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.
Ceramide (Cer) and glucosylceramide (GlcCer) were isolated from Satsuma mandarin (Citrus unshiu) fruits and characterized. 2,3-Dihydroxy fatty acids with C20 or longer acyl chains were found in Cer. ...GlcCers from the flesh of the fruit contained sphingosine (4-trans-sphingenine) as a major component. Notably, the Cer content was 1.5-fold higher than GlcCer content. The ratio of Cer plus GlcCer to the total lipid content in Satsuma mandarin was higher than that in the other citrus fruits analyzed in this study. Collectively, the pomace of the Satsuma mandarin fruit can be a good source of sphingolipids as functional components in foods.
A major question remaining in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xyloside α-1,3-xylosyltransferase (XXYLT1) is a ...retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly, the epidermal growth factor-like repeat acceptor substrate undergoes a large conformational change upon binding to the active site, providing a structural basis for substrate specificity. Our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations in which Notch dysregulation is known to cause cancer or developmental disorders.
Many genetic counseling (GC) studies have focused on anxiety status because clients of GC often feel anxious during their visits. Metacognition is known to be one of the causes of having an ...inappropriate thinking style. In this study, we examined the relationship between anxiety and the metacognitive status of GC clients according to their characteristics.
The participants were 106 clients who attended their first GC session in our hospital from November 2018 to March 2021. The survey items were the clients' characteristics, anxiety status at the time of the visit, and metacognitive status.
High state anxiety and high trait anxiety were observed in 34.9 and 11.3% of clients, respectively. Clients who were a relative or had a family history were significantly more likely to have high state anxiety. As for metacognitive status, only negative beliefs about thoughts concerning uncontrollability and danger were associated with having an anxiety status. Furthermore, multivariate analysis showed that negative beliefs about thoughts concerning uncontrollability and danger were an independent determinant of higher state anxiety, but not being a relative or having a family history. Metacognitive status scores were significantly lower in clients than in the control group.
State anxiety was shown to be more dependent on negative beliefs about thoughts concerning uncontrollability and danger of GC clients than their characteristics such as being a relative or having a family history. The results of this study will contribute to the development of new GC psychosocial support measures to address the anxiety of GC clients.
Fringes are glycosyltransferases that transfer a GlcNAc to
-fucose residues on Epidermal Growth Factor-like (EGF) repeats. Three Fringes exist in mammals: LUNATIC FRINGE (LFNG), MANIC FRINGE (MFNG), ...and RADICAL FRINGE (RFNG). Fringe modification of
-fucose on EGF repeats in the NOTCH1 (N1) extracellular domain modulates the activation of N1 signaling. Not all
-fucose residues of N1 are modified by all Fringes; some are modified by one or two Fringes and others not modified at all. The distinct effects on N1 activity depend on which Fringe is expressed in a cell. However, little data is available on the effect that more than one Fringe has on the modification of
-fucose residues and the resulting downstream consequence on Notch activation. Using mass spectral glycoproteomic site mapping and cell-based N1 signaling assays, we compared the effect of co-expression of N1 with one or more Fringes on modification of
-fucose and activation of N1 in three cell lines. Individual expression of each Fringe with N1 in the three cell lines revealed differences in modulation of the Notch pathway dependent on the presence of endogenous Fringes. Despite these cell-based differences, co-expression of several Fringes with N1 demonstrated a dominant effect of LFNG over MFNG or RFNG. MFNG and RFNG appeared to be co-dominant but strongly dependent on the ligands used to activate N1 and on the endogenous expression of Fringes. These results show a hierarchy of Fringe activity and indicate that the effect of MFNG and/or RFNG could be small in the presence of LFNG.
Rumi O-glucosylates the EGF repeats of a growing list of proteins essential in metazoan development, including Notch. Rumi is essential for Notch signaling, and Rumi dysregulation is linked to ...several human diseases. Despite Rumi's critical roles, it is unknown how Rumi glucosylates a serine of many but not all EGF repeats. Here we report crystal structures of Drosophila Rumi as binary and ternary complexes with a folded EGF repeat and/or donor substrates. These structures provide insights into the catalytic mechanism and show that Rumi recognizes structural signatures of the EGF motif, the U-shaped consensus sequence, C-X-S-X-(P/A)-C and a conserved hydrophobic region. We found that five Rumi mutations identified in cancers and Dowling-Degos disease are clustered around the enzyme active site and adversely affect its activity. Our study suggests that loss of Rumi activity may underlie these diseases, and the mechanistic insights may facilitate the development of modulators of Notch signaling.
Objectives
Neuropathy is considered a complication of rheumatoid arthritis (RA), whose underlying mechanisms are mainly entrapment, drug‐induced and rheumatoid vasculitis (RV). At Tokyo Women's ...Medical University, for the purpose of diagnosing vasculitic neuropathy, we carried out nerve and muscle biopsies in nine RA patients. Unexpectedly, we found three cases of demyelinating neuropathy, together with six cases of RV. Our aim was to investigate the neurophysiological features of demyelinating neuropathy in patients with RA, compared with those of patients with RV.
Methods
We reviewed the pathological, clinical and electrophysiological findings in patients with RA who underwent nerve and muscle biopsies. We compared patient demographics and nerve conduction study findings between patients with RV and other patients.
Results
The histological findings showed necrotizing vasculitis in six of nine patients. Vasculitis was absent in the other three patients, which showed evidence of demyelination and remyelination. The absence of a sensory nerve action potential and compound motor action potential were observed more frequently in the RV cohort. Sural sparing, shown as the sural‐to‐median sensory nerve action potential ratio, was significantly higher in demyelinating neuropathy patients. Treatment was corticosteroid and cyclophosphamide in RV patients, and intravenous immunoglobulin was administered to three demyelinating neuropathy patients. Treatment response was satisfactory in seven of the nine patients.
Conclusion
Demyelinating neuropathy was found more often in patients with RA neuropathy than expected. Sural nerve sparing, as well as the absence of sensory nerve action potential or compound motor action potential, are useful for the differential diagnosis of demyelinating neuropathy with RV in RA patients with neuropathy.
Demyelinating neuropathy was found more often in patients with RA neuropathy than expected. Particular nerve conduction studies parameters are useful for the differential diagnosis of RA patients with neuropathy.
•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.
Studies on evoked responses in Parkinson's disease (PD) may be useful for elucidating the etiology and quantitative evaluation of PD. However, in previous studies, the association between evoked ...responses and detailed motor symptoms or cognitive functions has not been clear. This study investigated the characteristics of the visual (VEF), auditory (AEF), and somatosensory (SEF) evoked magnetic fields in patients with Parkinson's disease (PD), and the correlations between evoked fields and the patient's clinical characteristics, motor symptoms, and cognitive functions. Twenty patients with PD and 10 healthy controls (HCs) were recruited as participants. We recorded VEF, AEF, and SEF, collected clinical characteristics, performed physical examinations, and administered 10 cognitive tests. We investigated differences in the latencies of the evoked fields between patients with PD and HCs. We also evaluated the correlation of the latencies with motor symptoms and cognitive functioning. There were significant differences between the two groups in 6 of the cognitive tests, all of which suggested mild cognitive impairment in patients with PD. The latencies of the VEF N75m, P100m, N145m, AEF P50m, P100m, and SEF P60m components were greater in the patients with PD than in the HCs. The latencies mainly correlated with medication and motor symptoms, less so with cognitive tests, with some elements of the correlations remaining significant after Bonferroni correction. In conclusion, the latencies of the VEF, AEF, and SEF were greater in PD patients than in HCs and were mainly correlated with medication and motor symptoms rather than cognitive functioning. Findings from this study suggest that evoked fields may reflect basal ganglia functioning and are candidates for assessing motor symptoms or the therapeutic effects of medication in patients with PD.
PDF
