms and imaging abnormalities characteristic of CADASIL. The location of glycine 73 in between C5-C6 disulfide bond of EGF-like domain 1 shows high conservation from humans to zebra fish. It has previously been suggested that the aggregate-prone property of mutant NOTCH3 contributes to a cytotoxic effect in the pathogenic mechanism underlying CADASIL. Here, we investigated the pathogenic mechanism of the new mutation in vitro using HEK293 cells transfected with either a wild-type (WT) or c.218G>C (p.G73A) NOTCH3ECD plasmids, and we found p.G73A NOTCH3ECD was more prone to form aggregation and resistant to degradation. Moreover, the p.G73A NOTCH3ECD compromised cell viability by promoting apoptosis. Two known CADASIL mutants R133C and R75P showed similar results with G73A mutants. Our study here identified G73A as a new mutation in NOTCH3 to cause CADASIL and revealed that the G73A mutation and two known mutants R75P and R133C decreased NOTCH3 protein turnover and induced cell death.

Glycosylation of Specific Notch EGF Repeats by O-Fut1 and Fringe Regulates Notch Signaling in Drosophila

SUMMARYFringe glycosyltransferases differentially modulate the binding of Notch receptors to Delta/DLL versus Serrate/Jagged ligands by adding GlcNAc to O-linked fucose on Notch epidermal growth factor-like (EGF) repeats. Although Notch has 22 O-fucosylation sites, the biologically relevant sites affecting Notch activity during animal development in vivo in the presence or absence of Fringe are not known. Using a variety of assays, we find important roles in Drosophila Notch signaling for GlcNAc-fucose-O glycans on three sites: EGF8, EGF9, and EGF12. O-Fucose monosaccharide on EGF12 (in the absence of Fringe) is essential for Delta-mediated lateral inhibition in embryos. However, wing vein development depends on the addition of GlcNAc to EGF8 and EGF12 by Fringe, with a minor contribution from EGF9. Fringe modifications of EGF8 and EGF12 together prevent Notch from cis-inhibiting Serrate, thereby promoting normal wing margin formation. Our work shows the combinatorial and context-dependent roles of GlcNAc-fucose-O glycans on these sites in Drosophila Notch-ligand interactions.

Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy

Membranous nephropathy is characterized by deposition of immune complexes along the glomerular basement membrane. PLA2R and THSD7A are target antigens in 70% and 1-5% of primary membranous nephropathy cases, respectively. In the remaining cases, the target antigen is unknown. Here, laser microdissection of glomeruli followed by mass spectrometry was used to identify novel antigen(s) in PLA2R-negative membranous nephropathy. An initial pilot mass spectrometry study in 35 cases of PLA2R-negative membranous nephropathy showed high spectral counts for neural tissue encoding protein with EGF-like repeats, NELL-1, in six cases. Mass spectrometry failed to detect NELL-1 in 23 PLA2R-associated membranous nephropathy and 88 controls. NELL-1 was localized by immunohistochemistry, which showed bright granular glomerular basement membrane staining for NELL-1 in all six cases. Next, an additional 23 NELL-1 positive cases of membranous nephropathy were identified by immunohistochemistry in a discovery cohort of 91 PLA2R-negative membranous nephropathy cases, 14 were confirmed by mass spectrometry. Thus, 29 of 126 PLA2R-negative cases were positive for NELL-1. PLA2R-associated membranous nephropathy and controls stained negative for NELL-1. We then identified five NELL-1 positive cases of membranous nephropathy out of 84 PLA2R and THSD7A-negative cases in two validation cohorts from France and Belgium. By confocal microscopy, both IgG and NELL-1 co-localized to the glomerular basement membrane. Western blot analysis showed reactivity to NELL-1 in five available sera, but no reactivity in control sera. Clinical and biopsy findings of NELL-1 positive membranous nephropathy showed features of primary membranous nephropathy. Thus, a subset of membranous nephropathy is associated with accumulation and co-localization of NELL-1 and IgG along the glomerular basement membrane, and with anti-NELL-1 antibodies in the serum. Hence, NELL-1 defines a distinct type of primary membranous nephropathy.

A modifier in the 129S2/SvPasCrl genome is responsible for the viability of Notch1[12f/12f] mice

BackgroundMouse NOTCH1 carries a highly conserved O-fucose glycan at Thr466 in epidermal growth factor-like repeat 12 (EGF12) of the extracellular domain. O-Fucose at this site has been shown by X-ray crystallography to be recognized by both DLL4 and JAG1 Notch ligands. We previously showed that a Notch1 Thr466Ala mutant exhibits very little ligand-induced NOTCH1 signaling in a reporter assay, whereas a Thr466Ser mutation enables the transfer of O-fucose and reverts the NOTCH1 signaling defect. We subsequently generated a mutant mouse with the Thr466Ala mutation termed Notch1[12f](Notch1tm2Pst). Surprisingly, homozygous Notch1[12f/12f] mutants on a mixed background were viable and fertile.ResultsWe now report that after backcrossing to C57BL/6 J mice for 11–15 generations, few homozygous Notch1[12f/12f] embryos were born. Timed mating showed that embryonic lethality occurred by embryonic day (E) ~E11.5, somewhat delayed compared to mice lacking Notch1 or Pofut1 (the O-fucosyltransferase that adds O-fucose to Notch receptors), which die at ~E9.5. The phenotype of C57BL/6 J Notch1[12f/12f] embryos was milder than mutants affected by loss of a canonical Notch pathway member, but disorganized vasculogenesis in the yolk sac, delayed somitogenesis and development were characteristic. In situ hybridization of Notch target genes Uncx4.1 and Dll3 or western blot analysis of NOTCH1 cleavage did not reveal significant differences at E9.5. However, qRT-PCR of head cDNA showed increased expression of Dll3, Uncx4.1 and Notch1 in E9.5 Notch1[12f/12f] embryos. Sequencing of cDNA from Notch1[12f/12f] embryo heads and Southern analysis showed that the Notch1[12f] locus was intact following backcrossing. We therefore looked for evidence of modifying gene(s) by crossing C57BL/6 J Notch1 [12f/+] mice to 129S2/SvPasCrl mice. Intercrosses of the F1 progeny gave viable F2 Notch1[12f/12f] mice.ConclusionWe conclude that the 129S2/SvPasCrl genome contains a dominant modifying gene that rescues the functions of NOTCH1[12f] in signaling. Identification of the modifying gene has the potential to illuminate novel factor(s) that promote Notch signaling when an O-fucose glycan is absent from EGF12 of NOTCH1.