Epidermal Growth Factor-like Research Articles

HSF1 Increases EOGT-Mediated Glycosylation of Notch1 to Promote IL-1β-Induced Inflammatory Injury of Chondrocytes.

Osteoarthritis (OA) is the most common arthritic disease in humans. Nevertheless, the pathogenic mechanism of OA remains unclear. This study aimed to explore that heat-shock transcription factor 1 (HSF1) facilitated interleukin-1 beta (IL-1β) chondrocyte injury by increasing Notch1 O-linked N-acetylglucosamine (O-GlcNAc) modification level. Human chondrocytes were incubated with 5 ng/ml interleukin-1 beta (IL-1β) for 24 h to establish OA cell model. The messenger RNA (mRNA) or protein expressions were assessed using reverse transcription-quantitative polymerase chain reaction, western blot, or immunofluorescence. Chondrocyte viability was examined by Cell Counting Kit-8 assay. Enzyme-linked immunosorbent assay was employed to detect the secretion levels of interleukin-6 (IL-6) and interleukin-8 (IL-8). Immunoprecipitation was adopted to detect Notch1 O-GlcNAc modification level. The interaction between HSF1 and epidermal growth factor-like (EGF) domain-specific O-GlcNAc transferase (EOGT) promoter was analyzed by dual-luciferase reporter gene and chromatin immunoprecipitation assays. Herein, our results demonstrated that HSF1, EOGT, Notch1, and Notch1 intracellular domain (NICD1) expressions in chondrocytes were markedly increased by IL-1β stimulation. EOGT elevated Notch1 expression in IL-1β-treated chondrocytes by increasing Notch1 O-GlcNAc modification level. EOGT silencing reduced IL-1β-induced chondrocyte inflammatory injury. In addition, HSF1 knockdown relieved IL-1β-induced chondrocyte inflammatory injury. Molecular interaction experiment proved that HSF1 transcriptionally activated EOGT expression in IL-1β-treated chondrocytes. HSF1 promoted IL-1β-induced inflammatory injury in chondrocytes by increasing EOGT-mediated glycosylation of Notch1.

In vivo evidence for GDP-fucose transport in the absence of transporter SLC35C1 and putative transporter SLC35C2

Slc35c1 encodes an antiporter that transports GDP-fucose into the Golgi and returns GMP to the cytoplasm. The closely related gene Slc35c2 encodes a putative GDP-fucose transporter and promotes Notch fucosylation and Notch signaling in cultured cells. Here, we show that HEK293T cells lacking SLC35C1 transferred reduced amounts of O-fucose to secreted epidermal growth factor-like (EGF) repeats from NOTCH1 or secreted thrombospondin type I repeats (TSRs) from thrombospondin 1. However, cells lacking SLC35C2 did not exhibit reduced fucosylation of these EGF repeats or TSRs. To investigate SLC35C2 functions in vivo, WW6 embryonic stem cells were targeted for Slc35c2. Slc35c2[-/-] mice were viable and fertile and exhibited no evidence of defective Notch signaling during skeletal or T cell development. By contrast, mice with inactivated Slc35c1 exhibited perinatal lethality and marked skeletal defects in late embryogenesis, typical of defective Notch signaling. Compound Slc35c1[-/-]Slc35c2[-/-] mutants were indistinguishable in skeletal phenotype from Slc35c1[-/-] embryos and neonates. Double mutants did not exhibit the exacerbated skeletal defects predicted if SLC35C2 was functionally important for Notch signaling in vivo. In addition, NOTCH1 immunoprecipitated from Slc35c1[-/-]Slc35c2[-/-] neonatal lung carried fucose detected by binding of Aleuria aurantia lectin. Given that the absence of both SLC35C1, a known GDP-fucose transporter, and SLC35C2, a putative GDP-fucose transporter, did not lead to afucosylated NOTCH1 nor to the severe Notch signaling defects and embryonic lethality expected if all GDP-fucose transport were abrogated, at least one more mechanism of GDP-fucose transport into the secretory pathway must exist in mammals.

EOGT enables residual Notch signaling in mouse intestinal cells lacking POFUT1

Notch signaling determines cell fates in mouse intestine. Notch receptors contain multiple epidermal growth factor-like (EGF) repeats modified by O-glycans that regulate Notch signaling. Conditional deletion of protein O-fucosyltransferase 1 (Pofut1) substantially reduces Notch signaling and markedly perturbs lineage development in mouse intestine. However, mice with inactivated Pofut1 are viable, whereas complete elimination of Notch signaling in intestine is lethal. Here we investigate whether residual Notch signaling enabled by EGF-domain-specific O-linked N-acetylglucosamine transferase (Eogt) permits mice conditionally lacking Pofut1 in intestine to survive. Mice globally lacking Eogt alone were grossly unaffected in intestinal development. In contrast, mice lacking both Eogt and Pofut1 died at ~ 28 days after birth with greater loss of body weight, a greater increase in the number of goblet and Paneth cells, and greater downregulation of the Notch target gene Hes1, compared to Pofut1 deletion alone. These data reveal that both O-fucose and O-GlcNAc glycans are fundamental to Notch signaling in the intestine and provide new insights into roles for O-glycans in regulating Notch ligand binding. Finally, EOGT and O-GlcNAc glycans provide residual Notch signaling and support viability in mice lacking Pofut1 in the intestine.

1H, 15N, 13C backbone and sidechain resonance assignments and secondary structure of mouse NOTCH1 EGF27.

NOTCH1 is a transmembrane receptor in metazoans that is linked to a variety of disorders. The receptor contains an extracellular domain (ECD) with 36 tandem epidermal growth factor-like (EGF) repeats. The ECD is responsible for intercellular signaling via protein-ligand interactions with neighboring cells. Each EGF repeat consists of approximately 40 amino acids and 3 conserved disulfide bonds. The Abruptex region (EGF24-29) is critical for NOTCH1 signaling and is known for its missense mutations. Certain EGF repeats are modified with the addition of O-linked glycans and many have calcium binding sites, which give each EGF repeat a unique function. It has been shown that the loss of the O-fucose site of EGF27 alters NOTCH1 activity. To investigate the role of glycosylation in the NOTCH1 signaling pathway, nuclear magnetic resonance spectroscopy has been employed to study the structures of EGF27 and its glycoforms. Here, we report the backbone and sidechain 1H, 15N, and 13C-resonance assignments of the unmodified EGF27 protein and the predicted secondary structure derived from the assigned chemical shifts.

Regulation of myeloid and lymphoid cell development by O-glycans on Notch.

Notch signaling via NOTCH1 stimulated by Delta-like ligand 4 (DLL4) is required for the development of T cells in thymus, and NOTCH2 stimulated by Notch ligand DLL1 is required for the development of marginal zone (MZ) B cells in spleen. Notch signaling also regulates myeloid cell production in bone marrow and is an essential contributor to the generation of early hematopoietic stem cells (HSC). The differentiation program in each of these cellular contexts is optimized by the regulation of Notch signaling strength by O-glycans attached to epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch receptors. There are three major types of O-glycan on NOTCH1 and NOTCH2 - O-fucose, O-glucose and O-GlcNAc. The initiating sugar of each O-glycan is added in the endoplasmic reticulum (ER) by glycosyltransferases POFUT1 (fucose), POGLUT1/2/3 (glucose) or EOGT (GlcNAc), respectively. Additional sugars are added in the Golgi compartment during passage through the secretory pathway to the plasma membrane. Of particular significance for Notch signaling is the addition of GlcNAc to O-fucose on an EGF repeat by the Fringe GlcNAc-transferases LFNG, MFNG or RFNG. Canonical Notch ligands (DLL1, DLL4, JAG1, JAG2) expressed in stromal cells bind to the extracellular domain of Notch receptors expressed in hematopoietic stem cells and myeloid and lymphoid progenitors to activate Notch signaling. Ligand-receptor binding is differentially regulated by the O-glycans on Notch. This review will summarize our understanding of the regulation of Notch signaling in myeloid and lymphoid cell development by specific O-glycans in mice with dysregulated expression of a particular glycosyltransferase and discuss how this may impact immune system development and malignancy in general, and in individuals with a congenital defect in the synthesis of the O-glycans attached to EGF repeats.

Secretory expression of mammalian NOTCH tandem epidermal growth factor-like repeats based on increased O-glycosylation

The Notch pathway represents evolutionarily conserved intercellular signaling essential for cell–to–cell communication during development. Dysregulation of Notch signaling has been implicated in various diseases, and its control represents a potential cancer treatment strategy. Notch signaling is initiated by the interaction of NOTCH receptors with their ligands on neighboring cells. Therefore, the truncated NOTCH ectodomain, composed mainly of tandem repeats of epidermal growth factor-like (EGF) domains, serves as a decoy molecule that competes for ligand binding and thus inhibits ligand-dependent Notch signaling. Although full-length NOTCH EGF repeats exhibited potent Notch inhibitory activity, they were poorly produced in the transfected cells. This study evaluated the effect of EGF domain-modifying glycosyltransferases on the secretion of NOTCH EGF repeats. Our results in HEK293T cells revealed that, unlike the effect on endogenous NOTCH receptors, overexpressed EGF domain-specific O-GlcNAc transferase (EOGT) markedly enhanced the secretion of NOTCH1 EGF repeats in an enzyme activity-dependent manner. The co-expression of protein O-glucosyltransferase 1 further manifested the effect of EOGT. The resultant changes in O-glycosylation of NOTCH3 were evaluated by label-free glycopeptide quantification. This study provides an experimental strategy to efficiently generate NOTCH EGF repeats by manipulating the expression of glycosyltransferases that alter the O-glycosylation of EGF domains.

Fringe GlcNAc-transferases differentially extend O-fucose on endogenous NOTCH1 in mouse activated T cells

NOTCH1 is a transmembrane receptor that initiates a cell–cell signaling pathway controlling various cell fate specifications in metazoans. The addition of O-fucose by protein O-fucosyltransferase 1 (POFUT1) to epidermal growth factor-like (EGF) repeats in the NOTCH1 extracellular domain is essential for NOTCH1 function, and modification of O-fucose with GlcNAc by the Fringe family of glycosyltransferases modulates Notch activity. Prior cell-based studies showed that POFUT1 modifies EGF repeats containing the appropriate consensus sequence at high stoichiometry, while Fringe GlcNAc-transferases (LFNG, MFNG, and RFNG) modify O-fucose on only a subset of NOTCH1 EGF repeats. Previous in vivo studies showed that each FNG affects naïve T cell development. To examine Fringe modifications of NOTCH1 at a physiological level, we used mass spectral glycoproteomic methods to analyze O-fucose glycans of endogenous NOTCH1 from activated T cells obtained from mice lacking all Fringe enzymes or expressing only a single FNG. While most O-fucose sites were modified at high stoichiometry, only EGF6, EGF16, EGF26, and EGF27 were extended in WT T cells. Additionally, cell-based assays of NOTCH1 lacking fucose at each of those O-fucose sites revealed small but significant effects of LFNG on Notch-Delta binding in the EGF16 and EGF27 mutants. Finally, in activated T cells expressing only LFNG, MFNG, or RFNG alone, the extension of O-fucose with GlcNAc in the same EGF repeats was diminished, consistent with cooperative interactions when all three Fringes were present. The combined data open the door for the analysis of O-glycans on endogenous NOTCH1 derived from different cell types.

Isolation of NELL 1 Aptamers for Rhabdomyosarcoma Targeting.

NELL1 (Neural epidermal growth factor-like (EGFL)-like protein) is an important biomarker associated with tissue and bone development and regeneration. NELL1 upregulation has been linked with metastasis and negative prognosis in rhabdomyosarcoma (RMS). Furthermore, multiple recent studies have also shown the importance of NELL1 in inflammatory bowel disease and membranous nephropathy, amongst other diseases. In this study, several anti-NELL1 DNA aptamers were selected from a randomized ssDNA pool using a fluorescence-guided method and evaluated for their binding affinity and selectivity. Several other methods such as a metabolic assay and confocal microscopy were also applied for the evaluation of the selected aptamers. The top three candidates were evaluated further, and AptNCan3 was shown to have a binding affinity up to 959.2 nM. Selectivity was examined in the RH30 RMS cells that overexpressed NELL1. Both AptNCan2 and AptNCan3 could significantly suppress metabolic activity in RMS cells. AptNCan3 was found to locate on the cell membrane and also on intracellular vesicles, which matched the location of NELL1 shown by antibodies in previous research. These results indicate that the selected anti-NELL1 aptamer showed strong and highly specific binding to NELL1 and therefore has potential to be used for in vitro or in vivo studies and treatments.

Identification, function, and biological relevance of POGLUT2 and POGLUT3.

O-glycosylation of Epidermal Growth Factor-like (EGF) repeats plays crucial roles in protein folding, trafficking and function. The Notch extracellular domain has been used as a model to study these mechanisms due to its many O-glycosylated EGF repeats. Three enzymes were previously known to O-glycosylate Notch EGF repeats: Protein O-Glucosyltransferase 1 (POGLUT1), Protein O-Fucosyltransferase 1 (POFUT1), and EGF Domain Specific O-Linked N-Acetylglucosamine Transferase (EOGT). All of these modifications affect Notch activity. Recently, POGLUT2 and POGLUT3 were identified as two novel O-glucosyltransferases that modify a few Notch EGF repeats at sites distinct from those modified by POGLUT1. Comparison of these modification sites revealed a putative consensus sequence which predicted modification of many extracellular matrix proteins including fibrillins (FBNs) and Latent TGFβ-binding proteins (LTBPs). Glycoproteomic analysis revealed that approximately half of the 47 EGF repeats in FBN1 and FBN2, and half of the 18 EGF repeats in LTBP1, are modified by POGLUT2 and/or POGLUT3. Cellular assays showed that loss of modifications by POGLUT2 and/or POGLUT3 significantly reduces FBN1 secretion. There is precedent for EGF modifications to affect protein-protein interactions, as has been demonstrated by research of POGLUT1 and POFUT1 modifications on Notch. Here we discuss the identification and characterization of POGLUT2 and POGLUT3 and the ongoing research that continues to elucidate the biological significance of these novel enzymes.

Glycans that regulate Notch signaling in the intestine.

Intestinal homeostasis is key to the maintenance of good health. The small intestine plays important roles in absorption, digestion, hormonal and immune functions. Crypt base columnar (CBC) stem cells residing at the bottom of crypts are nurtured by Paneth cells, and together create the stem cell niche, the foundation of intestinal homeostasis. CBC stem cells replicate to replenish their number, or differentiate into a variety of epithelial cells with specialized functions. Notch signaling is a cell-cell signaling pathway that regulates both the proliferation and differentiation of CBC stem cells. NOTCH1 and NOTCH2 stimulated by canonical Notch ligands DLL1 and DLL4 mediate Notch signaling in the intestine that, in concert with other signaling pathways including the WNT and BMP pathways, determines cell fates. Importantly, interactions between Notch receptors and canonical Notch ligands are regulated by O-glycans linked to Ser/Thr in epidermal growth factor-like (EGF) repeats of the Notch receptor extracellular domain (NECD). The O-glycans attached to NECD are key regulators of the strength of Notch signaling. Imbalances in Notch signaling result in altered cell fate decisions and may lead to cancer in the intestine. In this review, we summarize the impacts of mutations in Notch pathway members on intestinal development and homeostasis, with a focus on the glycosyltransferases that transfer O-glycans to EGF repeats of NOTCH1, NOTCH2, DLL1 and DLL4.

Eogt-catalyzed <i>O</i>-GlcNAcylation

O-GlcNAcylation is critical in human physiology. The dysregulation of O-GlcNAcylation on various intracellular proteins impacts cardiovascular diseases, the immune system, type 2 diabetes, neurodegeneration diseases, and cancers. In the past decade, extracellular O-GlcNAcylation was discovered from the conserved epidermal growth factor-like (EGF) domain in Drosophila and mammals. Extracellular O-GlcNAcylation is catalyzed by EGF domain O-GlcNAc transferase (EOGT) in the endoplasmic reticulum, and, unlike intracellular O-GlcNAcylation, it is further elongated with galactose and sialic acid in the Golgi apparatus. In this review, we retrospect the studies addressing the catalysis of EOGT, the conserved consensus sequence, the impacts of abnormal extracellular O-GlcNAcylation, and the diseases related to EOGT mutation.

Generation of Properly Folded Epidermal Growth Factor-Like (EGF) Repeats and Glycosyltransferases Enables In Vitro O-Glycosylation.

The epidermal growth factor-like (EGF) domain is an evolutionarily conserved motif found widely distributed among numerous secreted and membrane-anchored proteins, including the Notch receptors. Notch receptors include numerous EGF repeats tandemly connected in the extracellular domain. These EGF repeats must be properly folded in order for them to undergo the three different types of O-glycosylation associated with these extracellular proteins: O-fucose, O-glucose, and O-N-acetylglucosamine via glycosyltransferases POFUT1, POGLUT1, and EOGT. The O-glycosylation of the EGF repeats in the Notch receptors regulates the activation of Notch signaling and mutations in POFUT1, POGLUT1, and EOGT have been linked to specific human diseases. A large amount of EGF repeat and glycosyltransferase protein is required to construct an in vitro O-glycosylation system. Here, we describe how we prepared properly folded EGF repeats using two different bacterial expression vectors, generated recombinant glycosyltransferases, and performed in vitro O-glycosylation and subsequent product analysis by mass spectrometry. The methods described here are useful for investigating the enzymatic activities of mutated glycosyltransferases, revealing the structural basis of the O-glycosylation mechanism by co-crystallization of the glycosyltransferase-EGF repeat complexes, or identifying potential inhibitors of these glycosyltransferases.

High-Performance Smart Hydrogels with Redox-Responsive Properties Inspired by Scallop Byssus.

Smart hydrogels with versatile properties, including a tunable gelation time, nonswelling attributes, and biocompatibility, are in great need in the biomedical field. To meet this urgent demand, we explored novel biomaterials with the desired properties from sessile marine organisms. To this end, a novel protein, Sbp9, derived from scallop byssus was extensively investigated, which features typical epidermal growth factor-like (EGFL) multiple repetitive motifs. Our current work demonstrated that the key fragment of Sbp9 (calcium-binding domain (CBD) and 4 EGFL repeats (CE4)) was able to form a smart hydrogel driven by noncovalent interactions and facilitated by disulfide bonds. More importantly, this smart hydrogel demonstrates several desirable and beneficial features, which could offset the drawbacks of typical protein-based hydrogels, including (1) a redox-responsive gelation time (from <1 to 60 min); (2) tunable mechanical properties, nonswelling abilities, and an appropriate microstructure; and (3) good biocompatibility and degradability. Furthermore, proof-of-concept demonstrations showed that the newly discovered hydrogel could be used for anticancer drug delivery and cell encapsulation. Taken together, a smart hydrogel inspired by marine sessile organisms with desirable properties was generated and characterized and demonstrated to have extensive applicability potential in biomedical applications, including tissue engineering and drug release.

Regulation of Notch Signaling By O-Glycans during Lymphopoiesis and Myelopoiesis

IntroductionNotch signaling is essential for the optimal generation of T, B and myeloid cells. Epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch receptors are modified by O-fucose and O-GlcNAc glycans transferred by protein O-fucosyltransferase 1 (POFUT1) and EGF O-GlcNAc-transferase (EOGT), respectively. EOGT promotes Notch ligand binding and Notch signaling in the developing retina in mice, and its loss is the basis of Adam's Oliver Syndrome 4 (AOS4) in humans. The loss of POFUT1 in humans leads to the Dowling Degos Disease 2 (DDD2). Thus, O-fucose and O-GlcNAc glycans may have different functions in Notch signaling, reflected in different requirements for T, B or myeloid cell development.Methods:Eogt control, Eogt null, Pofut1 floxed and Vav1-iCre transgenic mice were used to generate single and compound mutant mice with inactive Eogt, Pofut1 or both Eogt and Pofut1. Antibody markers of hematopoietic stem progenitor cell (HSPC), lymphoid and myeloid subsets were used to identify different T, B and myeloid cell subsets by flow cytometry using the Cytek TM Aurora Flow Cytometer. FCS files were analyzed using FlowJo software (BD). CD45.1+ (B6.SJL-Ptprc a Pepc b/BoyJ #002014) congenic mice were used for bone marrow transfer. Expression of NOTCH1 and binding of soluble Notch ligands to DN T cells was analyzed by flow cytometry. Expression of different Notch target genes was determined using qRT-PCR anlaysis.Results:Eogt null mice exhibited altered production of certain T cell subsets in thymus and B cell subsets in spleen, most similar to alterations observed in mice lacking LFNG, MFNG and RFNG. This phenotype was cell-autonomous as Eogt null bone marrow cells transferred defective T and B cell development to irradiated recipient mice. More severe defects in hematopoiesis were observed in mice conditionally lacking Pofut1 in hematopoietic stem cells (HSC) via Vav1-iCre. However , deletion of both O-fucose and O-GlcNAc glycans together in Eogt:Pofut1 double knockout HSC, led to the most severe decrease in common lymphoid and myeloid progenitors in bone marrow. In thymus, the dramatic reduction in T cell subsets observed in Pofut1 cKO mice was reduced further (in DN2 subsets) in Eogt:Pofut1 dKO mice. In spleen, there was a significantly greater decrease in follicular B and other B cell populations, and a greater increase in CD11b/c+ and Gr1+ myeloid cells in Eogt:Pofut1 dKO mice. Splenomegaly occurred in both Pofut1 cKO and Eogt:Pofut1 dKO mice, with a greater increase in extramedullary hematopoiesis in the dKO spleen. Binding of soluble DLL4-Fc was >90% reduced in Pofut1 cKO DN T cells, and not further reduced in dKO cells, while NOTCH1 expression at the cell surface was only slightly reduced. The relative expression of Notch target genes including Hes1, CD25, cMyc, and Deltex1 was similarly reduced in Pofut1 cKO and Eogt:Pofut1 dKO DN T cell progenitors.Conclsuion:The combined data suggest that O-fucose and O-GlcNAc glycans act in an additive fashion to optimize Notch signaling in lymphoid and myeloid differentiation.Keywords: Notch Signaling, O-Glycans, Protein O-fucosyltransferase 1 (POFUT1), O-GlcNAc transferase (EOGT), Lymphopoiesis, Myelopoiesis. DisclosuresStanley: Aviceda Therapeutics, Inc.: Consultancy, Current holder of stock options in a privately-held company.

In silico structural analysis of truncated 2’ fluoro-RNA aptamer: Elucidating EGF-1 and EGF-2 binding domains on factor IX protein

A stable truncated 2’ fluoro-RNA aptamer contributes to the upstream interruption of the blood coagulation pathway by selectively binding with factor IX protein (FIX). This work followed a rational method for virtually analyzing structural orientation and binding interactions between aptamer and FIX. Three possible aptamer models from the same sequence were designed and evaluated for optimum binding with FIX. Molecular docking was performed to find a prime docking domain and the best docking conformation for a stable aptamer-FIX complex. Possible interactions are observed at PRO126-U9, LYS125-U9, LYS122-A7, ARG116-U9, GLY102-G22, THR101-G22, GLN74-G6, GLY60-G5, CGU36-U11, CGU40-A12, and ASP49-A10. A molecular dynamic simulation assessed the strength of the interaction between aptamer and FIX. It was revealed that the aptamer was effectively bound with the average amino acid fluctuation of 1 Å. The complex reached equilibrium within ten ns during the initial phase of the simulation and then remained stable over 100 ns. B factor analysis showed a moderate atomic displacement from the crystal structure, except at N-terminal from atom 2247−5938. Principal component analysis of post-dynamic trajectories further revealed the stability by the arrayed orientation of amino acids dihedral angles. Overall, 2’ fluoro-RNA aptamer exhibits stronger stability in their interaction with FIX. Noteworthy, the aptamer's stability as an inhibitor to deactivate the clotting cascade by specifically binding with epidermal growth factor-like (EGF) 1 and 2 domains was evidenced.

POGLUT2 and POGLUT3 O-glucosylate multiple EGF repeats in fibrillin-1, -2, and LTBP1 and promote secretion of fibrillin-1

Fibrillin-1 (FBN1) is the major component of extracellular matrix microfibrils, which are required for proper development of elastic tissues, including the heart and lungs. Through protein–protein interactions with latent transforming growth factor (TGF) β-binding protein 1 (LTBP1), microfibrils regulate TGF-β signaling. Mutations within the 47 epidermal growth factor-like (EGF) repeats of FBN1 cause autosomal dominant disorders including Marfan Syndrome, which is characterized by disrupted TGF-β signaling. We recently identified two novel protein O-glucosyltransferases, Protein O-glucosyltransferase 2 (POGLUT2) and 3 (POGLUT3), that modify a small fraction of EGF repeats on Notch. Here, using mass spectral analysis, we show that POGLUT2 and POGLUT3 also modify over half of the EGF repeats on FBN1, fibrillin-2 (FBN2), and LTBP1. While most sites are modified by both enzymes, some sites show a preference for either POGLUT2 or POGLUT3. POGLUT2 and POGLUT3 are homologs of POGLUT1, which stabilizes Notch proteins by addition of O-glucose to Notch EGF repeats. Like POGLUT1, POGLUT2 and 3 can discern a folded versus unfolded EGF repeat, suggesting POGLUT2 and 3 are involved in a protein folding pathway. In vitro secretion assays using the N-terminal portion of recombinant FBN1 revealed reduced FBN1 secretion in POGLUT2 knockout, POGLUT3 knockout, and POGLUT2 and 3 double-knockout HEK293T cells compared with wild type. These results illustrate that POGLUT2 and 3 function together to O-glucosylate protein substrates and that these modifications play a role in the secretion of substrate proteins. It will be interesting to see how disease variants in these proteins affect their O-glucosylation.

Structure of human factor VIIa-soluble tissue factor with calcium, magnesium and rubidium.

Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa-soluble tissue factor (TF) complex was solved at 1.8 Å resolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na+ and by ∼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163-180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.

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 N-acetylglucosamine (GlcNAc), and elongated with additional sugars. Notch receptors signal following binding of Notch ligands to the Notch receptor ECD, leading to release of the intracellular domain and activation of many Notch target genes. Notch signaling regulates cell fate decisions in many organs during development and differentiation. Dysregulation of Notch signaling leads to developmental disorders and to cancer. Lunatic (LFNG), Manic (MFNG) and Radical (RFNG) Fringe are GlcNAc transferases that extend Notch EGF repeats which already carry a fucose. EOGT is also a GlcNAc-transferase that transfers O-GlcNAc directly to EGF repeats. We are investigating roles for EOGT and each Fringe in T and B cell development using mice lacking various combinations of the Eogt, Lfng, Mfng and Rfng genes. We are also identifying individual roles for Fng genes using a co-culture assay of bone marrow hemopoietic stem cells (HSC) from compound mutant mice stimulated by OP9 stromal cells expressing one of the four Notch ligands, DLL1, DLL4, JAG1 or JAG2. The combined results show that the O-fucose glycans on NOTCH1 are key regulators of the strength of NOTCH1 signaling which, in turn, determines differentiation of HSC to T cell progenitors.

Current Views on the Roles of O-Glycosylation in Controlling Notch-Ligand Interactions.

The 100th anniversary of Notch discovery in Drosophila has recently passed. The Notch is evolutionarily conserved from Drosophila to humans. The discovery of human-specific Notch genes has led to a better understanding of Notch signaling in development and diseases and will continue to stimulate further research in the future. Notch receptors are responsible for cell-to-cell signaling. They are activated by cell-surface ligands located on adjacent cells. Notch activation plays an important role in determining the fate of cells, and dysregulation of Notch signaling results in numerous human diseases. Notch receptors are primarily activated by ligand binding. Many studies in various fields including genetics, developmental biology, biochemistry, and structural biology conducted over the past two decades have revealed that the activation of the Notch receptor is regulated by unique glycan modifications. Such modifications include O-fucose, O-glucose, and O-N-acetylglucosamine (GlcNAc) on epidermal growth factor-like (EGF) repeats located consecutively in the extracellular domain of Notch receptors. Being fine-tuned by glycans is an important property of Notch receptors. In this review article, we summarize the latest findings on the regulation of Notch activation by glycosylation and discuss future challenges.

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.