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Oliveira T, Zhang M, Chen CW, Packer NH, von Itzstein M, Heisterkamp N, Kolarich D. Remodelling of the glycome of B-cell precursor acute lymphoblastic leukemia cells developing drug-tolerance. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.08.22.609211. [PMID: 39229073 PMCID: PMC11370571 DOI: 10.1101/2024.08.22.609211] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/05/2024]
Abstract
Reduced responsiveness of precursor B-acute lymphoblastic leukemia (BCP-ALL) to chemotherapy can be first detected in the form of minimal residual disease leukemia cells that persist after 28 days of initial treatment. The ability of these cells to resist chemotherapy is partly due to the microenvironment of the bone marrow, which promotes leukemia cell growth and provides protection, particularly under these conditions of stress. It is unknown if and how the glycocalyx of such cells is remodelled during the development of tolerance to drug treatment, even though glycosylation is the most abundant cell surface post-translational modification present on the plasma membrane. To investigate this, we performed omics analysis of BCP-ALL cells that survived a 30-day vincristine chemotherapy treatment while in co-culture with bone marrow stromal cells. Proteomics showed decreased levels of some metabolic enzymes. Overall glycocalyx changes included a shift from Core-2 to less complex Core-1 O-glycans, and reduced overall sialylation, with a shift from α2-6 to α2-3 linked Neu5Ac. Interestingly, there was a clear increase in bisecting complex N-glycans with a concomitant increased mRNA expression of MGAT3 , the only enzyme known to form bisecting N-glycans. These small but reproducible quantitative differences suggest that individual glycoproteins become differentially glycosylated. Glycoproteomics confirmed glycosite-specific modulation of cell surface and lysosomal proteins in drug-tolerant BCP-ALL cells, including HLA-DRA, CD38, LAMP1 and PPT1. We conclude that drug-tolerant persister leukemia cells that grow under continuous chemotherapy stress have characteristic glycotraits that correlate with and perhaps contribute to their ability to survive and could be tested as neoantigens in drug-resistant leukemia.
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Sebzda T, Karwacki J, Cichoń A, Modrzejewska K, Heimrath J, Łątka M, Gnus J, Gburek J. Association of Serum Proteases and Acute Phase Factors Levels with Survival Outcomes in Patients with Colorectal Cancer. Cancers (Basel) 2024; 16:2471. [PMID: 39001534 PMCID: PMC11240471 DOI: 10.3390/cancers16132471] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2024] [Revised: 06/27/2024] [Accepted: 07/03/2024] [Indexed: 07/16/2024] Open
Abstract
Colorectal cancer (CRC) represents a substantial burden on global healthcare, contributing to significant morbidity and mortality worldwide. Despite advances in screening methodologies, its incidence remains high, necessitating continued efforts in early detection and treatment. Neoplastic invasion and metastasis are primary determinants of CRC lethality, emphasizing the urgency of understanding underlying mechanisms to develop effective therapeutic strategies. This study aimed to explore the potential of serum biomarkers in predicting survival outcomes in CRC patients, with a focus on cathepsin B (CB), leukocytic elastase (LE), total sialic acid (TSA), lipid-associated sialic acid (LASA), antitrypsin activity (ATA), C-reactive protein (CRP), and cystatin C (CC). We recruited 185 CRC patients and 35 healthy controls, assessing demographic variables, tumor characteristics, and 7 serum biomarker levels, including (1) CB, (2) LE, (3) TSA, (4) LASA, (5) ATA, (6) CRP, and (7) CC. Statistical analyses included ANOVA with Tukey's post hoc tests and MANOVA for continuous variables. Student's t-test was used for dependent samples, while non-parametric tests like Mann-Whitney U and Wilcoxon signed-rank tests were applied for variables deviating from the normal distribution. Categorical variables were assessed using chi-square and Kruskal-Wallis tests. Spearman's rank correlation coefficient was utilized to examine variable correlations. Survival analysis employed the Kaplan-Meier method with a log-rank test for comparing survival times between groups. Significant associations were observed between CB (p = 0.04), LE (p = 0.01), and TSA (p = 0.008) levels and survival outcomes in CRC patients. Dukes' classification stages also showed a significant correlation with survival (p = 0.001). However, no significant associations were found for LASA, ATA, CRP, and CC. Multivariate analysis of LE, TSA, and ATA demonstrated a notable correlation with survival (p = 0.041), notwithstanding ATA's lack of significance in univariate analysis (p = 0.13). CB, LE, and TSA emerged as promising diagnostic markers with prognostic value in CRC, potentially aiding in early diagnosis and treatment planning. Further research is needed to validate these findings and explore additional prognostic indicators.
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Affiliation(s)
- Tadeusz Sebzda
- Department of Pathophysiology, Wroclaw Medical University, 50-368 Wroclaw, Poland;
| | - Jakub Karwacki
- Department of Pathophysiology, Wroclaw Medical University, 50-368 Wroclaw, Poland;
- University Center of Excellence in Urology, Department of Minimally Invasive and Robotic Urology, Wroclaw Medical University, 50-556 Wroclaw, Poland
| | - Anna Cichoń
- Regional Specialist Hospital of St. Barbara, 41-200 Sosnowiec, Poland;
| | | | | | - Mirosław Łątka
- Department of Biomedical Engineering, Wroclaw University of Science and Technology, 50-370 Wroclaw, Poland;
| | - Jan Gnus
- Department of Physiotherapy, Wroclaw Medical University, 50-355 Wroclaw, Poland;
| | - Jakub Gburek
- Department of Pharmaceutical Biochemistry, Wroclaw Medical University, 50-556 Wroclaw, Poland
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Anim MT, Tuffour I, Willis R, Schell M, Ostlund T, Mahnashi MH, Halaweish F, Willand-Charnley R. Deacetylated Sialic Acid Sensitizes Lung and Colon Cancers to Novel Cucurbitacin-Inspired Estrone Epidermal Growth Factor Receptor (EGFR) Inhibitor Analogs. Molecules 2023; 28:6257. [PMID: 37687086 PMCID: PMC10488366 DOI: 10.3390/molecules28176257] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2023] [Revised: 08/14/2023] [Accepted: 08/19/2023] [Indexed: 09/10/2023] Open
Abstract
Cancers utilize sugar residues such as sialic acids (Sia) to improve their ability to survive. Sia presents a variety of functional group alterations, including O-acetylation on the C6 hydroxylated tail. Previously, sialylation has been reported to suppress EGFR activation and increase cancer cell sensitivity to Tyrosine Kinase Inhibitors (TKIs). In this study, we report on the effect of deacetylated Sia on the activity of three novel EGFR-targeting Cucurbitacin-inspired estrone analogs (CIEAs), MMA 294, MMA 321, and MMA 320, in lung and colon cancer cells. Acetylation was modulated by the removal of Sialate O-Acetyltransferase, also known as CAS1 Domain-containing protein (CASD1) gene via CRISPR-Cas9 gene editing. Using a variety of cell-based approaches including MTT cell viability assay, flow cytometry, immunofluorescence assay and in-cell ELISA we observed that deacetylated Sia-expressing knockout cells (1.24-6.49 μM) were highly sensitive to all CIEAs compared with the control cells (8.82-20.97 μM). Apoptosis and varied stage cell cycle arrest (G0/G1 and G2/M) were elucidated as mechanistic modes of action of the CIEAs. Further studies implicated overexpression of CIEAs' cognate protein target, phosphorylated EGFR, in the chemosensitivity of the deacetylated Sia-expressing knockout cells. This observation correlated with significantly decreased levels of key downstream proteins (phosphorylated ERK and mTOR) of the EGFR pathway in knockout cells compared with controls when treated with CIEAs. Collectively, our findings indicate that Sia deacetylation renders lung and colon cancer cells susceptible to EGFR therapeutics and provide insights for future therapeutic interventions.
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Affiliation(s)
- Mathias T. Anim
- Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA; (M.T.A.); (I.T.); (R.W.); (M.S.); (T.O.); (F.H.)
| | - Isaac Tuffour
- Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA; (M.T.A.); (I.T.); (R.W.); (M.S.); (T.O.); (F.H.)
| | - Rylan Willis
- Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA; (M.T.A.); (I.T.); (R.W.); (M.S.); (T.O.); (F.H.)
| | - Matthew Schell
- Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA; (M.T.A.); (I.T.); (R.W.); (M.S.); (T.O.); (F.H.)
| | - Trevor Ostlund
- Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA; (M.T.A.); (I.T.); (R.W.); (M.S.); (T.O.); (F.H.)
| | - Mater H. Mahnashi
- Department of Pharmaceutical Chemistry, Najran University, Najran P.O. Box 1988, Saudi Arabia;
| | - Fathi Halaweish
- Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA; (M.T.A.); (I.T.); (R.W.); (M.S.); (T.O.); (F.H.)
| | - Rachel Willand-Charnley
- Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA; (M.T.A.); (I.T.); (R.W.); (M.S.); (T.O.); (F.H.)
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Sebzda T, Gnus J, Dziadkowiec B, Latka M, Gburek J. Diagnostic usefulness of selected proteases and acute phase factors in patients with colorectal adenocarcinoma. World J Gastroenterol 2021; 27:6673-6688. [PMID: 34754160 PMCID: PMC8554409 DOI: 10.3748/wjg.v27.i39.6673] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/07/2021] [Revised: 07/07/2021] [Accepted: 09/02/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Uncontrolled growth and loss of control over basic metabolic functions, leading to invasive proliferation and metastases, are the salient traits of malignant tumors in general and colorectal cancer in particular. Invasion and metastases hinder effective tumor treatment. While surgical techniques and radiotherapy can be used to remove tumor focus, only chemotherapy can eliminate dispersed neoplastic cells. However, the efficacy of the latter method is limited in the advanced stages of the disease. Therefore, recognition of the mechanisms involved in neoplastic cell spreading is indispensable for developing effective therapies.
AIM To use a number of biomarkers involved in cancer progression and identify a panel that could be used for effective early diagnosis.
METHODS We recruited 185 patients with colorectal adenocarcinoma (98 men, 87 women with median age 63). Thirty-five healthy controls were sex and age-matched. Dukes’ staging was as follows: A = 22, B = 52, C = 72, D = 39. We analyzed patients' blood serum before surgery. We determined: (1) Cathepsin B (CB) with Barrett's method (fluorogenic substrate); (2) Leukocytic elastase (LE) in a complex with alpha 1 trypsin inhibitor (AAT) using the immunoenzymatic MERCK test; (3) Total sialic acid (TSA) with the colorimetric periodate-resorcinol method; (4) Lipid-bound sialic acid (LASA) with the colorimetric Taut's method; and (5) The antitrypsin activity (ATA) employing the colorimetric test.
RESULTS In patients, the values of the five biochemical parameters were as follows: CB = 16.1 ± 8.8 mU/L, LE = 875 ± 598 µg/L, TSA = 99 ± 31 mg%, LASA = 0.68 ± 0.33 mg%, and ATA = 3211 ± 1504 U/mL. Except for LASA, they were significantly greater than those of controls: CB = 11.4 ± 6.5 mU/L, LE = 379 ± 187 µg/L, TSA = 71.4 ± 15.1 mg%, LASA = 0.69 ± 0.28 mg%, and ATA = 2016 ± 690 U/mL. For CB and LASA, the differences between the four Dukes’ stages and controls were not statistically significant. The inter-stage differences for CB and LASA were also absent. The receiver operating characteristic (ROC) analysis revealed the potential diagnostic value of CB, TSA, and ATA. The area under ROC, sensitivity, and specificity for these three parameters were: 0.85, 72%, 90%; 0.75, 66%, 77%; and 0.77, 63%, 84%, respectively. The sensitivity and specificity for the three-parameter panel CB-TSA-ATA were equal to 88.2% and 100%, respectively.
CONCLUSION The increased value of CB, TSA, and ATA parameters are associated with tumor biology, invasion, and metastasis of colorectal cancer. The presented evidence suggests the potential value of the CB-TSA-ATA biochemical marker panel in early diagnostics.
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Affiliation(s)
- Tadeusz Sebzda
- Department of Pathophysiology, Wroclaw Medical University, Wroclaw 50-368, Poland
| | - Jan Gnus
- Department of Physiotherapy, Wroclaw Medical University, Wroclaw 50-355, Poland
| | - Barbara Dziadkowiec
- Department of Pathophysiology, Wroclaw Medical University, Wroclaw 50-368, Poland
| | - Miroslaw Latka
- Department of Biomedical Engineering, Wroclaw University of Science and Technology, Wroclaw 50-370, Poland
| | - Jakub Gburek
- Department of Pharmaceutical Biochemistry, Wroclaw Medical University, Wroclaw 50-556, Poland
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Visser EA, Moons SJ, Timmermans SBPE, de Jong H, Boltje TJ, Büll C. Sialic acid O-acetylation: From biosynthesis to roles in health and disease. J Biol Chem 2021; 297:100906. [PMID: 34157283 PMCID: PMC8319020 DOI: 10.1016/j.jbc.2021.100906] [Citation(s) in RCA: 63] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2021] [Revised: 06/16/2021] [Accepted: 06/18/2021] [Indexed: 02/06/2023] Open
Abstract
Sialic acids are nine-carbon sugars that frequently cap glycans at the cell surface in cells of vertebrates as well as cells of certain types of invertebrates and bacteria. The nine-carbon backbone of sialic acids can undergo extensive enzymatic modification in nature and O-acetylation at the C-4/7/8/9 position in particular is widely observed. In recent years, the detection and analysis of O-acetylated sialic acids have advanced, and sialic acid-specific O-acetyltransferases (SOATs) and O-acetylesterases (SIAEs) that add and remove O-acetyl groups, respectively, have been identified and characterized in mammalian cells, invertebrates, bacteria, and viruses. These advances now allow us to draw a more complete picture of the biosynthetic pathway of the diverse O-acetylated sialic acids to drive the generation of genetically and biochemically engineered model cell lines and organisms with altered expression of O-acetylated sialic acids for dissection of their roles in glycoprotein stability, development, and immune recognition, as well as discovery of novel functions. Furthermore, a growing number of studies associate sialic acid O-acetylation with cancer, autoimmunity, and infection, providing rationale for the development of selective probes and inhibitors of SOATs and SIAEs. Here, we discuss the current insights into the biosynthesis and biological functions of O-acetylated sialic acids and review the evidence linking this modification to disease. Furthermore, we discuss emerging strategies for the design, synthesis, and potential application of unnatural O-acetylated sialic acids and inhibitors of SOATs and SIAEs that may enable therapeutic targeting of this versatile sialic acid modification.
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Affiliation(s)
- Eline A Visser
- Institute for Molecules and Materials, Department of Synthetic Organic Chemistry, Radboud University Nijmegen, Nijmegen, the Netherlands
| | - Sam J Moons
- Institute for Molecules and Materials, Department of Synthetic Organic Chemistry, Radboud University Nijmegen, Nijmegen, the Netherlands
| | - Suzanne B P E Timmermans
- Institute for Molecules and Materials, Department of Synthetic Organic Chemistry, Radboud University Nijmegen, Nijmegen, the Netherlands
| | - Heleen de Jong
- Institute for Molecules and Materials, Department of Synthetic Organic Chemistry, Radboud University Nijmegen, Nijmegen, the Netherlands
| | - Thomas J Boltje
- Institute for Molecules and Materials, Department of Synthetic Organic Chemistry, Radboud University Nijmegen, Nijmegen, the Netherlands.
| | - Christian Büll
- Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; Hubrecht Institute, Utrecht, the Netherlands.
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Gray TE, Narayana K, Garner AM, Bakker SA, Yoo RKH, Fischer-Tlustos AJ, Steele MA, Zandberg WF. Analysis of the biosynthetic flux in bovine milk oligosaccharides reveals competition between sulfated and sialylated species and the existence of glucuronic acid-containing analogues. Food Chem 2021; 361:130143. [PMID: 34051596 DOI: 10.1016/j.foodchem.2021.130143] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2021] [Revised: 05/12/2021] [Accepted: 05/16/2021] [Indexed: 10/21/2022]
Abstract
We previously observed that sialylated bovine milk oligosaccharides (BMOs) decline in both absolute and relative abundances over the initial stages of bovine lactation, with initial evidence suggesting that this decline occurred due to increased concentrations of unique sulfated BMOs. Since both sulfated and sialylated BMOs have distinct bioactivites, a follow up study was launched in order to more clearly define relative changes in these classes of BMOs over the first week of lactation in dairy cattle. Capillary electrophoresis (CE) and several liquid chromatography mass spectrometry (LC-MS) methods, including a novel multiplexed tandem MS method, were used to profile the BMOs extracted from milk collected from the same 20 Holstein cows at milkings 1, 2, 3, 4, 8, and 14 post-partum. In addition to clearly validating that sulfated and sialylated BMOs exist in direct biosynthetic completion, our study has identified over 170 unique BMOs including 14 unique glucuronic acid-containing trisaccharides.
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Affiliation(s)
- Taylor E Gray
- Department of Chemistry, The University of British Columbia, Kelowna, BC V1V 1V7, Canada
| | - Kamal Narayana
- Department of Biology, The University of British Columbia, Kelowna, BC V1V 1V7, Canada
| | - Alexander M Garner
- Department of Biology, The University of British Columbia, Kelowna, BC V1V 1V7, Canada
| | - Samantha A Bakker
- Department of Chemistry, The University of British Columbia, Kelowna, BC V1V 1V7, Canada
| | - Rachael K H Yoo
- Department of Chemistry, The University of British Columbia, Kelowna, BC V1V 1V7, Canada
| | | | - Michael A Steele
- Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 1Y2, Canada.
| | - Wesley F Zandberg
- Department of Chemistry, The University of British Columbia, Kelowna, BC V1V 1V7, Canada.
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Barnard KN, Alford-Lawrence BK, Buchholz DW, Wasik BR, LaClair JR, Yu H, Honce R, Ruhl S, Pajic P, Daugherity EK, Chen X, Schultz-Cherry SL, Aguilar HC, Varki A, Parrish CR. Modified Sialic Acids on Mucus and Erythrocytes Inhibit Influenza A Virus Hemagglutinin and Neuraminidase Functions. J Virol 2020; 94:e01567-19. [PMID: 32051275 PMCID: PMC7163148 DOI: 10.1128/jvi.01567-19] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2019] [Accepted: 02/04/2020] [Indexed: 12/13/2022] Open
Abstract
Sialic acids (Sia) are the primary receptors for influenza viruses and are widely displayed on cell surfaces and in secreted mucus. Sia may be present in variant forms that include O-acetyl modifications at C-4, C-7, C-8, and C-9 positions and N-acetyl or N-glycolyl at C-5. They can also vary in their linkages, including α2-3 or α2-6 linkages. Here, we analyze the distribution of modified Sia in cells and tissues of wild-type mice or in mice lacking CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme, which synthesizes N-glycolyl (Neu5Gc) modifications. We also examined the variation of Sia forms on erythrocytes and in saliva from different animals. To determine the effect of Sia modifications on influenza A virus (IAV) infection, we tested for effects on hemagglutinin (HA) binding and neuraminidase (NA) cleavage. We confirmed that 9-O-acetyl, 7,9-O-acetyl, 4-O-acetyl, and Neu5Gc modifications are widely but variably expressed in mouse tissues, with the highest levels detected in the respiratory and gastrointestinal (GI) tracts. Secreted mucins in saliva and surface proteins of erythrocytes showed a high degree of variability in display of modified Sia between different species. IAV HAs from different virus strains showed consistently reduced binding to both Neu5Gc- and O-acetyl-modified Sia; however, while IAV NAs were inhibited by Neu5Gc and O-acetyl modifications, there was significant variability between NA types. The modifications of Sia in mucus may therefore have potent effects on the functions of IAV and may affect both pathogens and the normal flora of different mucosal sites.IMPORTANCE Sialic acids (Sia) are involved in numerous different cellular functions and are receptors for many pathogens. Sia come in chemically modified forms, but we lack a clear understanding of how they alter interactions with microbes. Here, we examine the expression of modified Sia in mouse tissues, on secreted mucus in saliva, and on erythrocytes, including those from IAV host species and animals used in IAV research. These Sia forms varied considerably among different animals, and their inhibitory effects on IAV NA and HA activities and on bacterial sialidases (neuraminidases) suggest a host-variable protective role in secreted mucus.
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Affiliation(s)
- Karen N Barnard
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
| | - Brynn K Alford-Lawrence
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
| | - David W Buchholz
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
| | - Brian R Wasik
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
| | - Justin R LaClair
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
| | - Hai Yu
- Department of Chemistry, University of California-Davis, Davis, California, USA
| | - Rebekah Honce
- Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
- Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, USA
| | - Stefan Ruhl
- Department of Oral Biology, University at Buffalo, Buffalo, New York, USA
| | - Petar Pajic
- Department of Oral Biology, University at Buffalo, Buffalo, New York, USA
| | - Erin K Daugherity
- Center for Animal Resources and Education, Cornell University, Ithaca, New York, USA
| | - Xi Chen
- Department of Chemistry, University of California-Davis, Davis, California, USA
| | - Stacey L Schultz-Cherry
- Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Hector C Aguilar
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
| | - Ajit Varki
- Glycobiology Research and Training Center, University of California, San Diego, California, USA
| | - Colin R Parrish
- Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
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Fernandes E, Sores J, Cotton S, Peixoto A, Ferreira D, Freitas R, Reis CA, Santos LL, Ferreira JA. Esophageal, gastric and colorectal cancers: Looking beyond classical serological biomarkers towards glycoproteomics-assisted precision oncology. Am J Cancer Res 2020; 10:4903-4928. [PMID: 32308758 PMCID: PMC7163443 DOI: 10.7150/thno.42480] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2019] [Accepted: 01/16/2020] [Indexed: 12/24/2022] Open
Abstract
Esophageal (OC), gastric (GC) and colorectal (CRC) cancers are amongst the digestive track tumors with higher incidence and mortality due to significant molecular heterogeneity. This constitutes a major challenge for patients' management at different levels, including non-invasive detection of the disease, prognostication, therapy selection, patient's follow-up and the introduction of improved and safer therapeutics. Nevertheless, important milestones have been accomplished pursuing the goal of molecular-based precision oncology. Over the past five years, high-throughput technologies have been used to interrogate tumors of distinct clinicopathological natures, generating large-scale biological datasets (e.g. genomics, transcriptomics, and proteomics). As a result, GC and CRC molecular subtypes have been established to assist patient stratification in the clinical settings. However, such molecular panels still require refinement and are yet to provide targetable biomarkers. In parallel, outstanding advances have been made regarding targeted therapeutics and immunotherapy, paving the way for improved patient care; nevertheless, important milestones towards treatment personalization and reduced off-target effects are also to be accomplished. Exploiting the cancer glycoproteome for unique molecular fingerprints generated by dramatic alterations in protein glycosylation may provide the necessary molecular rationale towards this end. Therefore, this review presents functional and clinical evidences supporting a reinvestigation of classical serological glycan biomarkers such as sialyl-Tn (STn) and sialyl-Lewis A (SLeA) antigens from a tumor glycoproteomics perspective. We anticipate that these glycobiomarkers that have so far been employed in non-invasive cancer prognostication may hold unexplored value for patients' management in precision oncology settings.
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Gachkar S, Oelkrug R, Herrmann B, Scanlan TS, Sun Q, Biebermann H, Hoefig CS, Schomburg L, Mittag J. N- and O-Acetylated 3-Iodothyronamines Have No Metabolic or Thermogenic Effects in Male Mice. Eur Thyroid J 2020; 9:57-66. [PMID: 32257954 PMCID: PMC7109410 DOI: 10.1159/000504887] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/07/2019] [Accepted: 11/19/2019] [Indexed: 12/13/2022] Open
Abstract
INTRODUCTION Injection of 3-iodothyronamine into experimental animals profoundly affects their metabolism and body temperature. As 3-iodothyronamine is rapidly acetylated in vivo after injection, it was hypothesized that the metabolites N- or O-acetyl-3-iodothyronamines could constitute the active hormones. METHODS Adult male mice were injected once daily with one of the metabolites (5 mg/kg body weight intraperitoneally dissolved in 60% DMSO in PBS) or solvent. Metabolism was monitored by indirect calorimetry, body temperature by infrared thermography, and body composition by nuclear magnetic resonance analysis. Signaling activities in brown fat or liver were assessed by studying target gene transcription by qPCR including uncoupling protein 1 or deiodinase type 1 or 2, and Western blot. RESULTS The markers of metabolism, body composition, or temperature tested were similar in the mice injected with solvent and those injected with one of the acetylated 3-iodothyronamines. CONCLUSIONS In our experimental setup, N- and O-acetyl-3-iodothyronamine do not constitute compounds contributing to the metabolic or temperature effects described for 3-iodothyronamine. The acetylation of 3-iodothyronamine observed in vivo may thus rather serve degradation and elimination purposes.
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Affiliation(s)
- Sogol Gachkar
- Molecular Endocrinology, Center of Brain, Behavior and Metabolism, University of Lübeck, Lübeck, Germany
| | - Rebecca Oelkrug
- Molecular Endocrinology, Center of Brain, Behavior and Metabolism, University of Lübeck, Lübeck, Germany
| | - Beate Herrmann
- Molecular Endocrinology, Center of Brain, Behavior and Metabolism, University of Lübeck, Lübeck, Germany
| | - Thomas S. Scanlan
- Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon, USA
| | - Qian Sun
- Institute for Experimental Endocrinology, Charité − Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
| | - Heike Biebermann
- Institute of Experimental Pediatric Endocrinology, Charité − Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
| | - Carolin S. Hoefig
- Institute for Experimental Endocrinology, Charité − Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
| | - Lutz Schomburg
- Institute for Experimental Endocrinology, Charité − Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
| | - Jens Mittag
- Molecular Endocrinology, Center of Brain, Behavior and Metabolism, University of Lübeck, Lübeck, Germany
- *Prof. Dr. Jens Mittag, Center of Brain, Behavior and Metabolism, Ratzeburger Allee 160, Haus 66, DE–23562 Lübeck (Germany), E-Mail
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Abstract
Sialic acids are cytoprotectors, mainly localized on the surface of cell membranes with multiple and outstanding cell biological functions. The history of their structural analysis, occurrence, and functions is fascinating and described in this review. Reports from different researchers on apparently similar substances from a variety of biological materials led to the identification of a 9-carbon monosaccharide, which in 1957 was designated "sialic acid." The most frequently occurring member of the sialic acid family is N-acetylneuraminic acid, followed by N-glycolylneuraminic acid and O-acetylated derivatives, and up to now over about 80 neuraminic acid derivatives have been described. They appeared first in the animal kingdom, ranging from echinoderms up to higher animals, in many microorganisms, and are also expressed in insects, but are absent in higher plants. Sialic acids are masks and ligands and play as such dual roles in biology. Their involvement in immunology and tumor biology, as well as in hereditary diseases, cannot be underestimated. N-Glycolylneuraminic acid is very special, as this sugar cannot be expressed by humans, but is a xenoantigen with pathogenetic potential. Sialidases (neuraminidases), which liberate sialic acids from cellular compounds, had been known from very early on from studies with influenza viruses. Sialyltransferases, which are responsible for the sialylation of glycans and elongation of polysialic acids, are studied because of their significance in development and, for instance, in cancer. As more information about the functions in health and disease is acquired, the use of sialic acids in the treatment of diseases is also envisaged.
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Affiliation(s)
- Roland Schauer
- Biochemisches Institut, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.
| | - Johannis P Kamerling
- Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands.
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11
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Hoefig CS, Zucchi R, Köhrle J. Thyronamines and Derivatives: Physiological Relevance, Pharmacological Actions, and Future Research Directions. Thyroid 2016; 26:1656-1673. [PMID: 27650974 DOI: 10.1089/thy.2016.0178] [Citation(s) in RCA: 68] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Thyronamines (3-T1AM, T0AM) are endogenous compounds probably derived from L-thyroxine or its intermediate metabolites. Combined activities of intestinal deiodinases and ornithine decarboxylase generate 3-T1AM in vitro. Alternatively, 3-T1AM might be formed by the thyroid gland and secreted into the blood. 3-T1AM and T0AM concentrations have been determined by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) in tissues, serum, and cell lines. However, large variations of 3-T1AM concentrations in human serum were reported by LC-MS/MS compared with a monoclonal antibody-based immunoassay. These differences might be caused by strong binding of the highly hydrophobic 3-T1AM to apolipoprotein B100. Pharmacological administration of 3-T1AM results in dose-dependent reversible effects on body temperature, cardiac function, energy metabolism, and neurological functions. The physiological relevance of these actions is unclear, but may occur at tissue concentrations close to the estimated endogenous concentrations of 3-T1AM or its metabolites T0AM or thyroacetic acid (TA1). A number of putative receptors, binding sites, and cellular target molecules mediating actions of the multi-target ligand 3-T1AM have been proposed. Among those are members of the trace amine associated receptor family, the adrenergic receptor ADRα2a, and the thermosensitive transient receptor potential melastatin 8 channel. Preclinical studies employing various animal experimental models are in progress, and more stable receptor-selective agonistic and antagonistic analogues of 3-T1AM are now available for testing. The potent endogenous thyroid hormone-derived biogenic amine 3-T1AM exerts marked cryogenic, metabolic, cardiac and central actions and represents a valuable lead compound linking endocrine, metabolic, and neuroscience research to advance development of new drugs.
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Affiliation(s)
- Carolin Stephanie Hoefig
- 1 Institut für Experimentelle Endokrinologie Charité, Universitätsmedizin Berlin , Berlin, Germany
| | - Riccardo Zucchi
- 2 Laboratory of Biochemistry, Department of Pathology, University of Pisa , Pisa, Italy
| | - Josef Köhrle
- 1 Institut für Experimentelle Endokrinologie Charité, Universitätsmedizin Berlin , Berlin, Germany
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Lee J, Katzenmaier EM, Kopitz J, Gebert J. Reconstitution of TGFBR2 in HCT116 colorectal cancer cells causes increased LFNG expression and enhanced N-acetyl-d-glucosamine incorporation into Notch1. Cell Signal 2016; 28:1105-13. [DOI: 10.1016/j.cellsig.2016.04.012] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2015] [Revised: 04/19/2016] [Accepted: 04/28/2016] [Indexed: 12/20/2022]
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Earley H, Lennon G, Balfe A, Kilcoyne M, Clyne M, Joshi L, Carrington S, Martin ST, Coffey JC, Winter DC, O’Connell PR. A Preliminary Study Examining the Binding Capacity of Akkermansia muciniphila and Desulfovibrio spp., to Colonic Mucin in Health and Ulcerative Colitis. PLoS One 2015; 10:e0135280. [PMID: 26491870 PMCID: PMC4619660 DOI: 10.1371/journal.pone.0135280] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2015] [Accepted: 07/20/2015] [Indexed: 01/30/2023] Open
Abstract
Background Akkermansia muciniphila and Desulfovibrio spp. are commensal microbes colonising the mucus gel layer of the colon. Both species have the capacity to utilise colonic mucin as a substrate. A. muciniphila degrades colonic mucin, while Desulfovibrio spp. metabolise the sulfate moiety of sulfated mucins. Altered abundances of these microorganisms have been reported in ulcerative colitis (UC). However their capacity to bind to human colonic mucin, and whether this binding capacity is affected by changes in mucin associated with UC, remain to be defined. Methods Mucin was isolated from resected colon from control patients undergoing resection for colonic cancer (n = 7) and patients undergoing resection for UC (n = 5). Isolated mucin was purified and printed onto mucin microarrays. Binding of reference strains and three clinical isolates of A. muciniphila and Desulfovibrio spp. to purified mucin was investigated. Results Both A. muciniphila and Desulfovibro spp. bound to mucin. The reference strain and all clinical isolates of A. muciniphila showed increased binding capacity for UC mucin (p < .005). The Desulfovibrio reference strain showed increased affinity for UC mucin. The mucin binding profiles of clinical isolates of Desulfovibrio spp. were specific to each isolate. Two isolates showed no difference in binding. One UC isolate bound with increased affinity to UC mucin (p < .005). Conclusion These preliminary data suggest that differences exist in the mucin binding capacity of isolates of A. muciniphila and Desulfovibrio spp. This study highlights the mucin microarray platform as a means of studying the ability of bacteria to interact with colonic mucin in health and disease.
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Affiliation(s)
- Helen Earley
- School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland
- Centre for Colorectal Disease, St Vincent’s University Hospital, Dublin 4, Ireland
| | - Grainne Lennon
- School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland
- Centre for Colorectal Disease, St Vincent’s University Hospital, Dublin 4, Ireland
| | - Aine Balfe
- School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland
- Centre for Colorectal Disease, St Vincent’s University Hospital, Dublin 4, Ireland
| | - Michelle Kilcoyne
- Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
- Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland
| | - Marguerite Clyne
- School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland
| | - Lokesh Joshi
- Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
| | - Stephen Carrington
- College of Life Sciences, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Dublin 4, Ireland
| | - Sean T. Martin
- Centre for Colorectal Disease, St Vincent’s University Hospital, Dublin 4, Ireland
| | | | - Desmond C. Winter
- Centre for Colorectal Disease, St Vincent’s University Hospital, Dublin 4, Ireland
| | - P. Ronan O’Connell
- School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland
- Centre for Colorectal Disease, St Vincent’s University Hospital, Dublin 4, Ireland
- * E-mail:
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14
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Baumann AMT, Bakkers MJG, Buettner FFR, Hartmann M, Grove M, Langereis MA, de Groot RJ, Mühlenhoff M. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate. Nat Commun 2015; 6:7673. [PMID: 26169044 PMCID: PMC4510713 DOI: 10.1038/ncomms8673] [Citation(s) in RCA: 90] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2015] [Accepted: 06/01/2015] [Indexed: 12/13/2022] Open
Abstract
Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host–pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1—a previously identified human candidate gene—is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. 9-O-Acetylation is one of the most common modifications of sialic acids, implicated in sialoglycan recognition and ganglioside biology. Here, the authors show that the key enzyme for the biosynthesis of 9-O-acetylated sialoglycans is CASD1, which uses CMP-activated sialic acid as acceptor substrate.![]()
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Affiliation(s)
- Anna-Maria T Baumann
- Institute of Cellular Chemistry, Hannover Medical School, D-30623 Hannover, Germany
| | - Mark J G Bakkers
- Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CH Utrecht, The Netherlands
| | - Falk F R Buettner
- Institute of Cellular Chemistry, Hannover Medical School, D-30623 Hannover, Germany
| | - Maike Hartmann
- Institute of Cellular Chemistry, Hannover Medical School, D-30623 Hannover, Germany
| | - Melanie Grove
- Institute of Cellular Chemistry, Hannover Medical School, D-30623 Hannover, Germany
| | - Martijn A Langereis
- Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CH Utrecht, The Netherlands
| | - Raoul J de Groot
- Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CH Utrecht, The Netherlands
| | - Martina Mühlenhoff
- Institute of Cellular Chemistry, Hannover Medical School, D-30623 Hannover, Germany
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15
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Mathew MP, Tan E, Saeui CT, Bovonratwet P, Liu L, Bhattacharya R, Yarema KJ. Metabolic glycoengineering sensitizes drug-resistant pancreatic cancer cells to tyrosine kinase inhibitors erlotinib and gefitinib. Bioorg Med Chem Lett 2015; 25:1223-7. [PMID: 25690786 PMCID: PMC5753412 DOI: 10.1016/j.bmcl.2015.01.060] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2015] [Revised: 01/23/2015] [Accepted: 01/26/2015] [Indexed: 02/02/2023]
Abstract
Metastatic human pancreatic cancer cells (the SW1990 line) that are resistant to the EGFR-targeting tyrosine kinase inhibitor drugs (TKI) erlotinib and gefitinib were treated with 1,3,4-O-Bu3ManNAc, a 'metabolic glycoengineering' drug candidate that increased sialylation by ∼2-fold. Consistent with genetic methods previously used to increase EGFR sialylation, this small molecule reduced EGF binding, EGFR transphosphorylation, and downstream STAT activation. Significantly, co-treatment with both the sugar pharmacophore and the existing TKI drugs resulted in strong synergy, in essence re-sensitizing the SW1990 cells to these drugs. Finally, 1,3,4-O-Bu3ManNAz, which is the azido-modified counterpart to 1,3,4-O-Bu3ManNAc, provided a similar benefit thereby establishing a broad-based foundation to extend a 'metabolic glycoengineering' approach to clinical applications.
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Affiliation(s)
- Mohit P Mathew
- Department of Biomedical Engineering and the Translational Tissue Engineering Center, The Johns Hopkins University, 5029 Robert H. & Clarice Smith Building, 400 North Broadway, Baltimore, MD 21231, USA
| | - Elaine Tan
- Department of Biomedical Engineering and the Translational Tissue Engineering Center, The Johns Hopkins University, 5029 Robert H. & Clarice Smith Building, 400 North Broadway, Baltimore, MD 21231, USA
| | - Christopher T Saeui
- Department of Biomedical Engineering and the Translational Tissue Engineering Center, The Johns Hopkins University, 5029 Robert H. & Clarice Smith Building, 400 North Broadway, Baltimore, MD 21231, USA
| | - Patawut Bovonratwet
- Department of Biomedical Engineering and the Translational Tissue Engineering Center, The Johns Hopkins University, 5029 Robert H. & Clarice Smith Building, 400 North Broadway, Baltimore, MD 21231, USA
| | - Lingshu Liu
- Department of Biomedical Engineering and the Translational Tissue Engineering Center, The Johns Hopkins University, 5029 Robert H. & Clarice Smith Building, 400 North Broadway, Baltimore, MD 21231, USA
| | - Rahul Bhattacharya
- Department of Biomedical Engineering and the Translational Tissue Engineering Center, The Johns Hopkins University, 5029 Robert H. & Clarice Smith Building, 400 North Broadway, Baltimore, MD 21231, USA
| | - Kevin J Yarema
- Department of Biomedical Engineering and the Translational Tissue Engineering Center, The Johns Hopkins University, 5029 Robert H. & Clarice Smith Building, 400 North Broadway, Baltimore, MD 21231, USA.
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16
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Park JJ, Lee M. Increasing the α 2, 6 sialylation of glycoproteins may contribute to metastatic spread and therapeutic resistance in colorectal cancer. Gut Liver 2013; 7:629-41. [PMID: 24312702 PMCID: PMC3848550 DOI: 10.5009/gnl.2013.7.6.629] [Citation(s) in RCA: 92] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/26/2013] [Revised: 10/02/2013] [Accepted: 10/02/2013] [Indexed: 12/13/2022] Open
Abstract
Abnormal glycosylation due to dysregulated glycosyltransferases and glycosidases is a key phenomenon of many malignancies, including colorectal cancer (CRC). In particular, increased ST6 Gal I (β-galactoside α 2, 6 sialyltransferase) and subsequently elevated levels of cell-surface α 2, 6-linked sialic acids have been associated with metastasis and therapeutic failure in CRC. As many CRC patients experience metastasis to the liver or lung and fail to respond to curative therapies, intensive research efforts have sought to identify the molecular changes underlying CRC metastasis. ST6 Gal I has been shown to facilitate CRC metastasis, and we believe that additional investigations into the involvement of ST6 Gal I in CRC could facilitate the development of new diagnostic and therapeutic targets. This review summarizes how ST6 Gal I has been implicated in the altered expression of sialylated glycoproteins, which have been linked to CRC metastasis, radioresistance, and chemoresistance.
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Affiliation(s)
- Jung-Jin Park
- Division of Life Science, Korea University College of Life Sciences and Biotechnology, Seoul, Korea
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17
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Sialic acid metabolism and sialyltransferases: natural functions and applications. Appl Microbiol Biotechnol 2012; 94:887-905. [PMID: 22526796 DOI: 10.1007/s00253-012-4040-1] [Citation(s) in RCA: 209] [Impact Index Per Article: 16.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2012] [Revised: 03/16/2012] [Accepted: 03/16/2012] [Indexed: 12/17/2022]
Abstract
Sialic acids are a family of negatively charged monosaccharides which are commonly presented as the terminal residues in glycans of the glycoconjugates on eukaryotic cell surface or as components of capsular polysaccharides or lipooligosaccharides of some pathogenic bacteria. Due to their important biological and pathological functions, the biosynthesis, activation, transfer, breaking down, and recycle of sialic acids are attracting increasing attention. The understanding of the sialic acid metabolism in eukaryotes and bacteria leads to the development of metabolic engineering approaches for elucidating the important functions of sialic acid in mammalian systems and for large-scale production of sialosides using engineered bacterial cells. As the key enzymes in biosynthesis of sialylated structures, sialyltransferases have been continuously identified from various sources and characterized. Protein crystal structures of seven sialyltransferases have been reported. Wild-type sialyltransferases and their mutants have been applied with or without other sialoside biosynthetic enzymes for producing complex sialic acid-containing oligosaccharides and glycoconjugates. This mini-review focuses on current understanding and applications of sialic acid metabolism and sialyltransferases.
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18
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Abstract
Sialic acids have a pivotal functional impact in many biological interactions such as virus attachment, cellular adhesion, regulation of proliferation, and apoptosis. A common modification of sialic acids is O-acetylation. O-Acetylated sialic acids occur in bacteria and parasites and are also receptor determinants for a number of viruses. Moreover, they have important functions in embryogenesis, development, and immunological processes. O-Acetylated sialic acids represent cancer markers, as shown for acute lymphoblastic leukemia, and they are known to play significant roles in the regulation of ganglioside-mediated apoptosis. Expression of O-acetylated sialoglycans is regulated by sialic acid-specific O-acetyltransferases and O-acetylesterases. Recent developments in the identification of the enigmatic sialic acid-specific O-acetyltransferase are discussed.
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Affiliation(s)
- Chitra Mandal
- Cancer and Cell Biology, Council of Scientific and Industrial Research - Indian Institute of Chemical Biology, 4 Raja S.C. Mallick Road, Kolkata, 700 032 India
| | - Reinhard Schwartz-Albiez
- Department of Translational Immunology, German Cancer Research Center, 69120 Heidelberg, Germany
| | - Reinhard Vlasak
- Department of Molecular Biology, University Salzburg, Billrothstr 11, 5020 Salzburg, Austria
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Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett's esophagus. Nat Med 2012; 18:315-21. [DOI: 10.1038/nm.2616] [Citation(s) in RCA: 199] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2010] [Accepted: 05/23/2011] [Indexed: 12/20/2022]
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Mandal C, Mandal C, Chandra S, Schauer R, Mandal C. Regulation of O-acetylation of sialic acids by sialate-O-acetyltransferase and sialate-O-acetylesterase activities in childhood acute lymphoblastic leukemia. Glycobiology 2011; 22:70-83. [DOI: 10.1093/glycob/cwr106] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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Wipfler D, Srinivasan GV, Sadick H, Kniep B, Arming S, Willhauck-Fleckenstein M, Vlasak R, Schauer R, Schwartz-Albiez R. Differentially regulated expression of 9-O-acetyl GD3 (CD60b) and 7-O-acetyl-GD3 (CD60c) during differentiation and maturation of human T and B lymphocytes. Glycobiology 2011; 21:1161-72. [PMID: 21507905 DOI: 10.1093/glycob/cwr050] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
GD3 (CD60a) and its 9-O-acetylated variant (CD60b) are intracellular regulators of apoptosis in T lymphocytes. Surface expressed 9-O-acetyl- and 7-O-acetyl-GD3 (CD60b and CD60c) may have a functional impact on activated T and B cells. In order to investigate the balance between surface and intracellular expression and synthesis and degradation of these glycosphingolipids in human lymphocytes of various differentiation stages, we analyzed (i) expression of GD3 molecules on native T and B cells and thymocytes by flow cytometry and (ii) activity and regulation of possible key enzymes for CD60a,b,c synthesis and degradation at the transcriptional level. Both, surface and cytoplasmic expression of CD60a and CD60c was highest in tonsillar T cells. In thymocytes, CD60c outweighs the other CD60 variants and was mainly found in the cytoplasm. All lymphocyte preparations contained sialate O-acetyltransferase activity producing 7-O-acetyl-GD3. Sialidase activity was highest in peripheral blood lymphocytes followed by thymocytes and tonsillar T and B cells. Transcription of GD3 synthase (ST8SiaI), the key enzyme for GD3 synthesis, was highest in tonsillar T cells, whereas transcriptional levels of sialidase NEU3 and O-acetylesterase H-Lse were lowest in activated T cells. This balance between enzymes of sialic acid metabolism may explain the strong overall staining intensity for all GD3 forms in T cells. Both CASD1, presumably encoding a sialic acid-specific O-acetyltransferase, and H-Lse showed highest transcription in peripheral B lymphocytes corresponding to the low expression of CD60b and c in these cells. Our data point to regulatory functions of these anabolic and catabolic key enzymes for the expression of GD3 and its O-acetylated variants in lymphocytes at a given differentiation stage.
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Affiliation(s)
- Dirk Wipfler
- German Cancer Research Center, D015 Translational Immunology, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
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Liu X, Afonso L. Is permethylation strategy always applicable to protein N-glycosylation study?: A case study on the O-acetylation of sialic acid in fish serum glycans. Methods Mol Biol 2010; 600:259-268. [PMID: 19882134 DOI: 10.1007/978-1-60761-454-8_18] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
O-Acetylation is one of the major modifications of sialic acids that significantly alters biological properties of the parent molecule. These O-acetylated forms are components of the cellular membrane and can affect physiological and pathological responses. Understanding the role of N-glycans in physiology is of increasing relevance to cellular biologists in various disciplines who study glycoproteomics yet lack information regarding the function of the attached glycans. However, permethylation, the most common mass spectrometric analytical means, leads to the loss of O-linked acetyl groups in sialic acids. In this chapter, we demonstrated that O-acetylation of sialic acid in Atlantic salmon serum N-glycan can be well investigated by capillary electrophoresis-mass spectrometry.
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Affiliation(s)
- Xin Liu
- Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada
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23
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Srinivasan GV, Schauer R. Assays of sialate-O-acetyltransferases and sialate-O-acetylesterases. Glycoconj J 2009; 26:935-44. [PMID: 18566887 DOI: 10.1007/s10719-008-9131-y] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2008] [Revised: 03/20/2008] [Accepted: 03/26/2008] [Indexed: 10/21/2022]
Abstract
The O-acetylation of sialic acids is one of the most frequent modifications of these monosaccharides and modulates many cell biological and pathological events. Sialic acid-specific O-acetyltransferases and O-acetylesterases are responsible for the metabolism of esterified sialic acids. Assays were developed for the analysis of the activities and specificities of these enzymes. The methods had to be varied in dependence on the substrate assayed, the kind of biological source, and the state of enzyme purity. With the new techniques the primary site of O-acetyl incorporation at C-7, catalyzed by the animal sialate-O-acetyltransferases studied, was ascertained. Correspondingly, this enzyme, for example from bovine submandibular gland, can be denominated as AcCoA:sialate-7-O-acetyltransferase (EC 2.3.1.45). Methods for assaying the activity of esterases de-O-acetylating sialic acids and their metabolic cooperation with the O-acetyltransferases are presented.
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Affiliation(s)
- G Vinayaga Srinivasan
- Biochemisches Institut, Christian-Albrechts-Universität, Olshausenstr. 40, 24098 Kiel, Germany
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Schnabl KL, Larcelet M, Thomson ABR, Clandinin MT. Uptake and fate of ganglioside GD3 in human intestinal Caco-2 cells. Am J Physiol Gastrointest Liver Physiol 2009; 297:G52-9. [PMID: 19423750 DOI: 10.1152/ajpgi.90599.2008] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Ganglioside GD3 is a glycosphingolipid found in colostrum, developing tissues, and tumors and is known to regulate cell growth, differentiation, apoptosis, and inflammation. Feeding a GD3-enriched diet to rats increases GD3 in intestinal lipid rafts and blood. The mechanism, efficiency, and fate of ganglioside absorption by human enterocytes have not been investigated. A model to study GD3 uptake by human intestinal cells was developed to test the hypothesis that enterocyte GD3 uptake is time and concentration dependent, with uptake efficiency and fate influenced by route of delivery. Caco-2 cells were exposed to GD3 on the apical or basolateral membrane (BLM) side for 6, 24, and 48 h. GD3 uptake, retention, transfer, and metabolism was determined. GD3 uptake across the apical and BLM was time and concentration dependent and reached a plateau. GD3 uptake across the BLM was more efficient than apical delivery. Apical GD3 was metabolized with some cell retention and transfer, whereas basolateral GD3 was mostly metabolized. This study demonstrates efficient GD3 uptake by enterocytes and suggests that the route of delivery influences ganglioside uptake and fate.
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Affiliation(s)
- Kareena L Schnabl
- Department of Medicine, Division of Gastroenterology, University of Alberta, Edmonton, Alberta T6G 2P5, Canada
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Makovitzky J, Richter S. The relevance of the aldehyde bisulfite toluidine blue reaction and its variants in the submicroscopic carbohydrate research. Acta Histochem 2009; 111:273-91. [PMID: 19157525 PMCID: PMC7172417 DOI: 10.1016/j.acthis.2008.11.027] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Carbohydrates are chemical compounds that contain only oxygen, hydrogen and carbon. They are classified by their number of sugar units: monosaccharides (such as glucose and fructose), and disaccharides (such as sucrose and lactose) are simple carbohydrates; oligosaccharides and polysaccharides (such as starch, glycogen and cellulose) are complex carbohydrates. Carbohydrates play a crucial role in diverse biological systems [Hricovín M. Structural aspects of carbohydrates and the relation with their biological properties. Curr Med Chem 2004;11:2565-83]. According to Roseman [Sugars of the cell membrane. In: Weissmann G, Clairborn E, editors. Cell membranes. Biochemistry, Cell Biology, Pathology. New York: H. P. Publ. Co; 1975. p. 55-64], two classes of glycoproteins are described. Free glycoproteins are localised in the surface coat of the membranes and form a thick mobile layer, without any association to the membrane itself. Functionally, however, they are located in a close association with the membrane (e.g. in the duodenal mucosa). The other group consists of the membrane glycoproteins, which are integral to the membranes and are located in the outer layer. The oligosaccharide chains are bound to the N-terminal part of proteins, and are situated in the hydrophilic zone. Glycoproteins have diverse functions. They are important in specific receptor functions, in immunological cell destruction and play a significant role in reactions with lectins, antibodies, as well as in cell association and mutual recognition of the cells. This paper focuses on aspects of a summary of polarisation optical investigations and biological functions of the following three groups of carbohydrates: oligosaccharides, glycoproteins and glycosaminoglycans.
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Affiliation(s)
- Josef Makovitzky
- Department of Neuropathology, University Heidelberg, Im Neuenheimer Feld 220, D-69120 Heidelberg, Germany.
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Payne CM, Bernstein C, Dvorak K, Bernstein H. Hydrophobic bile acids, genomic instability, Darwinian selection, and colon carcinogenesis. Clin Exp Gastroenterol 2008; 1:19-47. [PMID: 21677822 PMCID: PMC3108627 DOI: 10.2147/ceg.s4343] [Citation(s) in RCA: 102] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Sporadic colon cancer is caused predominantly by dietary factors. We have selected bile acids as a focus of this review since high levels of hydrophobic bile acids accompany a Western-style diet, and play a key role in colon carcinogenesis. We describe how bile acid-induced stresses cause cell death in susceptible cells, contribute to genomic instability in surviving cells, impose Darwinian selection on survivors and enhance initiation and progression to colon cancer. The most likely major mechanisms by which hydrophobic bile acids induce stresses on cells (DNA damage, endoplasmic reticulum stress, mitochondrial damage) are described. Persistent exposure of colon epithelial cells to hydrophobic bile acids can result in the activation of pro-survival stress-response pathways, and the modulation of numerous genes/proteins associated with chromosome maintenance and mitosis. The multiple mechanisms by which hydrophobic bile acids contribute to genomic instability are discussed, and include oxidative DNA damage, p53 and other mutations, micronuclei formation and aneuploidy. Since bile acids and oxidative stress decrease DNA repair proteins, an increase in DNA damage and increased genomic instability through this mechanism is also described. This review provides a mechanistic explanation for the important link between a Western-style diet and associated increased levels of colon cancer.
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Affiliation(s)
- Claire M Payne
- Department of Cell Biology and Anatomy, College of Medicine, University of Arizona, Tucson, Arizona, USA
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Bergfeld AK, Claus H, Lorenzen NK, Spielmann F, Vogel U, Mu Hlenhoff M. The polysialic acid-specific O-acetyltransferase OatC from Neisseria meningitidis serogroup C evolved apart from other bacterial sialate O-acetyltransferases. J Biol Chem 2008; 284:6-16. [PMID: 18986988 DOI: 10.1074/jbc.m807518200] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Neisseria meningitidis serogroup C is a major cause of bacterial meningitis and septicaemia. This human pathogen is protected by a capsule composed of alpha2,9-linked polysialic acid that represents an important virulence factor. In the majority of strains, the capsular polysaccharide is modified by O-acetylation at C-7 or C-8 of the sialic acid residues. The gene encoding the capsule modifying O-acetyltransferase is part of the capsule gene complex and shares no sequence similarities with other proteins. Here, we describe the purification and biochemical characterization of recombinant OatC. The enzyme was found as a homodimer, with the first 34 amino acids forming an efficient oligomerization domain that worked even in a different protein context. Using acetyl-CoA as donor substrate, OatC transferred acetyl groups exclusively onto polysialic acid joined by alpha2,9-linkages and did not act on free or CMP-activated sialic acid. Motif scanning revealed a nucleophile elbow motif (GXS286XGG), which is a hallmark of alpha/beta-hydrolase fold enzymes. In a comprehensive site-directed mutagenesis study, we identified a catalytic triad composed of Ser-286, Asp-376, and His-399. Consistent with a double-displacement mechanism common to alpha/beta-hydrolase fold enzymes, a covalent acetylenzyme intermediate was found. Together with secondary structure prediction highlighting an alpha/beta-hydrolase fold topology, our data provide strong evidence that OatC belongs to the alpha/beta-hydrolase fold family. This clearly distinguishes OatC from all other bacterial sialate O-acetyltransferases known so far because these are members of the hexapeptide repeat family, a class of acyltransferases that adopt a left-handed beta-helix fold and assemble into catalytic trimers.
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Affiliation(s)
- Anne K Bergfeld
- Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany
| | - Heike Claus
- Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany
| | - Nina K Lorenzen
- Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany
| | - Fabian Spielmann
- Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany
| | - Ulrich Vogel
- Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany
| | - Martina Mu Hlenhoff
- Department of Cellular Chemistry, Medical School Hannover, 30623 Hannover, Germany and the Institute for Hygiene and Microbiology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany.
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Mandal C, Srinivasan GV, Chowdhury S, Chandra S, Mandal C, Schauer R, Mandal C. High level of sialate-O-acetyltransferase activity in lymphoblasts of childhood acute lymphoblastic leukaemia (ALL): enzyme characterization and correlation with disease status. Glycoconj J 2008; 26:57-73. [PMID: 18677580 DOI: 10.1007/s10719-008-9163-3] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2008] [Revised: 06/13/2008] [Accepted: 06/16/2008] [Indexed: 11/27/2022]
Abstract
Previous studies had established an over-expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Here, we report the discovery and characterization of sialate-O-acetyltransferase enzyme in ALL-cell lines and lymphoblasts from bone marrow of children diagnosed with B- and T-ALL. We observed a positive correlation between the enhanced sialate-O-acetyltransferase activity and the enhanced expression of Neu5,9Ac(2)-GPs in these lymphoblasts. Sialate-O-acetyltransferase activity in cell lysates or microsomal fractions of lymphoblasts of patients was always higher than that in healthy donors reaching up to 22-fold in microsomes. Additionally, the V (max) of this enzymatic reaction with AcCoA was over threefold higher in microsomal fractions of lymphoblasts. The enzyme bound to the microsomal fractions showed high activity with CMP-N-acetylneuraminic acid, ganglioside GD3 and endogenous sialic acid as substrates. N-acetyl-7-O-acetylneuraminic acid was the main reaction product, as detected by radio-thin-layer chromatography and fluorimetrically coupled radio-high-performance liquid chromatography. CMP and coenzyme A inhibited the microsomal enzyme. Sialate-O-acetyltransferase activity increased at the diagnosis of leukaemia, decreased with clinical remission and sharply increased again in relapsed patients as determined by radiometric-assay. A newly-developed non-radioactive ELISA can quickly detect sialate-O-acetyltransferase, and thus, may become a suitable tool for ALL-monitoring in larger scale. This is the first report on sialate-O-acetyltransferase in ALL being one of the few descriptions of an enzyme of this type in human.
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Affiliation(s)
- Chandan Mandal
- Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Kolkata 700 032, India
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Liu X, Afonso L, Altman E, Johnson S, Brown L, Li J. O-acetylation of sialic acids in N-glycans of Atlantic salmon (Salmo salar) serum is altered by handling stress. Proteomics 2008; 8:2849-57. [DOI: 10.1002/pmic.200701093] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
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Lewis AL, Cao H, Patel SK, Diaz S, Ryan W, Carlin AF, Thon V, Lewis WG, Varki A, Chen X, Nizet V. NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharide in group B Streptococcus. J Biol Chem 2007; 282:27562-71. [PMID: 17646166 PMCID: PMC2588433 DOI: 10.1074/jbc.m700340200] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) was enhanced by CTP and Mg(2+), the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac(2) followed by CMP activation of Neu5Ac or activation of Neu5,9Ac(2) followed by de-O-acetylation of CMP-Neu5,9Ac(2). Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.
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Affiliation(s)
- Amanda L. Lewis
- Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, USA
- Department of Pediatrics, University of California, San Diego, La Jolla, California, USA
| | - Hongzhi Cao
- Department of Chemistry, University of California, Davis, CA 95616, USA
| | - Silpa K. Patel
- Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, USA
- Department of Pediatrics, University of California, San Diego, La Jolla, California, USA
| | - Sandra Diaz
- Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, USA
- Department of Medicine, University of California, San Diego, La Jolla, California, USA
- Department of Cellular & Molecular Medicine, University of California, San Diego, La Jolla, California, USA
| | - Wesley Ryan
- Department of Chemistry, University of California, Davis, CA 95616, USA
| | - Aaron F. Carlin
- Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, USA
- Department of Pediatrics, University of California, San Diego, La Jolla, California, USA
| | - Vireak Thon
- Department of Chemistry, University of California, Davis, CA 95616, USA
| | - Warren G. Lewis
- The Scripps Research Institute, Biochemistry Department, 10550 North Torrey Pines Road, La Jolla, California 92037, USA
- Genomics Institute of the Novartis Research Foundation, University of California, Davis, CA 95616, USA
| | - Ajit Varki
- Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, USA
- Department of Medicine, University of California, San Diego, La Jolla, California, USA
- Department of Cellular & Molecular Medicine, University of California, San Diego, La Jolla, California, USA
- Address Correspondence to: Ajit Varki, UCSD School of Medicine, La Jolla, CA 92093-0687 Phone: (858) 534-2214; Fax: (858) 534-5611;
| | - Xi Chen
- Department of Chemistry, University of California, Davis, CA 95616, USA
| | - Victor Nizet
- Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California, USA
- Department of Pediatrics, University of California, San Diego, La Jolla, California, USA
- School of Medicine, Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, USA
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