Epidermal Growth Factor-like Research Articles

A Pan-Cancer Study of Epidermal Growth Factor-Like Domains 6/7/8 as Therapeutic Targets in Cancer.

With highly homologous epidermal growth factor (EGF)-like (EGFL) domains, the members of the EGFL family play crucial roles in growth, invasion, and metastasis of tumors and are closely associated with the apoptosis of tumor cells and tumor angiogenesis. Furthermore, their contribution to immunoreaction and tumor microenvironment is highly known. In this study, a comprehensive analysis of EGFL6, −7, and −8 was performed on the basis of their expression profiles and their relationship with the rate of patient survival. Through a pan-cancer study, their effects were correlated with immune subtypes, tumor microenvironment, and drug resistance. Using The Cancer Genome Atlas pan-cancer data, expression profiles of EGFL6, −7, and −8, and their association with the patient survival rate and tumor microenvironment were analyzed in 33 types of cancers. The expression of the EGFL family was different in different cancer types, revealing the heterogeneity among cancers. The results showed that the expression of EGFL8 was lower than EGFL6 and EGFL7 among all cancer types, wherein EGFL7 had the highest expression. The univariate Cox proportional hazard regression model showed that EGFL6 and EGFL7 were the risk factors to predict poor prognosis of cancers. Survival analysis was then used to verify the relationship between gene expression and patient survival. Furthermore, EGFL6, EGFL7, and EGFL8 genes revealed a clear association with immune infiltrate subtypes; they were also related to the infiltration level of stromal cells and immune cells with different degrees. Moreover, they were negatively correlated with the characteristics of cancer stem cells measured by DNAs and RNAs. In addition, EGFL6, −7, and −8 were more likely to contribute to the resistance of cancer cells. Our systematic analysis of EGFL gene expression and their correlation with immune infiltration, tumor microenvironment, and prognosis of cancer patients emphasized the necessity of studying each EGFL member as a separate entity within each particular type of cancer. Simultaneously, EGFL6, −7, and −8 signals were verified as promising targets for cancer therapies, although further laboratory validation is still required.

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.

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).

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.

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.

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.

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.

Sodium‐site in serine protease domain of human coagulation factor IXa: evidence from the crystal structure and molecular dynamics simulations study

Essentials Consensus sequence and biochemical data suggest a Na+ -site in the factor (F) IXa protease domain. X-ray structure of the FIXa EGF2/protease domain at 1.37Å reveals a Na+ -site not observed earlier. Molecular dynamics simulations data support that Na+ ±Ca2+ promote FIXa protease domain stability. Sulfate ions found in the protease domain mimic heparin sulfate binding mode in FIXa. SUMMARY: Background Activated coagulation factor IX (FIXa) consists of a γ-carboxyglutamic acid domain, two epidermal growth factor-like (EGF) domains, and a C-terminal protease domain. Consensus sequence and biochemical data support the existence of a Na+ -site in the FIXa protease domain. However, soaking experiments or crystals grown in high concentration of ammonium sulfate did not reveal a Na+ -site in wild-type or mutant FIXa EGF2/protease domain structure. Objective Determine the structure of the FIXa EGF2/protease domain in the presence of Na+ ; perform molecular dynamics (MD) simulations to explore the role of Na+ in stabilizing FIXa structure. Methods Crystallography, MD simulations, and modeling heparin binding to FIXa. Results Crystal structure at 1.37-Å resolution revealed that Na+ is coordinated to carbonyl groups of residues 184A, 185, 221A, and 224 in the FIXa protease domain. The Na+ -site in FIXa is similar to that of FXa and is linked to the Asp189 S1-site. In MD simulations, Na+ reduced fluctuations in residues 217-225 (Na+ -loop) and 70-80 (Ca2+ -loop), whereas Ca2+ reduced fluctuations only in residues of the Ca2+ -loop. Ca2+ and Na+ together reduced fluctuations in residues of the Ca2+ -loop and Na+ -loop (residues 70-80, 183-194, and 217-225). Moreover, we observed four sulfate ions that make salt bridges with FIXa protease domain Arg/Lys residues, which have been implicated in heparin binding. Based upon locations of the sulfate ions, we modeled heparin binding to FIXa, which is similar to the heparin binding in thrombin. Conclusions The FIXa Na+ -site in association with Ca2+ contributes to stabilization of the FIXa protease domain. The heparin binding mode in FIXa is similar to that in thrombin.

Sirtilins – the new old members of the vitamin K‐dependent coagulation factor family

Essentials Blood coagulation is driven by vitamin K (VK)‐dependent proteases.We have identified and characterized ‘sirtilin’ as an additional VK‐dependent protease.Sirtilins emerged early in the evolution of the coagulation system of vertebrates.Ubiquitous occurrence might indicate an important functional role of sirtilins. SummaryBackgroundVitamin K (VK)‐dependent proteases are major players in blood coagulation, including both the initiation and the regulation of the cascade. Five different members of this protease family have been described, comprising the following coagulation factors: factor VII, FIX, FX, protein C (PC), and prothrombin (FII). FVII, FIX, FX and PC share a typical domain architecture, with an N‐terminal γ‐carboxyglutamate (Gla) domain, two epidermal growth factor‐like (EGF) domains, and a C‐terminal trypsin‐like serine protease (SP) domain.ObjectivesWe have identified uncharacterized proteins in snake genomes showing the typical Gla–EGF1–EGF2–SP domain architecture but relatively low sequence conservation compared to known VK‐dependent proteases. On the basis of sequence analysis, we hypothesized that these proteins are functional members of the VK‐dependent protease family.Methods/resultsUsing phylogenetic analyses, we confirmed the so‐called ‘sirtilins’ as an additional VK‐dependent protease class. These proteases were found in several vertebrates, including jawless fish, cartilaginous fish, bony fish, reptiles, birds, and marsupials, but not in other mammals. The recombinant zymogen form of Thamnophis sirtalis sirtilin was produced by in vitro renaturation, and was activated with human activated FXI. The activated form of sirtilin proteolytically cleaved peptide and protein substrates, including prothrombin. Mass spectrometry‐based substrate profiling of sirtilin revealed a narrower sequence specificity than those of FIX and FX.ConclusionsThe ubiquitous occurrence of sirtilins in many vertebrate classes might indicate an important functional role. Understanding the detailed functions of sirtilins might contribute to a deeper understanding of the evolution and function of the vertebrate coagulation system.

The discovered chimeric protein plays the cohesive role to maintain scallop byssal root structural integrity

Adhesion is essential for many marine sessile organisms. Unraveling the compositions and assembly of marine bioadheisves is the fundamental to understand their physiological roles. Despite the remarkable diversity of animal bioadhesion, our understanding of this biological process remains limited to only a few animal lineages, leaving the majority of lineages remain enigmatic. Our previous study demonstrated that scallop byssus had distinct protein composition and unusual assembly mechanism apart from mussels. Here a novel protein (Sbp9) was discovered from the key part of the byssus (byssal root), which contains two Calcium Binding Domain (CBD) and 49 tandem Epidermal Growth Factor-Like (EGFL) domain repeats. Modular architecture of Sbp9 represents a novel chimeric gene family resulting from a gene fusion event through the acquisition of CBD2 domain by tenascin like (TNL) gene from Na+/Ca2+exchanger 1 (NCX1) gene. Finally, free thiols are present in Sbp9 and the results of a rescue assay indicated that Sbp9 likely plays the cohesive role for byssal root integrity. This study not only aids our understanding of byssus assembly but will also inspire biomimetic material design.

Sensitized genetic backgrounds reveal differential roles for EGF repeat xylosyltransferases in Drosophila Notch signaling.

In multicellular organisms, glycosylation regulates various developmental signaling pathways including the Notch pathway. One of the O-linked glycans added to epidermal growth factor-like (EGF) repeats in animal proteins including the Notch receptors is the xylose-xylose-glucose-O oligosaccharide. Drosophila glucoside xylosyltransferase (Gxylt) Shams negatively regulates Notch signaling in specific contexts. Since Shams adds the first xylose residue to O-glucose, its loss-of-function phenotype could be due to the loss of the first xylose, the second xylose or both. To examine the contribution of the second xylose residues to Drosophila Notch signaling, we have performed biochemical and genetic analysis on CG11388, which is the Drosophila homolog of human xyloside xylosyltransferase 1 (XXYLT1). Experiments in S2 cells indicated that similar to human XXYLT1, CG11388 can add the second xylose to xylose-glucose-O glycans. Flies lacking both copies of CG11388 (Xxylt) are viable and fertile and do not show gross phenotypes indicative of altered Notch signaling. However, genetic interaction experiments show that in sensitized genetic backgrounds with decreased or increased Notch pathway components, loss of Xxylt promotes Delta-mediated activation of Notch. Unexpectedly, we find that in such sensitized backgrounds, even loss of one copy of the fly Gxylt shams enhances Delta-mediated Notch activation. Taken together, these data indicate that while the first xylose plays a key role in tuning the Delta-mediated Notch signaling in Drosophila, the second xylose has a fine-tuning role only revealed in sensitized genetic backgrounds.

Two novel protein O-glucosyltransferases that modify sites distinct from POGLUT1 and affect Notch trafficking and signaling

The Notch-signaling pathway is normally activated by Notch-ligand interactions. A recent structural analysis suggested that a novel O-linked hexose modification on serine 435 of the mammalian NOTCH1 core ligand-binding domain lies at the interface with its ligands. This serine occurs between conserved cysteines 3 and 4 of Epidermal Growth Factor-like (EGF) repeat 11 of NOTCH1, a site distinct from those modified by protein O-glucosyltransferase 1 (POGLUT1), suggesting that a different enzyme is responsible. Here, we identify two novel protein O-glucosyltransferases, POGLUT2 and POGLUT3 (formerly KDELC1 and KDELC2, respectively), which transfer O-glucose (O-Glc) from UDP-Glc to serine 435. Mass spectrometric analysis of NOTCH1 produced in HEK293T cells lacking POGLUT2, POGLUT3, or both genes showed that either POGLUT2 or POGLUT3 can add this novel O-Glc modification. EGF11 of NOTCH2 does not have a serine residue in the same location for this O-glucosylation, but EGF10 of NOTCH3 (homologous to EGF11 in NOTCH1 and -2) is also modified at the same position. Comparison of the sites suggests a consensus sequence for modification. In vitro assays with POGLUT2 and POGLUT3 showed that both enzymes modified only properly folded EGF repeats and displayed distinct acceptor specificities toward NOTCH1 EGF11 and NOTCH3 EGF10. Mutation of the O-Glc modification site on EGF11 (serine 435) in combination with sensitizing O-fucose mutations in EGF8 or EGF12 affected cell-surface presentation of NOTCH1 or reduced activation of NOTCH1 by Delta-like1, respectively. This study identifies a previously undescribed mechanism for fine-tuning the Notch-signaling pathway in mammals.

A perspective on structural and mechanistic aspects of protein O-fucosylation.

Protein O-fucosylation is an important post-translational modification (PTM) found in cysteine-rich repeats in proteins. Protein O-fucosyltransferases 1 and 2 (PoFUT1 and PoFUT2) are the enzymes responsible for this PTM and selectively glycosylate specific residues in epidermal growth factor-like (EGF) repeats and thrombospondin type I repeats (TSRs), respectively. Within the past six years, crystal structures of both enzymes have been reported, revealing important information on how they recognize protein substrates and achieve catalysis. Here, the structural information available today is summarized and how PoFUT1 and PoFUT2 employ different catalytic mechanisms is discussed.

Structural Divergence in O-GlcNAc Glycans Displayed on Epidermal Growth Factor-like Repeats of Mammalian Notch1.

Extracellular O-GlcNAc is a novel class of modification catalyzed by epidermal growth factor-like (EGF)-domain specific O-GlcNAc transferase (EOGT). In mammals, EOGT is required for ligand-mediated Notch signaling for vascular development. Previous studies have revealed that O-GlcNAc in mammalian cultured cells is subject to subsequent glycosylation, which may impose additional layers of regulation. This study aimed to analyze the O-GlcNAc glycans of Drosophila EGF20 as model substrates and mouse Notch1 EGF repeats by mass-spectrometry. The analysis of Drosophila EGF20 expressed in HEK293T cells revealed that the majority of the proteins are modified with an elongated form of O-GlcNAc glycan comprising terminal galactose or sialic acid residues. In contrast, recombinant Notch1 EGF repeats isolated from HEK293T cells revealed structural divergence of O-GlcNAc glycans among the different EGF domains. Although the majority of Notch1 EGF2 and EGF20 domains contained the extended forms of the glycan, the O-GlcNAc in many other domains mostly existed as a monosaccharide irrespective of the exogenous EOGT expression. Our results raised a hypothesis that an array of O-GlcNAc monosaccharides may impact the structure and function of Notch receptors.

The interface between the EGF1 and EGF2 domains is critical in integrin affinity regulation

It has been proposed that integrins adopt a bent, closed conformation with low ligand binding capability at resting state and switch into an extended, open conformation upon activation or interacting with extracellular matrix (ECM) ligand. In this study, we addressed how integrin conformational change at the β genu affects ligand binding and signaling. We discovered that swapping of the β3 epidermal growth factor-like (EGF) domain 1 and 2 with that of β8 greatly promoted ligand binding in β3 β8 chimeras. Sequence alignment indicated that β8 integrin uniquely lacks the interface between the EGF1 and 2. Disrupting this interface of the β3 integrin increased integrin ligand binding. Furthermore, the interface is critical for integrin affinity regulation but not downstream outside-in signaling.

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.

Structural Insights into Notch Receptor-Ligand Interactions.

Pioneering cell aggregation experiments from the Artavanis-Tsakonas group in the late 1980's localized the core ligand recognition sequence in the Drosophila Notch receptor to epidermal growth factor-like (EGF) domains 11 and 12. Since then, advances in protein expression, structure determination methods and functional assays have enabled us to define the molecular basis of the core receptor/ligand interaction and given new insights into the architecture of the Notch complex at the cell surface. We now know that Notch EGF11 and 12 interact with the Delta/Serrate/LAG-2 (DSL) and C2 domains of ligand and that membrane-binding, together with additional protein-protein interactions outside the core recognition domains, are likely to fine-tune generation of the Notch signal. Furthermore, structure determination of O-glycosylated variants of Notch alone or in complex with receptor fragments, has shown that these sugars contribute directly to the binding interface, as well as to stabilizing intra-molecular domain structure, providing some mechanistic insights into the observed modulatory effects of O-glycosylation on Notch activity.Future challenges lie in determining the complete extracellular architecture of ligand and receptor in order to understand (i) how Notch/ligand complexes may form at the cell surface in response to physiological cues, (ii) the role of lipid binding in stabilizing the Notch/ligand complex, (iii) the impact of O-glycosylation on binding and signalling and (iv) to dissect the different pathologies that arise as a consequence of mutations that affect proteins involved in the Notch 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.

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.