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

Takeuchi, Hideyuki; Schneider, Michael; Williamson, Daniel; Ito, Atsuko; Takeuchi, Megumi; Handford, Penny A.; Haltiwanger, Robert S.

doi:10.1073/pnas.1804005115

Cited by 72 publications

(86 citation statements)

References 38 publications

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“…The mammalian Notch signaling pathway can be regulated by O-glucosylation of EGF repeats of Notch receptors [16]. Takeuchi et al [11] demonstrated the transfer of O-glucose to Notch 1 receptor EGF 11 and Notch 3 EGF10 by either KDELC1 or KDELC2 but not to the Notch 2 receptor. In this study, we successfully proved that KDELC2 knockdown could interrupt KDELC1 expression, which implied that KDELC2 might have some regulations to KDELC1.…”

Section: Discussionmentioning

confidence: 99%

“…NECD comprises abundant surface-modified O-linked glycans, including O-glucose (O-Glc), O-fucose (O-Fuc), and O-GlcNAc [8,10]. These modifications strengthen the linkage between the Notch receptor and ligands and activate Notch signaling [11]. Initially, Notch receptors enter the endoplasmic reticulum (ER) and Golgi apparatus (Golgi) and are modified by some glycans and glycosyltransferases, including Rumi, Pofut1, O-glucosyltransferase 1 (Poglut1), Poglut2 (KDELC1), and Poglut3 (KDELC2) [12].…”

Section: Introductionmentioning

confidence: 99%

“…Initially, Notch receptors enter the endoplasmic reticulum (ER) and Golgi apparatus (Golgi) and are modified by some glycans and glycosyltransferases, including Rumi, Pofut1, O-glucosyltransferase 1 (Poglut1), Poglut2 (KDELC1), and Poglut3 (KDELC2) [12]. KDELC1 and KDELC2 aid transfer of O-glucose to Notch 1 EGF11 and Notch 3 EGF10 [11]. Recently, the Notch receptor activation relied on Pofut1 and Poglut1 expression [13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

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Molecular Mechanisms of KDELC2 on Glioblastoma Tumorigenesis and Temozolomide Resistance

Tsai

Chang

et al. 2020

Biomedicines

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The activation of the Notch pathway induces glioblastoma (GBM) development. Since KDEL (Lys-Asp-Glu-Leu) containing 2 (KDELC2) is involved in the Notch pathway, the detailed mechanism is still undetermined. The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases revealed that KDELC2 mRNA was associated with oncologic factors of GBM. U87, LN229, LNZ308, U118MG, and GBM8401 cells showed higher KDELC2 expression than normal brain tissues. The results of MTT, wound healing, and invasion assays proved that KDELC2 knockdown suppressed GBM-aggressive behaviors. The inhibitory properties of GBM stemness and angiogenesis under KDELC2 knockdown were evaluated by tumor spheroid and tube formation assays. Suppression of KDELC2 downregulated Notch factors’ expressions, including KDELC1, pofut1, Notch receptors 1–3, and HES-1. Immunoblot assay showed that KDELC2 knockdown promoted tumor apoptosis by downregulating PI3k/mTOR/Akt, MAPK/ERK, and NF-kB pathways. The combination of KDELC2 knockdown and temozolomide (TMZ) treatment had an optimal therapeutic effect by suppressing MGMT expression. Results of an orthotopic xenograft animal model and human tissue confirmed that KDELC2 correlated with glioma proliferation, advanced grades, and poor prognosis. Therefore, KDELC2 might be a potential pharmacological target to inhibit tumorigenesis, epithelial–mesenchymal transition, angiogenesis, and chemo-resistance of GBM.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular Mechanisms of KDELC2 on Glioblastoma Tumorigenesis and Temozolomide Resistance

Tsai

Chang

et al. 2020

Biomedicines

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“…Takeuchi and colleagues also identified two novel O -glucosyltransferases, POGLUT2 and POGLUT3, which are responsible for the transfer of UDP-glucose on the 11th EGF repeat on serine 435, a distinct site from that of POGLUT1 [ 17 ]. Mutations occurring in Ser435 affect the NOTCH1 cell-surface presentation and, consequently, its activation upon interaction with Dll1 [ 28 ].…”

Section: From Golgi To the Cell Membranementioning

confidence: 99%

Targeting Notch Trafficking and Processing in Cancers

Pagliaro

Sissi

Roti

2020

Cells

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The Notch family comprises a group of four ligand-dependent receptors that control evolutionarily conserved developmental and homeostatic processes and transmit signals to the microenvironment. NOTCH undergoes remodeling, maturation, and trafficking in a series of post-translational events, including glycosylation, ubiquitination, and endocytosis. The regulatory modifications occurring in the endoplasmic reticulum/Golgi precede the intramembrane γ-secretase proteolysis and the transfer of active NOTCH to the nucleus. Hence, NOTCH proteins coexist in different subcellular compartments and undergo continuous relocation. Various factors, including ion concentration, enzymatic activity, and co-regulatory elements control Notch trafficking. Interfering with these regulatory mechanisms represents an innovative therapeutic way to bar oncogenic Notch signaling. In this review, we briefly summarize the role of Notch signaling in cancer and describe the protein modifications required for NOTCH to relocate across different subcellular compartments. We focus on the functional relationship between these modifications and the corresponding therapeutic options, and our findings could support the development of trafficking modulators as a potential alternative to the well-known γ-secretase inhibitors.

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“…or O-fucosylation C 2 XXXX(S/T)C3 [88,129,130] consensus sequences (the superscript indicates the Cys residue within the EGF-like domain).However, recent studies uncovered new O-glucosyltransferases that can O-glucosylate Ser residues between the 3 rd and 4 th Cys of EGF-like domains[131], precisely where we map the new EGF-like 2 Hex1Pent2 O-glycan in rFIX. In fact, human Coagulation Factor X is O-glucosylated at S 106 in the EGF-like 2 domain, also between the 3 rd and 4 th Cys residues and in a position equivalent to the S 102 residue in rFIX[132].…”

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confidence: 97%

Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

Zacchi

Recinos

Pegg

et al. 2020

Preprint

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Coagulation factor IX (FIX) is a highly complex post-translationally modified human serum glycoprotein and a high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete g-carboxylation, is critical for rFIX clinical efficacy. Changes in bioreactor operating conditions can impact rFIX production and occupancy and structure of rFIX posttranslational modifications (PTMs). We hypothesized that monitoring the bioreactor cell culture supernatant with Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics would allow us to predict product yield and quality after purification. With the goal of optimizing rFIX production, we developed a suite of MS proteomics analytical methods and used these to investigate changes in rFIX yield, g-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. Our methods provided a detailed overview of the dynamics of site-specific PTM occupancy and abundance on rFIX during production, which accurately predicted the efficiency of purification and the quality of the purified product from different culture conditions. In addition, we identified new PTMs in rFIX, some of which were near the GLA domain and could impact rFIX GLA-dependent purification efficiency and protein function. The workflows presented here are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses. Figure 1: Coagulation Factor IX (FIX) structural domains and post-translational modifications (PTMs). Schematic of mature FIX with previously described PTMs in plasma derived FIX and/or recombinant FIX (figure modified from [13]). The GLA domain (red) contains 12 potential sites of g-carboxylation on E 7-40 and one O-glycan at T 38/39 . The EGF-like 1 domain (pink) contains two O-glycans on S 53 and S 61 , b-hydroxylation of D 64 , and phosphorylation of S 68 . The EGF-like 2 domain (violet) has no known PTMs. The short domain (white) linking the EGFlike 2 domain with the activation peptide (AP, green) contains one O-glycan at S 141 . The AP contains two N-glycans at N 157 and N 167 , four O-glycans at T 159 , T 169 , T 172 , and T 179 , sulfation of Y 155 , and phosphorylation of S 158 and T 159 . The serine protease domain (orange) contains one Nlinked glycan at N 258 .FIX is a highly post-translationally modified glycoprotein [7,14] (Fig. 1). Immature FIX is composed of six structural domains: an N-terminal propeptide, the GLA domain, two consecutive EGF-like domains followed by a short linker, the activation peptide (AP), and the C-terminal serine protease domain (Fig. 1). Most described PTMs are on or near the GLA, the EGF-like 1, and the AP domains. The propeptide and the AP are removed through proteolysis events, and both proteolysis are required for function [1,9,15]. FIX's propeptide is cleaved by the trans-Golgi protease PACE/Furin during transit through the secretory pathway [16,17]. FIX's AP is removed in the blood by Factor XIa as part of the c...

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

Cited by 72 publications

References 38 publications

Molecular Mechanisms of KDELC2 on Glioblastoma Tumorigenesis and Temozolomide Resistance

Molecular Mechanisms of KDELC2 on Glioblastoma Tumorigenesis and Temozolomide Resistance

Targeting Notch Trafficking and Processing in Cancers

Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

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