EGF-like Repeats Research Articles

Abstract 744: ADCT-701, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) targeting DLK1-expressing tumors

Abstract Delta-like 1 homolog protein (DLK-1) is an EGF-like membrane bound protein consisting of six tandem EGF-like repeats, a juxtamembrane region with a TACE (ADAM17)-mediated cleavage site, a transmembrane domain, and a short intracellular tail. DLK-1 is strongly expressed during fetal development, while its expression is highly restricted in adults. Conversely, DLK-1 gets re-expressed in several tumors, such as neuroblastoma, hepatocellular carcinoma (HCC), rhabdomyosarcoma, small cell lung cancer, myelodysplastic syndrome and acute myeloid leukemia. Interestingly, in HCC DLK-1 has been shown to be a marker of cancer stem cells, a subpopulation of cells responsible for tumor initiation, growth, metastasis, and recurrence. Altogether, DLK-1 represents an attractive target for an antibody-drug conjugate (ADC) approach based on its selective expression in a wide range of malignancies and restricted expression in healthy organs, as well as its expression on HCC cancer stem cells. ADCT-701 is an ADC composed of a humanized IgG1 antibody against human DLK-1, site-specifically conjugated using GlycoconnectTM technology to PL1601, which contains a valine-alanine cleavable linker and the PBD dimer cytotoxin SG3199. The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-701 in human cancer cell lines and patient-derived xenograft (PDX) models and to determine its safety and tolerability in the rat. In vitro, ADCT-701 demonstrated potent cytotoxicity in a panel of human cancer cell lines of different origin and levels of DLK-1, while its potency was strongly reduced in DLK-1-negative cell lines. In vivo, ADCT-701 showed potent anti-tumor activity in the DLK1-expressing neuroblastoma-derived SK-N-FI xenograft in which a single dose of ADCT-701 at 0.5 or 1 mg/kg showed dose-dependent anti-tumor activity compared to the vehicle- and isotype control ADC-treated mice. At 1 mg/kg, ADCT-701 resulted in 1/9 partial responders (PR) and 4/9 complete responder (CR), one of which was tumor-frees survivors (TFS) at the end of the study on day 60. Moreover, ADCT-701 showed dose-dependent anti-tumor activity in a HCC PDX model when tested as a single dose at 0.1, 0.3 or 1 mg/kg. At the highest dose tested, ADCT-701 resulted in 3/8 PR and 2/8 CR, while none of the mice treated with the vehicle or the isotype-control ADC (1 mg/kg, single dose) showed any activity. ADCT-701 was stable, well tolerated and showed a favorable pharmacokinetic profile in the rat with a half-life of 9 days and a maximum tolerated dose of at least 5 mg/kg. In conclusion, ADCT-701 demonstrated potent and specific in vitro and in vivo anti-tumor activity in DLK-1-expressing cancer-derived models and it was stable and well tolerated in the rat, warranting further development of this ADC into the clinic. Citation Format: Francesca Zammarchi, Karin Havenith, Simon Chivers, Paul W. Hogg, Charlie Britten, Sandamali Dissanayake, Peter Tyrer, Francois Bertelli, Ian Hutchinson, Luke Masterson, Phil Howard, John A. Hartley, Patrick H. van Berkel. ADCT-701, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) targeting DLK1-expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 744.

Protein O-Glucosyltransferase 1 Expression Influences Formation of Differentiated Myotubes in C2C12 Cell Line.

The protein O-glucosyltransferase 1 (Poglut1) links O-glucose to epidermal growth factor-like repeats harboring the C1XSX(P/A)C2 consensus sequence. Poglut1 is a ubiquitous endoplasmic reticulum-resident protein largely found in metazoans, but only about 50 proteins possess this consensus sequence. Among them, Notch receptors have multiple O-glucosylation sites and their activation depends on this status. In adult skeletal muscle, Notch signaling contributes to the maintenance of satellite cell (SC) quiescence and the proliferation of myoblasts after SC activation. To address the role of Poglut1 in myogenesis, we created two stable C2C12 cell lines where Poglut1 was downexpressed by 42% and 81%, and assessed their ability to differentiate. We showed that Poglut1 knockdown reduced Notch signaling and largely affected the key regulators of myogenic differentiation, with PAX7 decrease and MYOD increase. This perturbed Pax7/MyoD expression balance led to a premature myogenic differentiation and an increase in myotube size, accentuated in case of strong Poglut1 downexpression. Differences observed between myotubes of the two Poglut1 knockdown cell lines could reflect dissimilar fusion defects. We concluded that Poglut1 contributes to myogenesis by regulating Notch signaling and defining, directly or indirectly, the proportion of cells that commit differentiation.

Variant in human POFUT1 reduces enzymatic activity and likely causes a recessive microcephaly, global developmental delay with cardiac and vascular features

Protein O-fucosyltransferase-1 (POFUT1) adds O-fucose monosaccharides to epidermal growth factor-like (EGF) repeats found on approximately 100 mammalian proteins, including Notch receptors. Haploinsufficiency of POFUT1 has been linked to adult-onset Dowling Degos Disease (DDD) with hyperpigmentation defects. Homozygous deletion of mouse Pofut1 results in embryonic lethality with severe Notch-like phenotypes including defects in somitogenesis, cardiogenesis, vasculogenesis and neurogenesis, but the extent to which POFUT1 is required for normal human development is not yet understood. Here we report a patient with a congenital syndrome consisting of severe global developmental delay, microcephaly, heart defects, failure to thrive and liver disease with a previously unreported homozygous NM_015352.1: c.485C>T variant (p.Ser162Leu) in POFUT1 detected by exome sequencing. Both parents are heterozygotes and neither manifests any signs of DDD. No other detected variant explained the phenotype. This variant eliminated a conserved N-glycosylation sequon at Asn160 in POFUT1 and profoundly decreased POFUT1 activity in patient fibroblasts compared to control fibroblasts. Purified p.Ser162Leu mutant protein also showed much lower POFUT1 activity with a lower affinity for EGF acceptor substrate than wild type POFUT1. Eliminating the N-glycan sequon by replacing Asn160 with Gln had little effect on POFUT1 activity, suggesting that loss of the glycan is not responsible for the defect. Furthermore, the p.Ser162Leu mutant showed weaker ability to rescue Notch activity in cell-based assays. These results suggest that this N-glycan of POFUT1 is not required for its proper enzymatic function, and that the p.Ser162Leu mutation of POFUT1 likely causes global developmental delay, microcephaly with vascular and cardiac defects.

Computational Study of PCSK9-EGFA Complex with Effective Polarizable Bond Force Field.

Inhibiting of Proprotein Convertase Subtilisin/Kexin-type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR) binding is an effective way for reducing Low Density Lipoprotein cholesterol (LDL-C). Understanding the interaction between PCSK9 and LDLR is useful for PCSK9 inhibitor design. In this work, MD simulations with the standard (non-polarizable) AMBER force field and effective polarizable bond (EPB) force field were performed for wild type and four mutants of PCSK9 and EGFA (Epidermal Growth Factor-like repeat A) domain of LDLR complexes. These four mutants are gain-of-function mutants. The analysis of hydrogen bond dynamics and the relative binding free energy indicates that EPB is more reliable in simulating protein dynamics and predicting relative binding affinity. Structures sampled from MD simulations with the standard AMBER force field deviate too far away from crystal structures. Many important interaction components between of PCSK9 and EGFA no longer exist in the simulation with the Amber force field. For comparison, simulation using EPB force field gives more stable structures as shown by hydrogen bond analysis and produced relative binding free energies that are consistent with experimental results. Our study suggests that inclusion of polarization effects in MD simulation is important for studying the protein-protein interaction.

The extracellular matrix protein Edil3 stimulates osteoblast differentiation through the integrin α5β1/ERK/Runx2 pathway.

Epidermal growth factor-like repeats and discoidin I-like domain 3 (Edil3) is an extracellular matrix protein containing an Arg-Gly-Asp (RGD) motif that binds integrin. Recently, Edil3 has been implicated in various biological processes, including angiogenesis and cellular differentiation. It can inhibit inflammatory bone destruction. The objective of this study was to explore the role of Edil3 in osteoblast differentiation and its underlying molecular mechanisms. In wild-type mice, high expression levels of Edil3 mRNA were observed in isolated calvaria and tibia/femur bones. Immunohistochemical analysis showed that Edil3 protein was localized along periosteum and calcified regions surrounding bone tissues. When murine calvaria-derived MC3T3-E1 cells were cultured in osteogenic medium containing 50 μg/ml ascorbic acid and 5 mM β-glycerophosphate, Edil3 mRNA and protein expression levels were increased. Treatment with Edil3 protein in growth media increased expression levels of alkaline phosphatase and osteocalcin gene and phosphorylation level of extracellular signal-regulated kinase (ERK). Edil3 treatment with osteogenic medium induced mineralization. Treatment with a neutralizing antibody against α5β1 and MEK inhibitor U0126 inhibited Edil3-enhanced osteogenic marker gene expression and mineral deposition. Edil3 increased protein expression levels of transcription factor runt-related transcription factor2 (Runx2). Edil3-induced Runx2 protein expression was suppressed by pretreatment with U0126. Taken together, these results suggest that Edil3 may stimulate osteoblast differentiation and matrix mineralization by increasing expression of Runx2 through α5β1 integrin /ERK pathway.

Inhibition of Delta-induced Notch signaling using fucose analogs.

Notch is a cell-surface receptor that controls cell fate decisions and is regulated by O-glycans attached to epidermal growth factor-like (EGF) repeats in its extracellular domain. Protein O-fucosyltransferase 1 (Pofut1) modifies EGF repeats with O-fucose and is essential for Notch signaling. Constitutive activation of Notch signaling has been associated with a variety of human malignancies. Therefore, tools for inhibiting Notch activity are being developed as cancer therapeutics. Towards this end, we screened L-fucose analogs for their effects on Notch signaling. Two analogs, 6-alkynyl and 6-alkenyl fucose, were substrates of Pofut1 and were incorporated directly into Notch EGF repeats in cells. Both analogs were potent inhibitors of binding to and activation of Notch1 by Notch ligands Dll1 and Dll4, but not by Jag1. Mutagenesis and modeling studies suggest that incorporation of the analogs into EGF8 of Notch1 markedly reduces the ability of Delta ligands to bind and activate Notch1.

Molecular Chaperone Receptors.

Extracellular heat shock proteins (HSP) play important roles in cell signaling and immunity. Many of these effects are mediated by surface receptors expressed on a wide range of cell types. We have investigated the nature of such proteins by cloning candidate receptors into cells (CHO-K1) with the rare property of being null for HSP binding. Using this approach we have discovered that Hsp70 binds avidly to at least two classes of receptors including: (1) c-type lectin receptors (CLR) and (2) scavenger receptors (SR). However, the structural nature of the receptor-ligand interactions is not clear at this time. Hsp70 can bind to LOX-1 (a member of both the CLR and SR), with the c-type lectin binding domain (CTLD) as well as the SR family members SREC-I and FEEL-1/CLEVER-1/STABILIN-1, which by contrast have arrays of EGF-like repeats in their extracellular domains. In this chapter we will discuss: (1) methods for discovery of HSP receptors, (2) approaches to the study of individual receptors in cells that contain multiple such receptors, and (3) methods for investigating HSP receptor function in vivo.

Evolocumab

Evolocumab

Identification and characterization of a novel Sso7d scaffold-based binder against Notch1

Notch signaling has important functions in regulating cell growth and development, misregulation of which has been implicated in various cancers. Monoclonal antibodies (mAbs) targeting Notch protein activity have already moved into clinical trials. However due to the limitations associated with cost and productivity of mAbs, there has been a surge in the development of complementary approaches that are based on non-antibody scaffolds. Non-antibody scaffolds are small proteins that are stable and can be engineered to develop high-affinity binders against specific targets of interest. Here we describe the isolation and characterization of a novel Notch1-binding protein, N9, obtained by screening of a combinatorial library based on the ultra-stable Sso7d scaffold. N9 targets the extracellular EGF-like repeats (ELR) 11–13 in Notch1, and therefore serves as a competitive inhibitor for Notch ligands to decrease expression of Notch target genes. We demonstrate that N9 recognizes surface expression of Notch1 on the plasma membrane and binds preferentially to cell lines misexpressing Notch1. Although N9 was selected against Notch1, we also observe cross-reactivity against other Notch receptors, including Notch2/3. Finally, we demonstrate that N9 inhibits proliferation and generation of tumorspheres in Notch expressing cancer cell lines, suggesting its potential as a therapeutic agent in Notch-associated malignancies.

O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking

Glycosylation in the endoplasmic reticulum (ER) is closely associated with protein folding and quality control. We recently described a non-canonical ER quality control mechanism for folding of thrombospondin type 1 repeats by protein O-fucosyltransferase 2 (POFUT2). Epidermal growth factor-like (EGF) repeats are also small cysteine-rich protein motifs that can be O-glycosylated by several ER-localized enzymes, including protein O-glucosyltransferase 1 (POGLUT1) and POFUT1. Both POGLUT1 and POFUT1 modify the Notch receptor on multiple EGF repeats and are essential for full Notch function. The fact that POGLUT1 and POFUT1 can distinguish between folded and unfolded EGF repeats raised the possibility that they participate in a quality control pathway for folding of EGF repeats in proteins such as Notch. Here, we demonstrate that cell-surface expression of endogenous Notch1 in HEK293T cells is dependent on the presence of POGLUT1 and POFUT1 in an additive manner. In vitro unfolding assays reveal that addition of O-glucose or O-fucose stabilizes a single EGF repeat and that addition of both O-glucose and O-fucose enhances stability in an additive manner. Finally, we solved the crystal structure of a single EGF repeat covalently modified by a full O-glucose trisaccharide at 2.2 Å resolution. The structure reveals that the glycan fills up a surface groove of the EGF with multiple contacts with the protein, providing a chemical basis for the stabilizing effects of the glycans. Taken together, this work suggests that O-fucose and O-glucose glycans cooperatively stabilize individual EGF repeats through intramolecular interactions, thereby regulating Notch trafficking in cells.

Generation of an induced pluripotent stem cell line (CSCRMi001-A) from a patient with a new type of limb-girdle muscular dystrophy (LGMD) due to a missense mutation in POGLUT1 (Rumi)

Recently, a new type of limb-girdle muscular dystrophy (LGMD type 2Z) has been identified due to a missense mutation in POGLUT1 (protein O-glucosyltransferase-Rumi), an enzyme capable of adding glucose to a distinct serine residue of epidermal growth factor-like repeats containing a C-X-S-X-(P/A)-C consensus sequence such as Notch receptors. Affected patients demonstrate reduced Notch signaling, decreased muscle stem cell pool and hypoglycosylation of α-dystroglycan, leading to LGMD phenotype. Here we report the generation and characterization of an iPSC line (CSCRMi001-A) from a LGMD-2Z patient with missense mutation in POGLUT1 which can be used for in vitro disease modeling.

Prognostic Significance of EDIL3 Expression and Correlation with Mesenchymal Phenotype and Microvessel Density in Lung Adenocarcinoma

We examined the prognostic significance of Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3) expression and its correlations with mesenchymal phenotype and microvessel density in non-small cell lung carcinoma (NSCLC). A total of 268 NSCLC specimens were evaluated retrospectively by immunohistochemical staining for EDIL3, EMT markers (e-cadherin, β-catenin, and vimentin), and CD31 to measure microvessel density. EDIL3, e-cadherin, β-catenin, and vimentin were expressed in 16%, 22.8%, 3.7%, and 10.1% of the specimens, respectively. The mRNA level of EDIL3 in tumor was correlated with the level of EDIL3 protein expression using immunohistochemistry. In lung adenocarcinoma patients, EDIL3 expression was significantly correlated with low e-cadherin expression, high vimentin expression, and increased microvessel density (P < 0.001, P = 0.001, and P = 0.023, respectively). In lung squamous cell carcinoma patients, EDIL3 expression was significantly correlated with low e-cadherin expression and high vimentin expression (P = 0.021 and P = 0.002, respectively). In lung adenocarcinoma patients, EDIL3 was an independent prognostic factor for overall survival in a multivariate analysis (hazard ratio: 2.552, P = 0.004). EDIL3 is significantly correlated with mesenchymal phenotype, angiogenesis, and tumor progression in lung adenocarcinoma.

The emerging role of NPNT in tissue injury repair and bone homeostasis.

Nephronectin (NPNT), a highly conserved extracellular matrix protein, plays an important role in regulating cell adhesion, differentiation, spreading, and survival. NPNT protein belongs to the epidermal growth factor (EGF)-like superfamily and exhibits several common structural determinants; including EGF-like repeat domains, MAM domain (Meprin, A5 Protein, and Receptor Protein-Tyrosine Phosphatase µ), RGD motif (Arg-Gly-Asp) and a coiled-coil domain. It regulates integrins-mediated signaling pathways via the interaction of its RGD motif with integrin α8β1. Recent studies revealed that NPNT is involved in kidney development, renal injury repair, atrioventricular canal differentiation, pulmonary function, and muscle cell niche maintenance. Moreover, NPNT regulates osteoblast differentiation and mineralization, as well as osteogenic angiogenesis. Altered expression of NPNT has been linked with the progression of certain types of cancers, such as spontaneous breast tumor metastasis and malignant melanoma. Interestingly, NPNT gene expression can be regulated by a range of external factors such as tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), oncostatin M (OSM), bone morphogenic protein 2 (BMP2), Wnt3a, Vitamin D3 , and microRNA-378 (miR378). Further understanding the cellular and molecular mechanisms by which NPNT regulates tissue homeostasis in an organ-specific manner is critical in exploring NPNT as a therapeutic target for tissue regeneration and tissue engineering.

EOGT and O-GlcNAc on secreted and membrane proteins.

Here, we describe a recently discovered O-GlcNAc transferase termed EOGT for EGF domain-specific O-GlcNAc transferase. EOGT transfers GlcNAc (N-acetylglucosamine) to Ser or Thr in secreted and membrane proteins that contain one or more epidermal growth factor-like repeats with a specific consensus sequence. Thus, EOGT is distinct from OGT, the O-GlcNAc transferase, that transfers GlcNAc to Ser/Thr in proteins of the cytoplasm or nucleus. EOGT and OGT are in separate cellular compartments and have mostly distinct substrates, although both can act on cytoplasmic (OGT) and lumenal (EOGT) domains of transmembrane proteins. The present review will describe known substrates of EOGT and biological roles for EOGT in Drosophila and humans. Mutations in EOGT that give rise to Adams-Oliver Syndrome in humans will also be discussed.

O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals.

The glycosyltransferase EOGT transfers O-GlcNAc to a consensus site in epidermal growth factor-like (EGF) repeats of a limited number of secreted and membrane proteins, including Notch receptors. In EOGT-deficient cells, the binding of DLL1 and DLL4, but not JAG1, canonical Notch ligands was reduced, and ligand-induced Notch signaling was impaired. Mutagenesis of O-GlcNAc sites on NOTCH1 also resulted in decreased binding of DLL4. EOGT functions were investigated in retinal angiogenesis that depends on Notch signaling. Global or endothelial cell-specific deletion of Eogt resulted in defective retinal angiogenesis, with a mild phenotype similar to that caused by reduced Notch signaling in retina. Combined deficiency of different Notch1 mutant alleles exacerbated the abnormalities in Eogt-/- retina, and Notch target gene expression was decreased in Eogt-/-endothelial cells. Thus, O-GlcNAc on EGF repeats of Notch receptors mediates ligand-induced Notch signaling required in endothelial cells for optimal vascular development.

Does Evolocumab, as a PCSK9 Inhibitor, Ameliorate the Lipid Profile in Familial Hypercholesterolemia Patients? A Meta-Analysis of Randomized Controlled Trials.

Proprotein convertase subtilisin-kexin type 9 (PCSK9) is a member of regulatory serine proteases which is mostly expressed in liver. In the physiological condition, LDL-C binds to LDL receptors (LDLRs) and via endocytosis, LDLRs are degraded. PCSK9 binds to the epidermal growth factor-like repeat A (EGFA) domain of extracellular LDLRs, and then physiological recycling of LDLRs from surface of liver is cancelled, resulting in elevation of circulating LDL-C in plasma. To evaluate whether evolucomab, as PCSK9 inhibitor monoclonal antibody, ameliorates lipid profile in familial hypercholesterolemia (FH) patients, this meta-analysis has been conducted. PubMed, Web of Science (ISI) and Scopus databases were searched for studies which had investigated the efficacy of evolucomab. Types of outcome investigated were percentage changes from baseline of the lipid profile. Our meta-analysis shows that evolucomab at the dosage of 420 mg monthly could decrease LDL-C by 54.71%, TC by 35.08%, VLDL-C by 28.37 %, ratio of TC to HDL-C by 39.14 %, triglycerides by 12.11 %, and increased HDL-C by 6.06% from baseline compared to placebo at the end of study in FH patients. Our findings indicate that evolocumab could be a hopeful agent for challenging patients, such as statin intolerance or patients who fail to attain the target goal of LDL-C despite consumption of maximum doses of statins.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

Tooth development is initiated by epithelial-mesenchymal interactions via basement membrane (BM) and growth factors. In the present study, we found that nephronectin (Npnt), a component of the BM, is highly expressed in the developing tooth. Npnt localizes in the BM on the buccal side of the tooth germ and shows an expression pattern opposite that of the dental epithelial stem cell marker Sox2. To identify the roles of Npnt during tooth development, we performed knockdown and overexpression experiments using ex vivo organ and dental epithelial cell cultures. Our findings showed that loss of Npnt induced ectopic Sox2-positive cells and reduced tooth germ size. Over expression of Npnt showed increased proliferation, whereas the number of Sox2-positive cells was decreased in dental epithelial cells. Npnt contains 5 EGF-like repeat domains, as well as an RGD sequence and MAM domain. We found that the EGF-like repeats are critical for Sox2 expression and cell proliferation. Furthermore, Npnt activated the EGF receptor (EGFR) via the EGF-like repeat domains and induced the PI3K-Akt signaling pathway. Our results indicate that Npnt plays a critical scaffold role in dental epithelial stem cell differentiation and proliferation, and regulates Sox2 expression during tooth development.

Structure of human POFUT1, its requirement in ligand-independent oncogenic Notch signaling, and functional effects of Dowling-Degos mutations.

Protein O-fucosyltransferase-1(POFUT1), which transfers fucose residues to acceptor sites on serine and threonine residues of epidermal growth factor-like repeats of recipient proteins, is essential for Notch signal transduction in mammals. Here, we examine the consequences of POFUT1 loss on the oncogenic signaling associated with certain leukemia-associated mutations of human Notch1, report the structures of human POFUT1 in free and GDP-fucose bound states, and assess the effects of Dowling-Degos mutations on human POFUT1 function. CRISPR-mediated knockout of POFUT1 in U2OS cells suppresses both normal Notch1 signaling, and the ligand-independent signaling associated with leukemogenic mutations of Notch1. Normal and oncogenic signaling are rescued by wild-type POFUT1 but rescue is impaired by an active-site R240A mutation. The overall structure of the human enzyme closely resembles that of the Caenorhabditis elegans protein, with an overall backbone RMSD of 0.93 Å, despite primary sequence identity of only 39% in the mature protein. GDP-fucose binding to the human enzyme induces limited backbone conformational movement, though the side chains of R43 and D244 reorient to make direct contact with the fucose moiety in the complex. The reported Dowling-Degos mutations of POFUT1, except for M262T, fail to rescue Notch1 signaling efficiently in the CRISPR-engineered POFUT1-/- background. Together, these studies identify POFUT1 as a potential target for cancers driven by Notch1 mutations and provide a structural roadmap for its inhibition.

The Crumbs_C isoform of Drosophila shows tissue- and stage-specific expression and prevents light-dependent retinal degeneration.

ABSTRACTDrosophila Crumbs (Crb) is a key regulator of epithelial polarity and fulfils a plethora of other functions, such as growth regulation, morphogenesis of photoreceptor cells and prevention of retinal degeneration. This raises the question how a single gene regulates such diverse functions, which in mammals are controlled by three different paralogs. Here, we show that in Drosophila different Crb protein isoforms are differentially expressed as a result of alternative splicing. All isoforms are transmembrane proteins that differ by just one EGF-like repeat in their extracellular portion. Unlike Crb_A, which is expressed in most embryonic epithelia from early stages onward, Crb_C is expressed later and only in a subset of embryonic epithelia. Flies specifically lacking Crb_C are homozygous viable and fertile. Strikingly, these flies undergo light-dependent photoreceptor degeneration despite the fact that the other isoforms are expressed and properly localised at the stalk membrane. This allele now provides an ideal possibility to further unravel the molecular mechanisms by which Drosophila crb protects photoreceptor cells from the detrimental consequences of light-induced cell stress.

Deciphering the Fringe-Mediated Notch Code: Identification of Activating and Inhibiting Sites Allowing Discrimination between Ligands

Fringe proteins are β3-N-acetylglucosaminyltransferases that modulate Notch activity by modifying O-fucose residues on epidermal growth factor-like (EGF) repeats of Notch. Mammals have three Fringes: Lunatic, Manic, and Radical. While Lunatic and Manic Fringe inhibit Notch1 activation from Jagged1 and enhance activation from Delta-like 1, Radical Fringe enhances signaling from both. We used a mass spectrometry approach to determine whether the variable effects of Fringes on Notch1 result from generation of unique glycosylation patterns on Notch1. We found that Lunatic and Manic Fringe modified similar sites on Notch1, while Radical Fringe modified a subset. Fringe modifications at EGF8 and EGF12 enhanced Notch1 binding to and activation from Delta-like 1, while modifications at EGF6 and EGF36 (added by Manic and Lunatic but not Radical) inhibited Notch1 activation from Jagged1. Combined, these results suggest that Fringe modifications "mark" different regions in the Notch1 extracellular domain for activation or inhibition.