scholarly journals Role of Site-Specific Glycosylation in the I-Like Domain of Integrin β1 in Small Extracellular Vesicle-Mediated Malignant Behavior and FAK Activation

2021 ◽  
Vol 22 (4) ◽  
pp. 1770 ◽  
Author(s):  
Lin Cao ◽  
Yurong Wu ◽  
Xiuxiu Wang ◽  
Xiang Li ◽  
Zengqi Tan ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Biological Function ◽  
Extracellular Vesicle ◽  
Wild Type ◽  
Mcf7 Cells ◽  
Integrin Β1 ◽  
Site Specific ◽  
Glycosylation Sites ◽  
Mutant Cells

Integrin β1 plays an essential role in the crosstalk between tumor cells and their microenvironment. Aberrant N-glycosylation of integrin β1 was documented to alter integrin β1 expression, dimerization, and biological function. However, the biological function of site-specific N-glycosylation of integrin β1 on extracellular vesicles is not fully understood. In this study, we mutated putative N-glycosylation sites in different domains of integrin β1. Removal of the N-glycosylation sites on the I-like domain of integrin β1 (termed the Δ4–6 β1 mutant) suppressed focal adhesion kinase (FAK) signaling, cell migration, and adhesion compared with other β1 mutants. Cell adhesion, migration, and activation of FAK were suppressed in recipient MCF7 cells co-cultured with Δ4–6 mutant cells and treated with small extracellular vesicles (sEVs) from Δ4–6 mutant cells. Notably, the wild-type and β1 mutant were both present in sEVs, and could be transferred to recipient cells via sEVs, resulting in changes of cell behavior. Our findings demonstrate the important roles of N-glycosylation of the I-like domain of integrin β1. Moreover, the vesicular Δ4–6 β1 mutant can regulate integrin-mediated functions in recipient cells via sEVs.

scholarly journals Extracellular Vesicles Derived fromAcholeplasma laidlawiiPG8

2011 ◽  
Vol 11 ◽  
pp. 1120-1130 ◽  
Author(s):  
Vladislav M. Chernov ◽  
Olga A. Chernova ◽  
Alexey A. Mouzykantov ◽  
Irina R. Efimova ◽  
Gulnara F. Shaymardanova ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Human Peripheral Blood ◽  
Genetic Material ◽  
Membrane Vesicles ◽  
Extracellular Vesicle ◽  
Ultrastructural Organization ◽  
Gram Negative Bacteria ◽  
The Common ◽  
Intensive Formation

Extracellular vesicle production is believed to be a ubiquitous process in bacteria, but the data on such a process in Mollicutes are absent. We report the isolation of ultramicroforms – extracellular vesicles from supernatants ofAcholeplasma laidlawiiPG8 (ubiquitous mycoplasma; the main contaminant of cell culture). Considering sizes, morphology, and ultrastructural organization, the ultramicroforms ofA. laidlawiiPG8 are similar to membrane vesicles of Gram-positive and Gram-negative bacteria. We demonstrate thatA. laidlawiiPG8 vesicles contain genetic material and proteins, and are mutagenic to lymphocytes of human peripheral blood. We show thatMycoplasma gallisepticumS6, the other mycoplasma, also produce similar structures, which suggests that shedding of the vesicles might be the common phenomenon in Mollicutes. We found that the action of stress conditions results in the intensive formation of ultramicroforms in mycoplasmas. The role of vesicular formation in mycoplasmas remains to be studied.

scholarly journals Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

1998 ◽  
Vol 18 (11) ◽  
pp. 6805-6815 ◽  
Author(s):  
Jens Solsbacher ◽  
Patrick Maurer ◽  
F. Ralf Bischoff ◽  
Gabriel Schlenstedt
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Wild Type ◽  
Importin Α ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, theSaccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei ofcse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.

scholarly journals Factor XII in PMM2-CDG patients: role of N-glycosylation in the secretion and function of the first element of the contact pathway

2020 ◽  
Vol 15 (1) ◽  
Author(s):  
Raquel López-Gálvez ◽  
María Eugenia de la Morena-Barrio ◽  
Alberto López-Lera ◽  
Monika Pathak ◽  
Antonia Miñano ◽  
...  
Keyword(s):  
Factor Xii ◽  
Clinical Effects ◽  
Wild Type ◽  
Vascular Events ◽  
Contact Phase ◽  
Glycosylation Sites ◽  
Expression Model ◽  
Contact Pathway ◽  
And Function

Abstract Background Congenital disorders of glycosylation (CDG) are rare diseases with impaired glycosylation and multiorgan disfunction, including hemostatic and inflammatory disorders. Factor XII (FXII), the first element of the contact phase, has an emerging role in hemostasia and inflammation. FXII deficiency protects against thrombosis and the p.Thr309Lys variant is involved in hereditary angioedema through the hyperreactivity caused by the associated defective O-glycosylation. We studied FXII in CDG aiming to supply further information of the glycosylation of this molecule, and its functional and clinical effects. Plasma FXII from 46 PMM2-CDG patients was evaluated by coagulometric and by Western Blot in basal conditions, treated with N-glycosydase F or activated by silica or dextran sulfate. A recombinant FXII expression model was used to validate the secretion and glycosylation of wild-type and variants targeting the two described FXII N-glycosylation sites (p.Asn230Lys; p.Asn414Lys) as well as the p.Thr309Lys variant. Results PMM2-CDG patients had normal FXII levels (117%) but high proportions of a form lacking N-glycosylation at Asn414. Recombinant FXII p.Asn230Lys, and p.Asn230Lys&p.Asn414Lys had impaired secretion and increased intracellular retention compared to wild-type, p.Thr309Lys and p.Asn414Lys variants. The hypoglycosylated form of PMM2-CDG activated similarly than FXII fully glycosylated. Accordingly, no PMM2-CDG had angioedema. FXII levels did not associate to vascular events, but hypoglycosylated FXII, like hypoglycosylated transferrin, antithrombin and FXI levels did it. Conclusions N-glycosylation at Asn230 is essential for FXII secretion. PMM2-CDG have high levels of FXII lacking N-glycosylation at Asn414, but this glycoform displays similar activation than fully glycosylated, explaining the absence of angioedema in CDG.

scholarly journals TET-mediated 5-methylcytosine oxidation in tRNA promotes translation

2020 ◽  
pp. jbc.RA120.014226
Author(s):  
Hui Shen ◽  
Robert Jordan Ontiveros ◽  
Michael C Owens ◽  
Monica Yun Liu ◽  
Uday Ghanty ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Biological Function ◽  
Catalytic Domain ◽  
Embryonic Stem ◽  
Wild Type ◽  
Oxidation Activity ◽  
Tet Enzymes ◽  
Mediated Oxidation

Oxidation of 5-methylcytosine (5mC) in DNA by the Ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNAs). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison to wild type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma.

scholarly journals Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan.

1996 ◽  
Vol 132 (6) ◽  
pp. 1199-1208 ◽  
Author(s):  
A Bartolazzi ◽  
A Nocks ◽  
A Aruffo ◽  
F Spring ◽  
I Stamenkovic
Keyword(s):  
Cell Adhesion ◽  
Regulatory Mechanism ◽  
Cell Attachment ◽  
Glycosylation Site ◽  
Side Chain ◽  
Site Specific ◽  
Coated Substrate ◽  
Glycosylation Sites ◽  
Coated Surfaces

CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44-hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44-mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N-linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.

Human acetyl-CoA:glucosamine-6-phosphate N-acetyltransferase 1 has a relaxed donor specificity and transfers acyl groups up to four carbons in length

2016 ◽  
Vol 94 (2) ◽  
pp. 197-204 ◽  
Author(s):  
Inka Brockhausen ◽  
Dileep G. Nair ◽  
Min Chen ◽  
Xiaojing Yang ◽  
John S. Allingham ◽  
...  
Keyword(s):  
Amino Acids ◽  
Articular Cartilage ◽  
Enzymatic Synthesis ◽  
Biomedical Applications ◽  
Binding Pocket ◽  
Wild Type ◽  
Acetyl Coenzyme A ◽  
Site Specific ◽  
Acetyl Group

Glucosamine-6-phosphate N-acetyltransferase1 (GNA1) catalyses the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to glucosamine-6-phosphate (GlcN6P) to form N-acetylglucosamine-6-phosphate (GlcNAc6P), which is an essential intermediate in UDP-GlcNAc biosynthesis. An analog of GlcNAc, N-butyrylglucosamine (GlcNBu) has shown healing properties for bone and articular cartilage in animal models of arthritis. The goal of this work was to examine whether GNA1 has the ability to transfer a butyryl group from butyryl-CoA to GlcN6P to form GlcNBu6P, which can then be converted to GlcNBu. We developed fluorescent and radioactive assays and examined the donor specificity of human GNA1. Acetyl, propionyl, n-butyryl, and isobutyryl groups were all transferred to GlcN6P, but isovaleryl-CoA and decanoyl-CoA did not serve as donor substrates. Site-specific mutants were produced to examine the role of amino acids potentially affecting the size and properties of the AcCoA binding pocket. All of the wild type and mutant enzymes showed activities of both acetyl and butyryl transfer and can therefore be used for the enzymatic synthesis of GlcNBu for biomedical applications.

scholarly journals Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity

2021 ◽  
Vol 17 (3) ◽  
pp. e1009356
Author(s):  
Takaaki Koma ◽  
Cheng Huang ◽  
Adrian Coscia ◽  
Steven Hallam ◽  
John T. Manning ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Rational Design ◽  
Critical Role ◽  
Case Fatality ◽  
Hemorrhagic Fever ◽  
Wild Type ◽  
Virus Growth ◽  
Glycosylation Sites

Several arenaviruses cause hemorrhagic fevers in humans with high case fatality rates. A vaccine named Candid#1 is available only against Junin virus (JUNV) in Argentina. Specific N-linked glycans on the arenavirus surface glycoprotein (GP) mask important epitopes and help the virus evade antibody responses. However the role of GPC glycans in arenavirus pathogenicity is largely unclear. In a lethal animal model of hemorrhagic fever-causing Machupo virus (MACV) infection, we found that a chimeric MACV with the ectodomain of GPC from Candid#1 vaccine was partially attenuated. Interestingly, mutations resulting in acquisition of N-linked glycans at GPC N83 and N166 frequently occurred in late stages of the infection. These glycosylation sites are conserved in the GPC of wild-type MACV, indicating that this is a phenotypic reversion for the chimeric MACV to gain those glycans crucial for infection in vivo. Further studies indicated that the GPC mutant viruses with additional glycans became more resistant to neutralizing antibodies and more virulent in animals. On the other hand, disruption of these glycosylation sites on wild-type MACV GPC rendered the virus substantially attenuated in vivo and also more susceptible to antibody neutralization, while loss of these glycans did not affect virus growth in cultured cells. We also found that MACV lacking specific GPC glycans elicited higher levels of neutralizing antibodies against wild-type MACV. Our findings revealed the critical role of specific glycans on GPC in arenavirus pathogenicity and have important implications for rational design of vaccines against this group of hemorrhagic fever-causing viruses.

scholarly journals U2AF1(S34F) Mutant Hematopoietic Cells Require Expression of Wild-Type U2af1 for Survival

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 941-941
Author(s):  
Brian Wadugu ◽  
Amanda Heard ◽  
Joseph Bradley ◽  
Matthew Ndonwi ◽  
Jin J Shao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Splicing ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Poly I:C ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Mutant Cells ◽  
Marrow Cells

Abstract Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tumor-derived extracellular vesicles regulate tumor-infiltrating regulatory T cells via the inhibitory immunoreceptor CD300a

2020 ◽  
Author(s):  
Yuta Nakazawa ◽  
Kazumasa Kanemaru ◽  
Chigusa Nakahashi-Oda ◽  
Akira Shibuya
Keyword(s):  
Dendritic Cells ◽  
Treg Cell ◽  
Cell Activation ◽  
Extracellular Vesicle ◽  
Treg Cells ◽  
Wild Type ◽  
Cell Numbers ◽  
Tumor Microenvironments ◽  
Deficient Mice

AbstractAlthough tumor-infiltrating regulatory T (Treg) cells play a pivotal role in tumor immunity, how Treg cell activation are regulated in tumor microenvironments remains unclear. Here, we found that mice deficient in the inhibitory immunoreceptor CD300a on their dendritic cells (DCs) have increased numbers of Treg cells in tumors and greater tumor growth compared with wild-type mice after transplantation of B16 melanoma. Pharmacological impairment of extracellular vesicle (EV) release decreased Treg cell numbers in CD300a-deficient mice. Coculture of DCs with tumor-derived EV (TEV) induced the internalization of CD300a and the incorporation of EVs into endosomes, in which CD300a inhibited TEV-mediated TLR3-TRIF signaling for activation of the IFN-β-Treg cells axis. We also show that higher expression of CD300A was associated with decreased tumor-infiltrating Treg cells and longer survival time in patients with melanoma. Our findings reveal the role of TEV and CD300a on DCs in Treg cell activation in the tumor microenvironment.

scholarly journals Quantitative glycoproteomics reveals new classes of STT3A- and STT3B-dependent N-glycosylation sites

2019 ◽  
Vol 218 (8) ◽  
pp. 2782-2796 ◽  
Author(s):  
Natalia A. Cherepanova ◽  
Sergey V. Venev ◽  
John D. Leszyk ◽  
Scott A. Shaffer ◽  
Reid Gilmore
Keyword(s):  
Protein Translocation ◽  
Site Occupancy ◽  
Glycosylation Site ◽  
Wild Type ◽  
New Class ◽  
Cysteine Rich Protein ◽  
Glycosylation Sites ◽  
Flanking Sequences ◽  
Dependent Site ◽  
Mutant Cells

Human cells express two oligosaccharyltransferase complexes (STT3A and STT3B) with partially overlapping functions. The STT3A complex interacts directly with the protein translocation channel to mediate cotranslational glycosylation, while the STT3B complex can catalyze posttranslocational glycosylation. We used a quantitative glycoproteomics procedure to compare glycosylation of roughly 1,000 acceptor sites in wild type and mutant cells. Analysis of site occupancy data disclosed several new classes of STT3A-dependent acceptor sites including those with suboptimal flanking sequences and sites located within cysteine-rich protein domains. Acceptor sites located in short loops of multi-spanning membrane proteins represent a new class of STT3B-dependent site. Remarkably, the lumenal ER chaperone GRP94 was hyperglycosylated in STT3A-deficient cells, bearing glycans on five silent sites in addition to the normal glycosylation site. GRP94 was also hyperglycosylated in wild-type cells treated with ER stress inducers including thapsigargin, dithiothreitol, and NGI-1.

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