Comprehensive O-GlcNAc glycoproteomics on NOTCH1 EGF repeats refined sequons for O-GlcNAcylation and uncovered unique Lewis X epitopes in mammals

The O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms of O-glycans on epidermal growth factor-like (EGF) repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specific O-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongated O-GlcNAc glycans have not been extensively explored. To extend our view of the specificity and variety of the glycan modification, we conducted a comprehensive analysis of O-GlcNAc glycans on NOTCH1 in mammals. Mass spectrometric analysis of NOTCH1 fragments expressed in HEK293T cells revealed that several EGF domains with putative O-GlcNAcylation sites were hardly modified with O-GlcNAc. Although amino acid residues before the modification site are preferentially occupied with aromatic residues, Phe and Tyr are preferrable to Trp for the apparent modification with O-GlcNAc. Furthermore, a minor form of fucosylated O-GlcNAc glycans was detected in a subset of EGF domains. Fucosylation of O-GlcNAc glycans was enhanced by FUT1, FUT2, or FUT9 expression. The FUT9-dependent Lewis X epitope was confirmed by immunoblotting using an anti-Lewis X antibody. As expected from the similarity in the glycan structures, the Lexis X antigen was detected on O-fucose glycans. Notably, the Lewis X structure on O-glycans was identified in endogenous NOTCH1 isolated from MCF7 cells. Our results refined the putative consensus sequence for the EOGT-dependent extracellular O-GlcNAc modification in mammals and revealed the structural diversity of functional Notch O-glycans.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1804005115 ◽

2018 ◽

Vol 115 (36) ◽

pp. E8395-E8402 ◽

Cited By ~ 25

Author(s):

Hideyuki Takeuchi ◽

Michael Schneider ◽

Daniel B. Williamson ◽

Atsuko Ito ◽

Megumi Takeuchi ◽

...

Keyword(s):

Notch Signaling ◽

Signaling Pathway ◽

Consensus Sequence ◽

Notch Signaling Pathway ◽

Fine Tuning ◽

In Vitro Assays ◽

Spectrometric Analysis ◽

Ligand Interactions ◽

Novel Protein

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.

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In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

Journal of Fungi ◽

10.3390/jof7060439 ◽

2021 ◽

Vol 7 (6) ◽

pp. 439

Author(s):

Tecla Ciociola ◽

Walter Magliani ◽

Tiziano De Simone ◽

Thelma A. Pertinhez ◽

Stefania Conti ◽

...

Keyword(s):

In Silico ◽

Mammalian Cells ◽

Galleria Mellonella ◽

Ex Vivo ◽

Consensus Sequence ◽

In Silico Analysis ◽

Significant Activity ◽

Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

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

Biomolecules ◽

10.3390/biom11020309 ◽

2021 ◽

Vol 11 (2) ◽

pp. 309

Author(s):

Wataru Saiki ◽

Chenyu Ma ◽

Tetsuya Okajima ◽

Hideyuki Takeuchi

Keyword(s):

Notch Signaling ◽

100Th Anniversary ◽

Notch Receptor ◽

Notch Receptors ◽

Evolutionarily Conserved ◽

Future Challenges ◽

Ligand Interactions ◽

Epidermal Growth ◽

Notch Activation ◽

Human Specific

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.

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Poly(ADP-ribosyl)ation of p53 In Vitro and In Vivo Modulates Binding to its DNA Consensus Sequence

Neoplasia ◽

10.1038/sj.neo.7900155 ◽

2001 ◽

Vol 3 (3) ◽

pp. 179-188 ◽

Cited By ~ 30

Author(s):

Cynthia M. Simbulan-Rosenthal ◽

Dean S. Rosenthal ◽

RuiBai Luo ◽

Raed Samara ◽

Mira Jung ◽

...

Keyword(s):

Consensus Sequence

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Metabolism of rat bone proteoglycans in vivo

Biochemical Journal ◽

10.1042/bj2160589 ◽

1983 ◽

Vol 216 (3) ◽

pp. 589-596 ◽

Cited By ~ 53

Author(s):

C W Prince ◽

F Rahemtulla ◽

W T Butler

Keyword(s):

Extraction Procedure ◽

Total Radioactivity ◽

Mineralized Tissue ◽

Guanidinium Chloride ◽

Disaccharide Composition ◽

A Minor ◽

Papain Digestion ◽

Rat Bone

Former evaluations of the role of proteoglycans in mineralization have neglected to address the possibility that the metabolism of proteoglycans may be of significance in this regard. This problem was studied by using radiolabeling in vivo of rat calvaria with [35Sulphate for 2-72 h and a sequential extraction procedure to yield two pools of newly synthesized proteoglycans: one obtained from non-mineralized tissue by extraction with guanidinium chloride (GdmCl) and another obtained only after demineralization with EDTA. Total radioactivity in calvaria was maximal after 12 h of incorporation, but by 36 h had declined to a level that was about 55-65% of maximum. Radioactivity in the GdmCl extract declined steadily after 12 h, whereas that in the EDTA extract remained constant until 36 h, when it began to increase. Each extract contained a minor proteoglycan that eluted at the void volume (Vo) of a Sepharose CL-6B column. Unlike in the EDTA extract, this proteoglycan gradually disappeared from the GdmCl extract. Each extract also contained a major, smaller proteoglycan, with a Kav. of 0.24 and 0.36 in the GdmCl and EDTA extracts respectively. Papain digestion of each extract yielded glycosaminoglycan chains with Kav. values of 0.32 and 0.50 on CL-6B in the GdmCl and EDTA extracts respectively. Digestion of each extract with chondroitinase ABC and chondroitinase AC showed that the glycosaminoglycans were of similar disaccharide composition, with about 85% being 4-sulphated and the remainder 6-sulphated and/or iduronic acid-containing. These data suggest that about 45% of the newly synthesized proteoglycans are removed from the tissue during the course of mineralization.

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Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.5073 ◽

1989 ◽

Vol 9 (11) ◽

pp. 5073-5080 ◽

Cited By ~ 314

Author(s):

M Kozak

Keyword(s):

Context Effects ◽

Consensus Sequence ◽

Negative Regulator ◽

Late Event ◽

Free Translation ◽

Initiator Codon ◽

Translation Systems ◽

Short Versions

The context requirements for recognition of an initiator codon were evaluated in vitro by monitoring the relative use of two AUG codons that were strategically positioned to produce long (pre-chloramphenicol acetyl transferase [CAT]) and short versions of CAT protein. The yield of pre-CAT initiated from the 5'-proximal AUG codon increased, and synthesis of CAT from the second AUG codon decreased, as sequences flanking the first AUG codon increasingly resembled the eucaryotic consensus sequence. Thus, under prescribed conditions, the fidelity of initiation in extracts from animal as well as plant cells closely mimics what has been observed in vivo. Unexpectedly, recognition of an AUG codon in a suboptimal context was higher when the adjacent downstream sequence was capable of assuming a hairpin structure than when the downstream region was unstructured. This finding adds a new, positive dimension to regulation by mRNA secondary structure, which has been recognized previously as a negative regulator of initiation. Translation of pre-CAT from an AUG codon in a weak context was not preferentially inhibited under conditions of mRNA competition. That result is consistent with the scanning model, which predicts that recognition of the AUG codon is a late event that occurs after the competition-sensitive binding of a 40S ribosome-factor complex to the 5' end of mRNA. Initiation at non-AUG codons was evaluated in vitro and in vivo by introducing appropriate mutations in the CAT and preproinsulin genes. GUG was the most efficient of the six alternative initiator codons tested, but GUG in the optimal context for initiation functioned only 3 to 5% as efficiently as AUG. Initiation at non-AUG codons was artifactually enhanced in vitro at supraoptimal concentrations of magnesium.

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Potassium depletion improves myocardial potassium uptake in vivo

AJP Cell Physiology ◽

10.1152/ajpcell.00580.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. C135-C141 ◽

Cited By ~ 7

Author(s):

Henning Bundgaard

Keyword(s):

Uptake Rate ◽

High Plasma ◽

Potassium Depletion ◽

Uptake Capacity ◽

K Uptake ◽

Impulse Generation ◽

Uptake Rates ◽

Clinical Interest ◽

A Minor

Potassium depletion (KD) is a very common clinical entity often associated with adverse cardiac effects. KD is generally considered to reduce muscular Na-K-ATPase density and secondarily reduce K uptake capacity. In KD rats we evaluated myocardial Na-K-ATPase density, ion content, and myocardial K reuptake. KD for 2 wk reduced plasma K to 1.8 ± 0.1 vs. 3.5 ± 0.2 mM in controls ( P < 0.01, n = 7), myocardial K to 80 ± 1 vs. 86 ± 1 μmol/g wet wt ( P < 0.05, n = 7), increased Mg, and induced a tendency to increased Na. Myocardial Na-K-ATPase α2-subunit abundance was reduced by ∼30%, whereas increases in α1- and K-dependent pNPPase activity of 24% ( n = 6) and 13% ( n = 6), respectively, were seen. This indicates an overall upregulation of the myocardial Na-K pump pool. KD rats tolerated a higher intravenous KCl dose. KCl infusion until animals died increased myocardial K by 34% in KD rats and 18% in controls ( P < 0.05, n = 6 for both) but did not induce different net K uptake rates between groups. However, clamping plasma K at ∼5.5 mM by KCl infusion caused a higher net K uptake rate in KD rats (0.22 ± 0.04 vs. 0.10 ± 0.03 μmol·g wet wt−1·min−1; P < 0.05, n = 8). In conclusion, a minor KD-induced decrease in myocardial K increased Na-K pump density and in vivo increased K tolerance and net myocardial K uptake rate during K repletion. Thus the heart is protected from major K losses and accumulates considerable amounts of K during exposure to high plasma K. This is of clinical interest, because a therapeutically induced rise in myocardial K may affect contractility and impulse generation-propagation and may attenuate increased myocardial Na, the hallmark of heart failure.

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Flagellar Morphogenesis: Protein Targeting and Assembly in the Paraflagellar Rod of Trypanosomes

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8191 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8191-8200 ◽

Cited By ~ 63

Author(s):

Philippe Bastin ◽

Thomas H. MacRae ◽

Susan B. Francis ◽

Keith R. Matthews ◽

Keith Gull

Keyword(s):

Cell Cycle ◽

Trypanosoma Brucei ◽

Protein Targeting ◽

Fluorescent Protein ◽

Green Fluorescent Protein Reporter ◽

Mutant Proteins ◽

Green Fluorescent ◽

A Minor ◽

Fluorescent Protein Reporter

ABSTRACT The paraflagellar rod (PFR) of the African trypanosomeTrypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.

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Identification of target genes and a unique cis element regulated by IRF-8 in developing macrophages

Blood ◽

10.1182/blood-2005-01-0080 ◽

2005 ◽

Vol 106 (6) ◽

pp. 1938-1947 ◽

Cited By ~ 96

Author(s):

Tomohiko Tamura ◽

Pratima Thotakura ◽

Tetsuya S. Tanaka ◽

Minoru S. H. Ko ◽

Keiko Ozato

Keyword(s):

Transcription Factor ◽

Cystatin C ◽

Target Genes ◽

De Novo ◽

Consensus Sequence ◽

Binding Motif ◽

Microarray Gene Expression ◽

Cathepsin C ◽

Cis Element

Abstract Interferon regulatory factor-8 (IRF-8)/interferon consensus sequence–binding protein (ICSBP) is a transcription factor that controls myeloid-cell development. Microarray gene expression analysis of Irf-8-/- myeloid progenitor cells expressing an IRF-8/estrogen receptor chimera (which differentiate into macrophages after addition of estradiol) was used to identify 69 genes altered by IRF-8 during early differentiation (62 up-regulated and 7 down-regulated). Among them, 4 lysosomal/endosomal enzyme-related genes (cystatin C, cathepsin C, lysozyme, and prosaposin) did not require de novo protein synthesis for induction, suggesting that they were direct targets of IRF-8. We developed a reporter assay system employing a self-inactivating retrovirus and analyzed the cystatin C and cathepsin C promoters. We found that a unique cis element mediates IRF-8–induced activation of both promoters. Similar elements were also found in other IRF-8 target genes with a consensus sequence (GAAANN[N]GGAA) comprising a core IRF-binding motif and an Ets-binding motif; this sequence is similar but distinct from the previously reported Ets/IRF composite element. Chromatin immunoprecipitation assays demonstrated that IRF-8 and the PU.1 Ets transcription factor bind to this element in vivo. Collectively, these data indicate that IRF-8 stimulates transcription of target genes through a novel cis element to specify macrophage differentiation.

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Structural defects in rat liver deoxyribonucleic acid. Endogenous single-strained regions in comparison with damage induced in vivo by a carcinogen

Biochemical Journal ◽

10.1042/bj1790341 ◽

1979 ◽

Vol 179 (2) ◽

pp. 341-352 ◽

Cited By ~ 17

Author(s):

B W Stewart ◽

P H Huang ◽

M J Brian

Keyword(s):

Rat Liver ◽

Structural Damage ◽

Deae Cellulose ◽

Minor Component ◽

Structural Defects ◽

Structural Abnormality ◽

Denaturation Kinetics ◽

Limited Digestion ◽

A Minor

Rat liver DNA may be separated into two fractions by stepwise elution from benzoylated-DEAE-cellulose with NaCl and caffeine solutions respectively. Other studies using bacterical and yeast DNA suggested that the first fraction contains native DNA, whereas the second may exhibit some degree of single-stranded character. In the present experiments, chromatography of DNA was monitored by labelling in vivo with [methyl-3H]thymidine in rats previously subjected to partial hepatectomy. In animals killed up to 1 h after thymidine injection, radioactivity eluted in the second fraction was inversely related to the incorporation time, being greatest when animals were killed 10 min after radioisotope injection. However, for most experiments, animals were allowed to survive 2-4 weeks after surgery before use, analysis being made on non-dividing DNA. Under these conditions, the proportion of caffeine-eluted DNA was decreased by subjecting the preparation to shear, before chromatography. A procedure that resulted in 12% of the recovered radioactivity being eluted with caffeine was adopted for experiments involving comparisons of the two DNA fractions. Under these conditions, cross-contamination could be detected by rechromatography, but this did not preclude distinction being made between the two fractions in terms of DNA structure. NaCl-eluted DNA did not bind to nitrocellulose filters. Caffeine-eluted DNA was retained by the filters and released by washing with 3mM-Tris/HCl, pH9.4. The fractions did not differ in terms of isopycnic centrifugation in CsCl. The NaCl-eluted fraction migrated as a single band in polyacrylamide gels, and this pattern was not modified by prior digestion with Neurospora crassa endonuclease. In contrast, caffeine-eluted DNA contained a minor component having a wide molecular-weight distribution and was subject to limited digestion by the endonuclease. The kinetics of denaturation of NaCi-eluted DNA in the presence of formaldehyde, in common with unfractionated DNA, were consistent with double-stranded structure. The same analysis of caffeine-eluted DNA revealed structural abnormality equivalent to two defects per 10000 base-pairs. The data are consistent with the minor fraction of rat liver DNA, separated by using benzoylated-DEAE-cellulose, containing regions of local denaturation. We previously showed that administration of the hepatocarcinogen dimethylnitrosamine is associated with an increase in the proportion of caffeine-eluted DNA. In terms of most analysis, differences between DNA fraction from nitrosamine-treated rats were similar to differences exhibited by preparations from control animals. However, structural analysis using denaturation kinetics indicated defects in both the NaCl- and caffeine-eluted DNA isolated from nitrosamine-treated rats. The two fractions differed from each other in that caffeine-eluted DNA exhibited a degree of structural damage far greater than that detected in any preparation from control animals...

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