Cancer-associated Notch receptor variants lead to O-fucosylation defects that deregulate Notch signaling.

NOTCH1 is a transmembrane receptor that initiates a signaling pathway involved in embryonic development of adult tissue homeostasis. The extracellular domain of NOTCH1 is composed largely of epidermal growth factor-like repeats (EGFs), many of which can be O-fucosylated at a specific consensus sequence by protein O-fucosyltransferase 1 (POFUT1). O-fucosylation of NOTCH1 is necessary for its function. The Notch pathway is deregulated in many cancers, and alteration of POFUT1 has been reported in several cancers, but further investigation is needed to assess whether there is deregulation of the Notch pathway associated with mutations that affect O-fucosylation in cancers. Using Biomuta and COSMIC databases, we selected nine NOTCH1 variants that could cause a change in O-fucosylation of key EGFs. Mass spectral glycoproteomic site mapping was used to identify alterations in O-fucosylation of EGFs containing the mutations. Cell-based NOTCH-1 signaling assays, ligand-binding assays, and cellsurface analysis were used to determine the effect of each mutation on Notch activation. Two variants led to a gain of function (GOF), six to a loss of function (LOF), and one had minimal effects. Most GOF and LOF were associated with a change in O-fucosylation. Finally, by comparing our results with known NOTCH1 alterations in cancers from which our mutations originated, we were able to establish a correlation between our results and the known GOF or LOF of NOTCH1 in these cancers. This study shows that point mutations in N1 can lead to alterations in O-fucosylation that deregulate the Notch pathway and be associated with cancer processes.

[Does Early Olaparib Administration Improve Prognosis in Patients with HER2-Negative Metastatic Breast Cancer and BRCA1 and/or BRCA2 Pathogenic Variants?-A Case Report].

We herein describe our experience with patients who had been diagnosed with BRCA1/2 pathogenic variants and metastatic breast cancer. Three patients who experienced postoperative recurrences had received chemotherapy before recurrence, while an additional patient with stage Ⅳ disease at diagnosis required chemotherapy before olaparib administration. Prior anthracycline and/or taxane-based therapies needed prior to administration of poly(adenosine diphosphate ribose) polymerase inhibitors might still be controversial in terms of patient benefits.

ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.

Peters plus syndrome (MIM #261540 PTRPLS), characterized by defects in eye development, prominent forehead, hypertelorism, short stature and brachydactyly, is caused by mutations in the ß3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose ß1-3fucose disaccharide to properly folded thrombospondin type 1 repeats (TSRs). Forty-nine proteins are predicted to be modified by POFUT2, and nearly half are members of the ADAMTS superfamily. Previous studies suggested that O-linked fucose is essential for folding and secretion of POFUT2-modified proteins and that B3GLCT-mediated extension to the disaccharide is essential for only a subset of targets. To test this hypothesis and gain insight into the origin of PTRPLS developmental defects, we developed and characterized two mouse B3glct knockout alleles. Using these models, we tested the role of B3GLCT in enabling function of ADAMTS9 and ADAMTS20, two highly conserved targets whose functions are well characterized in mouse development. The mouse B3glct mutants developed craniofacial and skeletal abnormalities comparable to PTRPLS. In addition, we observed highly penetrant hydrocephalus, white spotting and soft tissue syndactyly. We provide strong genetic and biochemical evidence that hydrocephalus and white spotting in B3glct mutants resulted from loss of ADAMTS20, eye abnormalities from partial reduction of ADAMTS9 and cleft palate from loss of ADAMTS20 and partially reduced ADAMTS9 function. Combined, these results provide compelling evidence that ADAMTS9 and ADAMTS20 were differentially sensitive to B3GLCT inactivation and suggest that the developmental defects in PTRPLS result from disruption of a subset of highly sensitive POFUT2/B3GLCT targets such as ADAMTS20.

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.

Modulation of the NOTCH1 Pathway by LUNATIC FRINGE Is Dominant over That of MANIC or RADICAL FRINGE.

Fringes are glycosyltransferases that transfer a GlcNAc to O-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 O-fucose on EGF repeats in the NOTCH1 (N1) extracellular domain modulates the activation of N1 signaling. Not all O-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 O-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 O-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.

[Relationship between HER2 Gene Amplification and the Therapeutic Effect of Pertuzumab in HER2-Positive Breast Cancer].

OBJECTIVE: We examined the therapeutic effect of adding of pertuzumab in combination with trastuzumab as neoadjuvant chemotherapy for HER2-positive breast cancer. PARTICIPANTS: Overall, 27 patients with HER2-positive breast cancer who had completed neoadjuvant chemotherapy and undergone surgery between January 2014 and January 2020 were included in the study. We performed a retrospective analysis of the relationship between HER2 gene amplification and the therapeutic effect of pertuzumab in this group. For the anti-HER2 treatment, only trastuzumab was administered to all patients until December 2018, and then a combination of trastuzumab and pertuzumab was administered to 4 patients from January 2019. RESULTS: The case distribution based on histological therapeutic effect was: 4 patients with Grade 1, 12 patients with Grade 2, and 11 patients with Grade 3. Patients with immunohistochemistry scores of 3+ and high signal ratios of HER2/ CEP17 in FISH testing tended to have better therapeutic outcomes than other patients. DISCUSSION: Outcomes similar to those observed in this study were reported in other cases where pertuzumab was used for anti-HER2 treatment. We expected that the therapeutic effects of pertuzumab would be better in patients with high HER2 expression. However, further research is required to understand the effects of pertuzumab in patients with cT1c that were not included in the Neo Sphere trial. The therapeutic effects of adding pertuzumab to the treatment in patients with cT1c and lymph node-positive breast cancer were indicated by comparison with our cases.

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.

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 analysis of Notch-regulating Rumi reveals basis for pathogenic mutations.

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.

Genetic and biochemical evidence that gastrulation defects in Pofut2 mutants result from defects in ADAMTS9 secretion.

Protein O-fucosyltransferase 2 (POFUT2) adds O-linked fucose to Thrombospondin Type 1 Repeats (TSR) in 49 potential target proteins. Nearly half the POFUT2 targets belong to the A Disintegrin and Metalloprotease with ThromboSpondin type-1 motifs (ADAMTS) or ADAMTS-like family of proteins. Both the mouse Pofut2 RST434 gene trap allele and the Adamts9 knockout were reported to result in early embryonic lethality, suggesting that defects in Pofut2 mutant embryos could result from loss of O-fucosylation on ADAMTS9. To address this question, we compared the Pofut2 and Adamts9 knockout phenotypes and used Cre-mediated deletion of Pofut2 and Adamts9 to dissect the tissue-specific role of O-fucosylated ADAMTS9 during gastrulation. Disruption of Pofut2 using the knockout (LoxP) or gene trap (RST434) allele, as well as deletion of Adamts9, resulted in disorganized epithelia (epiblast, extraembryonic ectoderm, and visceral endoderm) and blocked mesoderm formation during gastrulation. The similarity between Pofut2 and Adamts9 mutants suggested that disruption of ADAMTS9 function could be responsible for the gastrulation defects observed in Pofut2 mutants. Consistent with this prediction, CRISPR/Cas9 knockout of POFUT2 in HEK293T cells blocked secretion of ADAMTS9. We determined that Adamts9 was dynamically expressed during mouse gastrulation by trophoblast giant cells, parietal endoderm, the most proximal visceral endoderm adjacent to the ectoplacental cone, extraembryonic mesoderm, and anterior primitive streak. Conditional deletion of either Pofut2 or Adamts9 in the epiblast rescues the gastrulation defects, and identified a new role for O-fucosylated ADAMTS9 during morphogenesis of the amnion and axial mesendoderm. Combined, these results suggested that loss of ADAMTS9 function in the extra embryonic tissue is responsible for gastrulation defects in the Pofut2 knockout. We hypothesize that loss of ADAMTS9 function in the most proximal visceral endoderm leads to slippage of the visceral endoderm and altered characteristics of the extraembryonic ectoderm. Consequently, loss of input from the extraembryonic ectoderm and/or compression of the epiblast by Reichert's membrane blocks gastrulation. In the future, the Pofut2 and Adamts9 knockouts will be valuable tools for understanding how local changes in the properties of the extracellular matrix influence the organization of tissues during mammalian development.

Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism.

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.

A retrospective analysis on the utility and complications of upper arm ports in 433 cases at a single institute.

BACKGROUND: We have employed upper arm central venous ports (UACVPs) since 2006 for long-term intravenous chemotherapy (CTx) or fluid supplementation. We evaluated the long-term availability of CVPs implanted in the upper arm to determine whether UACVPs could be one of the treatment options besides chest CVPs in terms of CVP-related complications. METHODS: We reviewed the medical records of all patients who underwent subcutaneous implantation of UACVPs at Kyoto University Hospital from 1 April, 2006 to 30 June, 2009. We assessed the indwelling duration of the UACVPs and the incidences of early and late UACVP-related complications. RESULTS: A total of 433 patients underwent subcutaneous implantation of UACVPs during this time period. The cumulative follow-up period was 251,538 catheter days, and the median duration of UACVP indwelling was 439.0 days (1-2, 24). There was no UACVP-related mortality throughout the study period. A total of 83 UACVP-related complications occurred (19.2 %), including 43 cases of infection (9.9 %, 0.17/1000 catheter days), ten cases of catheter-related thrombosis (2.3 %, 0.040/1000 catheter days), ten cases of occlusion (2.3 %, 0.040/1000 catheter days), nine cases of catheter dislocation (2.0 %, 0.036/1000 catheter days), five cases of port leakage (1.2 %, 0.019/1000 catheter days), four cases of skin dehiscence (0.9 %, 0.015/1000 catheter days) and two cases of port chamber twist (0.5 %, 0.008/1000 catheter days). The removal-free one-year port availability was estimated at 87.8 %. CONCLUSIONS: UACVPs were of long-term utility, with complication rates comparable to those of chest CVPs previously reported.

Orally administered S-1 suppresses circulating endothelial cell counts in metastatic breast cancer patients.

BACKGROUND: S-1 is an oral cytotoxic preparation that contains tegafur. Gamma-butyrolactone (GBL) is a metabolite of tegafur that is known to suppress vascular endothelial growth factor (VEGF)-mediated angiogenic activity. The aim of this study was to determine the change in circulating endothelial cell (CEC) counts, GBL levels, and angiogenesis-related factors during S-1 administration in metastatic breast cancer (MBC) patients. METHODS: Patients with HER2-negative MBC were eligible. S-1 was administered orally twice daily in a 4 week on/2 week off cycle until disease progression or unacceptable toxicity occurred. Blood was collected on the following: days 1, 43, 85 (before each cycle of S-1 administration), days 15, 57 (1 h after S-1 administration), and day 29. The CellSearch(®) system was used to count the CECs. The gas chromatographic-mass spectrometric method was used to measure plasma GBL and 5-FU levels. Levels of VEGF were assayed by enzyme-linked immunosorbent assay. RESULTS: A total of 18 patients were enrolled. The plasma GBL levels on days 15 and 57 were 41.3 ± 15.8 and 41.0 ± 11.2 ng/mL, respectively. The CEC levels decreased on day 15, and significantly low levels were maintained until day 85 (P = 0.002 vs day 1). The plasma VEGF levels significantly decreased on day 15 (P = 0.012 vs day 1) and had a tendency to decrease until day 57. CONCLUSIONS: This exploratory study showed that GBL levels increased, VEGF levels decreased, and CEC levels were suppressed during S-1 administration. S-1 appears to have anti-angiogenic activity.

Disease modeling and gene correction of LGMDR21 iPSCs elucidates the role of POGLUT1 in skeletal muscle maintenance, regeneration, and the satellite cell niche.

Autosomal recessive limb-girdle muscular dystrophy 21 (LGMDR21) is caused by pathogenic variants in protein O-glucosyltransferase 1 (POGLUT1), which is responsible for O-glucosylation of specific epidermal growth factor (EGF) repeats found in ∼50 mammalian proteins, including Notch receptors. Previous data from patient biopsies indicated that impaired Notch signaling, reduction of muscle stem cells, and accelerated differentiation are probably involved in disease etiopathology. Using patient induced pluripotent stem cells (iPSCs), their corrected isotypes, and control iPSCs, gene expression profiling indicated dysregulation of POGLUT1, NOTCH, muscle development, extracellular matrix (ECM), cell adhesion, and migration as involved pathways. They also exhibited reduced in vitro POGLUT1 enzymatic activity and NOTCH signaling as well as defective myogenesis, proliferation, migration and differentiation. Furthermore, in vivo studies demonstrated significant reductions in engraftment, muscle stem cell formation, PAX7 expression, and maintenance, along with an increased percentage of mislocalized PAX7+ cells in the interstitial space. Gene correction in patient iPSCs using CRISPR-Cas9 nickase led to the rescue of the main in vitro and in vivo phenotypes. These results demonstrate the efficacy of iPSCs and gene correction in disease modeling and rescue of the phenotypes and provide evidence of the involvement of muscle stem cell niche localization, PAX7 expression, and cell migration as possible mechanisms in LGMDR21.

Lymphatic mapping with fluorescence navigation using indocyanine green and axillary surgery in patients with primary breast cancer.

The indocyanine green fluorescence (ICGf) navigation method provides real-time lymphatic mapping and sentinel lymph node (SLN) visualization, which enables the removal of SLNs and their associated lymphatic networks. In this study, we investigated the features of the drainage pathways detected with the ICGf navigation system and the order of metastasis in axillary nodes. From April 2008 to February 2010, 145 patients with clinically node-negative breast cancer underwent SLN surgery with ICGf navigation. The video-recorded data from 79 patients were used for lymphatic mapping analysis. We analyzed 145 patients with clinically node-negative breast cancer who underwent SLN surgery with the ICGf navigation system. Fluorescence-positive SLNs were identified in 144 (99%) of 145 patients. Both single and multiple routes to the axilla were identified in 47% of cases using video-recorded lymphatic mapping data. An internal mammary route was detected in 6% of the cases. Skip metastasis to the second or third SLNs was observed in 6 of the 28 node-positive patients. We also examined the strategy of axillary surgery using the ICGf navigation system. We found that, based on the features of nodal involvement, 4-node resection could provide precise information on the nodal status. The ICGf navigation system may provide a different lymphatic mapping result than computed tomography lymphography in clinically node-negative breast cancer patients. Furthermore, it enables the identification of lymph nodes that do not accumulate indocyanine green or dye adjacent to the SLNs in the sequence of drainage. Knowledge of the order of nodal metastasis as revealed by the ICGf system may help to personalize the surgical treatment of axilla in SLN-positive cases, although additional studies are required.

Inappropriate Metacognitive Status Increases State Anxiety in Genetic Counseling Clients.

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Characterization of Ceramides and Glucosylceramides of the Satsuma Mandarin(Citrus unshiu) Fruit.

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.

O-fucosylation of thrombospondin type 1 repeats is essential for ECM remodeling and signaling during bone development.

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-ß 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.

Asparagine Tautomerization in Glycosyltransferase Catalysis. The Molecular Mechanism of Protein O-Fucosyltransferase 1.

O-glycosylation is a post-translational protein modification essential to life. One of the enzymes involved in this process is protein O-fucosyltransferase 1 (POFUT1), which fucosylates threonine or serine residues within a specific sequence context of epidermal growth factor-like domains (EGF-LD). Unlike most inverting glycosyltransferases, POFUT1 lacks a basic residue in the active site that could act as a catalytic base to deprotonate the Thr/Ser residue of the EGF-LD acceptor during the chemical reaction. Using quantum mechanics/molecular mechanics (QM/MM) methods on recent crystal structures, as well as mutagenesis experiments, we uncover the enzyme catalytic mechanism, revealing that it involves proton shuttling through an active site asparagine, conserved among species, which undergoes tautomerization. This mechanism is consistent with experimental kinetic analysis of Caenorhabditis elegans POFUT1 Asn43 mutants, which ablate enzyme activity even if mutated to Asp, the canonical catalytic base in inverting glycosyltransferases. These results will aid inhibitor development for Notch-associated O-glycosylation disorders.