Notch signaling is a cellular pathway regulating cell-fate determination and adult tissue homeostasis. Little is known about how canonical Notch ligands or Fringe enzymes differentially affect NOTCH1 ...and NOTCH2. Using cell-based Notch signaling and ligand-binding assays, we evaluated differences in NOTCH1 and NOTCH2 responses to Delta-like (DLL) and Jagged (JAG) family members and the extent to which Fringe enzymes modulate their activity. In the absence of Fringes, DLL4–NOTCH1 activation was more than twice that of DLL4–NOTCH2, whereas all other ligands activated NOTCH2 similarly or slightly more than NOTCH1. However, NOTCH2 showed less sensitivity to the Fringes. Lunatic fringe (LFNG) enhanced NOTCH2 activation by DLL1 and -4, and Manic fringe (MFNG) inhibited NOTCH2 activation by JAG1 and -2. Mass spectral analysis showed that O-fucose occurred at high stoichiometry at most consensus sequences of NOTCH2 and that the Fringe enzymes modified more O-fucose sites of NOTCH2 compared with NOTCH1. Mutagenesis studies showed that LFNG modification of O-fucose on EGF8 and -12 of NOTCH2 was responsible for enhancement of DLL1–NOTCH2 activation, similar to previous reports for NOTCH1. In contrast to NOTCH1, a single O-fucose site mutant that substantially blocked the ability of MFNG to inhibit NOTCH2 activation by JAG1 could not be identified. Interestingly, elimination of the O-fucose site on EGF12 allowed LFNG to inhibit JAG1-NOTCH2 activation, and O-fucosylation on EGF9 was important for trafficking of both NOTCH1 and NOTCH2. Together, these studies provide new insights into the differential regulation of NOTCH1 and NOTCH2 by Notch ligands and Fringe enzymes.
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.
Fringe 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.
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•Fringe regulates fly Notch signaling by adding GlcNAc to O-fucose on Notch EGF8, -9, and -12•O-Fucose monosaccharide on Notch EGF12 is essential for fly embryonic neurogenesis•Fringe-modified EGF8, -9, and -12 promote Delta-Notch signaling in wing vein formation•Fringe-modified EGF8 and -12 prevent cis-inhibition of Serrate by Notch in vivo
POFUT1/O-Fut1 and Fringe glycosyltransferases regulate Notch signaling by adding fucose and GlcNAc, respectively, to Notch EGF repeats. Using in vitro and in vivo experiments, Pandey et al. define the critical target sites of these enzymes on Drosophila Notch and determine the distinct roles of each sugar in Notch-dependent processes.
Abstract
Peters plus syndrome, characterized by defects in eye and skeletal development with isolated cases of ventriculomegaly/hydrocephalus, is caused by mutations in the β3-glucosyltransferase ...(B3GLCT) gene. In the endoplasmic reticulum, B3GLCT adds glucose to O-linked fucose on properly folded thrombospondin type 1 repeats (TSRs). The resulting glucose–fucose disaccharide is proposed to stabilize the TSR fold and promote secretion of B3GLCT substrates, with some substrates more sensitive than others to loss of glucose. Mouse B3glct mutants develop hydrocephalus at high frequency. In this study, we demonstrated that B3glct mutant ependymal cells had fewer cilia basal bodies and altered translational polarity compared to controls. Localization of mRNA encoding A Disintegrin and Metalloproteinase with ThromboSpondin type 1 repeat 20 (ADAMTS20) and ADAMTS9 suggested that reduced function of these B3GLCT substrates contributed to ependymal cell abnormalities. In addition, we showed that multiple B3GLCT substrates (Adamts3, Adamts9 and Adamts20) are expressed by the subcommissural organ, that subcommissural organ-spondin ((SSPO) also known as SCO-spondin) TSRs were modified with O-linked glucose–fucose and that loss of B3GLCT reduced secretion of SSPO in cultured cells. In the B3glct mutant, intracellular levels of SSPO were reduced and BiP levels increased, suggesting a folding defect. Secreted SSPO colocalized with BiP, raising the possibility that abnormal extracellular assembly of SSPO into Reissner’s fiber also contributed to impaired CSF flow in mutants. Combined, these studies underscore the complexity of the B3glct mutant hydrocephalus phenotype and demonstrate that impaired cerebrospinal fluid (CSF) flow likely stems from the collective effects of the mutation on multiple processes.
A 5-year multicenter retrospective cohort study was conducted across six hospitals in Niigata, Japan. Patients (n = 179) with bacteremia due to extended-spectrum β-lactamase (ESBL)producing organisms ...were included in the study. The rates of appropriate carbapenem prescription were 61% (n = 41) in patients aged 65–84 years and 89% (n = 31) in those aged ≥ 85 years. Patients aged ≥ 85 years were significantly more likely to receive carbapenem than their younger counterparts. After propensity score matching, 65 patients were assigned to two groups based on age (65–84 years or ≥ 85 years). Multivariate regression analysis showed that other sites of infection had a positive association with 30-day mortality (odds ratio OR, 27.50; 95% confidence interval CI, 2.90–260.00) and biliary tract infection tended to have a positive association with 30-day mortality (OR, 8.90; 95% CI, 0.88– 89.90) compared with urinary tract infection. However, an age ≥ 85 years was not associated with 30-day mortality. Elderly patients aged ≥ 85 years were more likely to be treated with carbapenem; however, old age was not associated with 30-day mortality when bacteremia was caused by ESBLproducing organisms. These results may help clinicians justify withholding carbapenem in patients aged ≥ 85 years.
•Deletion of Pofut2 (Protein O-fucosyltransferase 2) in mouse developing limb mesenchyme blocks O-fucosylation of Thrombospondin type-1 repeats (TSR) and significantly shortens limb bones.•Loss of ...TSR O-fucosylation alters collagen and fibrillins distribution during primary endochondral ossification.•Altered ECM remodeling enhances TGF-β and reduces BMP signaling in developing chondrocytes leading to a reduced hypertrophic zone.•Changes in the ECM composition and signaling likely result from simultaneous reduction in the function and/or level of multiple POFUT2 substrates including members of the ADAMTS family.
Many extracellular matrix (ECM) associated proteins that influence ECM properties have Thrombospondin type 1 repeats (TSRs) which are modified with O-linked fucose. The O-fucose is added in the endoplasmic reticulum to folded TSRs by the enzyme Protein O-fucosyltransferase-2 (POFUT2) and is proposed to promote efficient trafficking of substrates. The importance of this modification for function of TSR-proteins is underscored by the early embryonic lethality of mouse embryos lacking Pofut2. To overcome early lethality and investigate the impact of the Pofut2 knockout on the secretion of POFUT2 substrates and on extracellular matrix properties in vivo, we deleted Pofut2 in the developing limb mesenchyme using Prrx1-Cre recombinase. Loss of Pofut2 in the limb mesenchyme caused significant shortening of the limbs, long bones and tendons and stiff joint resembling the musculoskeletal dysplasias in human and in mice with mutations in ADAMTS or ADAMTSL proteins. Limb shortening was evident at embryonic day 14.5 where loss of O-fucosylation led to an accumulation of fibrillin 2 (FBN2), decreased BMP and IHH signaling, and increased TGF-β signaling. Consistent with these changes we saw a decrease in the size of the hypertrophic zone with lower levels of Collagen-X. Unexpectedly, we observed minimal effects of the Pofut2 knockout on secretion of two POFUT2 substrates, CCN2 or ADAMTS17, in the developing bone. In contrast, CCN2 and two other POFUT2 substrates important for bone development, ADAMTS6 and 10, showed a decrease in secretion from POFUT2-null HEK293T cells in vitro. These combined results suggest that the impact of the Pofut2 mutation is cell-type specific. In addition, these observations raise the possibility that the O-fucose modification on TSRs extends beyond promoting efficient trafficking of POFUT2 substrates and has the potential to influence their function in the extracellular environment.
Abstract
Successful hematopoietic progenitor cell (HPC) transplant therapy is improved by mobilizing HPCs from the bone marrow niche in donors. Notch receptor–ligand interactions are known to retain ...HPCs in the bone marrow, and neutralizing antibodies against Notch ligands, Jagged-1 or Delta-like ligand (DLL4), or NOTCH2 receptor potentiates HPC mobilization. Notch–ligand interactions are dependent on posttranslational modification of Notch receptors with O-fucose and are modulated by Fringe-mediated extension of O-fucose moieties. We previously reported that O-fucosylglycans on Notch are required for Notch receptor–ligand engagement controlling hematopoietic stem cell quiescence and retention in the marrow niche. Here, we generated recombinant fragments of NOTCH1 or NOTCH2 extracellular domain carrying the core ligand-binding regions (EGF11–13) either as unmodified forms or as O-fucosylglycan-modified forms. We found that the addition of O-fucose monosaccharide or the Fringe-extended forms of O-fucose to EGF11–13 showed substantial increases in binding to DLL4. Furthermore, the O-fucose and Fringe-extended NOTCH1 EGF11–13 protein displayed much stronger binding to DLL4 than the NOTCH2 counterpart. When assessed in an in vitro 3D osteoblastic niche model, we showed that the Fringe-extended NOTCH1 EGF11–13 fragment effectively released lodged HPC cells with a higher potency than the NOTCH2 blocking antibody. We concluded that O-fucose and Fringe-modified NOTCH1 EGF11–13 protein can be utilized as effective decoys for stem cell niche localized ligands to potentiate HPC egress and improve HPC collection for hematopoietic cell therapy.
Abstract Babesiosis has attracted attention as a zoonotic disease. The disease is caused in immunocompetent individuals almost solely by Babesia microti , a rodent babesia. Most cases of human ...babesiosis by B. microti have been reported in the endemic foci of the Northeastern coastal areas and upper Midwest regions of the United States, while some sporadic cases have recently been reported in several Asian countries including Japan. Our previous surveys identified that four small subunit ribosomal RNA gene (SSUrDNA) types of B. microti parasitize Japanese rodents. Indirect fluorescent antibody test (IFAT) is often performed for the diagnosis of babesiosis together with microscopical examination of thin blood smears and PCR. We established IFAT against four SSUrDNA-types of B. microti using erythrocytes of SCID mice or Syrian hamsters infected with each SSUrDNA-type B. microti. The results of IFAT for sera of ICR mice or Syrian hamsters infected with each SSUrDNA-type B. microti demonstrated that the four SSUrDNA-types of B. microti have different serotypes. Here, we report technical or practical procedures of IFAT, which gains sufficiently stable results, including procedures of continuous in vivo culture of B. microti.
We hypothesized that quick Sequential Organ Failure Assessment (qSOFA) would be associated with 30-day mortality in bacteremia caused by extended-spectrum β-lactamase (ESBL)-producing bacteria and ...might be a selection criterion for the use of carbapenem as initial empirical therapy. A multicenter retrospective study was conducted in six hospitals. All patients who had bacteremia due to ESBL-producing bacteria were included in the study. Multivariable logistic regression analysis was performed to analyze 30-day mortality as the main outcome. A total of 203 adult patients were identified with bacteremia caused by ESBL-producing Escherichia coli, Klebsiella pneumoniae, or Proteus mirabilis. In multivariate logistic regression analysis, bacteremia caused by ESBL-producing K. pneumoniae or P. mirabilis (odds ratio OR 5.07, 95% confidence interval CI 1.64–15.56), underlying liver disease (OR 3.38, 95% CI 1.09–10.00), and underlying solid cancer (OR 3.45, 95% CI 1.27–9.69) were associated with 30-day mortality. In a subgroup analysis, empirical non-carbapenem therapy was associated with 30-day mortality in bacteremia caused by ESBL-producing K. pneumoniae or P. mirabilis. Our results suggest that the qSOFA score is not a selection criterion for the use of carbapenem in initial empirical therapy.
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