Imperial College London

ProfessorStuartHaslam

Faculty of Natural SciencesDepartment of Life Sciences

Professor in Structural Glycobiology
 
 
 
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Contact

 

+44 (0)20 7594 5222s.haslam

 
 
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Location

 

101ASir Ernst Chain BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
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288 results found

Cao H, Mathur A, Robertson C, Antonopoulos A, Henderson S, Girard L-P, Wong JH, Davie A, Wright S, Brewin J, Rees DC, Dell A, Haslam SM, Vickers MAet al., 2022, Measurement of erythrocyte membrane mannoses to assess splenic function, British Journal of Haematology, Vol: 198, Pages: 155-164, ISSN: 0007-1048

Red blood cells (RBCs) lose plasma membrane in the spleen as they age, but the cells and molecules involved are yet to be identified. Sickle cell disease and infection by Plasmodium falciparum cause oxidative stress that induces aggregates of cross-linked proteins with N-linked high-mannose glycans (HMGs). These glycans can be recognised by mannose-binding lectins, including the mannose receptor (CD206), expressed on macrophages and specialised phagocytic endothelial cells in the spleen to mediate the extravascular haemolysis characteristic of these diseases. We postulated this system might also mediate removal of molecules and membrane in healthy individuals. Surface expression of HMGs on RBCs from patients who had previously undergone splenectomy was therefore assessed: high levels were indeed observable as large membrane aggregates. Glycomic analysis by mass spectrometry identified a mixture of Man5-9GlcNAc2 structures. HMG levels correlated well with manual pit counts (r = 0.75–0.85). To assess further whether HMGs might act as a splenic reticuloendothelial function test, we measured levels on RBCs from patients with potential functional hyposplenism, some of whom exhibited high levels that may indicate risk of complications.

Journal article

Marbiah M, Kotidis P, Donini R, Gómez IA, Jimenez Del Val I, Haslam SM, Polizzi KM, Kontoravdi Cet al., 2022, Rapid antibody glycoengineering in Chinese hamster ovary cells., Journal of Visualized Experiments, Vol: 184, Pages: 1-19, ISSN: 1940-087X

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, t

Journal article

Mountain K, MacIntyre D, Chan D, Hyde A, Lee Y, Brown R, David A, Dell A, Ten F, Haslam S, Liu Y, Lewis H, Norman J, Stock S, Teoh TG, Terzidou V, Kundu S, Bennett P, Sykes Let al., 2022, Blood group antigens influence host-microbe interactions and risk of early preterm birth, Publisher: WILEY, Pages: 55-56, ISSN: 1470-0328

Conference paper

Duncombe L, Howells L, Haughey A, Taylor AV, Kaveh D, Erdenliğ Gϋrbilek S, Dell A, Hitchen PG, Haslam SM, Mandal SS, Ganesh NV, Bundle DR, McGiven Jet al., 2022, The tip of brucella O-Polysaccharide is a potent epitope in response to brucellosis infection and enables short synthetic antigens to be superior diagnostic reagents, Microorganisms, Vol: 10, Pages: 1-19, ISSN: 2076-2607

Brucellosis is a global disease and the world’s most prevalent zoonosis. All cases in livestock and most cases in humans are caused by members of the genus Brucella that possess a surface O-polysaccharide (OPS) comprised of a rare monosaccharide 4-deoxy-4-formamido-D-mannopyranose assembled with α1,2 and α1,3 linkages. The OPS of the bacterium is the basis for serodiagnostic tests for brucellosis. Bacteria that also contain the same rare monosaccharide can induce antibodies that cross-react in serological tests. In previous work we established that synthetic oligosaccharides, representing elements of the Brucella A and M polysaccharide structures, were excellent antigens to explore the antibody response in the context of infection, immunisation and cross reaction. These studies suggested the existence of antibodies that are specific to the tip of the Brucella OPS. Sera from naturally and experimentally Brucella abortus-infected cattle as well as from cattle experimentally infected with the cross-reactive bacterium Yersinia enterocolitica O:9 and field sera that cross react in conventional serological assays were studied here with an expanded panel of synthetic antigens. The addition of chemical features to synthetic antigens that block antibody binding to the tip of the OPS dramatically reduced their polyclonal antibody binding capability providing conclusive evidence that the OPS tip (non-reducing end) is a potent epitope. Selected short oligosaccharides, including those that were exclusively α1,2 linked, also demonstrated superior specificity when evaluated with cross reactive sera compared to native smooth lipopolysaccharide (sLPS) antigen and capped native OPS. This surprising discovery suggests that the OPS tip epitope, even though common to both Brucella and Y. enterocolitica O:9, has more specific diagnostic properties than the linear portion of the native antigens. This finding opens the way to the development of improved serological

Journal article

Kundu S, Lee Y, Sykes L, Chan D, Lewis H, Brown R, Kindinger L, Dell A, Feizi T, Haslam S, Liu Y, Marchesi J, MacIntyre D, Bennett Pet al., 2022, The Effect of Secretor Status and the Vaginal Microbiome on Birth Outcome, Publisher: SPRINGER HEIDELBERG, Pages: 197-197, ISSN: 1933-7191

Conference paper

Edwards E, Livanos M, Krueger A, Dell A, Haslam SM, Mark Smales C, Bracewell DGet al., 2022, Strategies to control therapeutic antibody glycosylation during bioprocessing: synthesis and separation., Biotechnology and Bioengineering, Vol: 119, ISSN: 0006-3592

Glycosylation can be a critical quality attribute (CQA) in biologic manufacturing. In particular, it has implications on the half-life, immunogenicity and pharmacokinetics of therapeutic monoclonal antibodies (mAbs) and must be closely monitored throughout drug development and manufacturing. To address this, advances have been made primarily in upstream processing, including mammalian cell line engineering to yield more predictably glycosylated mAbs, and the addition of media supplements during fermentation to manipulate the metabolic pathways involved in glycosylation. A more robust approach would be a conjoined upstream-downstream processing strategy. This could include implementing novel downstream technologies, such as the use of Fc gamma-based affinity ligands for the separation of mAb glycovariants. This review highlights the importance of controlling therapeutic antibody glycosylation patterns, the challenges faced in terms of glycosylation during mAb biosimilar development, current efforts both upstream and downstream to control glycosylation and their limitations, and the need for research in the downstream space in order to establish holistic and consistent manufacturing processes for the production of antibody therapies. This article is protected by copyright. All rights reserved.

Journal article

Kotidis P, Marbiah M, Donini R, Gómez IA, Del Val IJ, Haslam SM, Polizzi KM, Kontoravdi Cet al., 2022, Rapid Antibody Glycoengineering in CHO Cells Via RNA Interference and CGE-LIF N-Glycomics., Methods Mol Biol, Vol: 2370, Pages: 147-167

The impact of the glycan distribution on the in vivo function and half-life of monoclonal antibodies has long motivated the genetic engineering of producer cells to achieve structures that enhance efficacy, safety and stability. To facilitate glycoengineering of IgG-producing Chinese hamster ovary cells, we present a rapid protocol that involves the use of RNA interference for the knockdown of genes of interest coupled with capillary gel electrophoresis and laser-induced fluorescence detection (CGE-LIF) for fast, high-throughput glycan analysis. We apply this methodology to the Fut8 gene, responsible for the addition of core fucose, which is a typical target for increasing antibody-dependent cellular cytotoxicity.

Journal article

Kawahara R, Chernykh A, Alagesan K, Bern M, Cao W, Chalkley RJ, Cheng K, Choo MS, Edwards N, Goldman R, Hoffmann M, Hu Y, Huang Y, Kim JY, Kletter D, Liquet B, Liu M, Mechref Y, Meng B, Neelamegham S, Nguyen-Khuong T, Nilsson J, Pap A, Park GW, Parker BL, Pegg CL, Penninger JM, Phung TK, Pioch M, Rapp E, Sakalli E, Sanda M, Schulz BL, Scott NE, Sofronov G, Stadlmann J, Vakhrushev SY, Woo CM, Wu H-Y, Yang P, Ying W, Zhang H, Zhang Y, Zhao J, Zaia J, Haslam SM, Palmisano G, Yoo JS, Larson G, Khoo K-H, Medzihradszky KF, Kolarich D, Packer NH, Thaysen-Andersen Met al., 2021, Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis (vol 18, pg 1304, 2021), NATURE METHODS, Vol: 19, Pages: 130-130, ISSN: 1548-7091

Journal article

Chakraborty A, Perez M, Mohammed NBB, Antonopoulos A, Ortega L, Wells M, Carroll JD, Staudinger C, Marrero C, Jimenez R, Wilmott JS, Thompson JF, Wang W, Scolyer RA, Murphy GF, Haslam SM, Dimitroff CJet al., 2021, hypoxia controls the glycobiological signature and related pro-tumorigenic properties of metastatic melanomas, Publisher: OXFORD UNIV PRESS INC, Pages: 1683-1684, ISSN: 0959-6658

Conference paper

Peswani AR, Narkpuk J, Krueger A, Bracewell DG, Lekcharoensuk P, Haslam SM, Dell A, Jaru-Ampornpan P, Robinson Cet al., 2021, Novel constructs and 1-step chromatography protocols for the production of Porcine Circovirus 2d (PCV2d) and Circovirus 3 (PCV3) subunit vaccine candidates, FOOD AND BIOPRODUCTS PROCESSING, Vol: 131, Pages: 125-135, ISSN: 0960-3085

Journal article

Kawahara R, Chernykh A, Alagesan K, Bern M, Cao W, Chalkley RJ, Cheng K, Choo MS, Edwards N, Goldman R, Hoffmann M, Hu Y, Huang Y, Kim JY, Kletter D, Liquet B, Liu M, Mechref Y, Meng B, Neelamegham S, Nguyen-Khuong T, Nilsson J, Pap A, Park GW, Parker BL, Pegg CL, Penninger JM, Phung TK, Pioch M, Rapp E, Sakalli E, Sanda M, Schulz BL, Scott NE, Sofronov G, Stadlmann J, Vakhrushev SY, Woo CM, Wu H-Y, Yang P, Ying W, Zhang H, Zhang Y, Zhao J, Zaia J, Haslam SM, Palmisano G, Yoo JS, Larson G, Khoo K-H, Medzihradszky KF, Kolarich D, Packer NH, Thaysen-Andersen Met al., 2021, Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis, NATURE METHODS, Vol: 18, Pages: 1304-+, ISSN: 1548-7091

Journal article

Ezeabikwa B, Mondal N, Antonopoulos A, Haslam SM, Matsumoto Y, Martin-Caraballo M, Lehoux S, Mandalasi M, Ishaque A, Heimburg-Molinaro J, Cummings RD, Nyame AKet al., 2021, Major differences in glycosylation and Fucosyltransferase expression in low-grade versus high-grade bladder cancer cell lines., Glycobiology, Vol: 31, Pages: 1444-1463, ISSN: 0959-6658

Bladder cancer is the ninth most frequently diagnosed cancer worldwide, and there is a need to develop new biomarkers for staging and prognosis of this disease. Here we report that cell lines derived from low-grade and high-grade bladder cancers exhibit major differences in expression of glycans in surface glycoproteins. We analyzed protein glycosylation in three low-grade bladder cancer cell lines RT4 (grade-1-2), 5637 (grade-2), and SW780 (grade-1), and three high-grade bladder cancer cell lines J82COT (grade-3), T24 (grade-3), and TCCSUP (grade-4), with primary bladder epithelial cells, A/T/N, serving as a normal bladder cell control. Using a variety of approaches including flow cytometry, immunofluorescence, glycomics, and gene expression analysis, we observed that the low-grade bladder cancer cell lines RT4, 5637, and SW780 express high levels of the fucosylated Lewis x (Lex) antigen (CD15) (Galβ1-4(Fucα1-3) GlcNAcβ1-R), while normal bladder epithelial A/T/N cells lack Lex expression. T24 and TCCSUP cells also lack Lex, whereas J82COT cells express low levels of Lex. Glycomics analyses revealed other major differences in fucosylation and sialylation of N-glycans between these cell types. O-glycans are highly differentiated, as RT4 cells synthesize core 2-based O-glycans that are lacking in the T24 cells. These differences in glycan expression correlated with differences in RNA expression levels of their cognate glycosyltransferases, including α1-3/4-fucosyltransferase genes. These major differences in glycan structures and gene expression profiles between low- and high-grade bladder cancer cells suggest that glycans and glycosyltransferases are candidate biomarkers for grading bladder cancers.

Journal article

Baksmeier C, Blundell P, Steckel J, Schultz V, Gu Q, Da Silva Filipe A, Kohl A, Linnington C, Lu D, Dell A, Haslam S, Wang J, Czajkowsky D, Goebels N, Pleass RJet al., 2021, Modified recombinant human IgG1-Fc is superior to natural IVIG at inhibiting immune-mediated demyelination., Immunology, Vol: 464, Pages: 90-105, ISSN: 0019-2805

Intravenous immunoglobulin (IVIG) is an established treatment for numerous autoimmune conditions. Although Fc fragments derived from IVIG have shown efficacy in controlling immune thrombocytopenia (ITP) in children, the mechanisms of action are unclear and controversial. The aim of this study is to dissect IVIG effector mechanisms using further adapted Fc fragments on demyelination in an ex vivo model of the central nervous system (CNS)-immune interface. Using organotypic cerebellar slice cultures (OSC) from transgenic mice we induced extensive immune-mediated demyelination and oligodendrocyte loss with an antibody specific for myelin oligodendrocyte glycoprotein (MOG) and complement. Protective effects of adapted Fc fragments were assessed by live imaging of GFP expression, immunohistochemistry and confocal microscopy. Cysteine and glycan adapted Fc fragments protected OSC from demyelination in a dose-dependent manner where equimolar concentrations of either IVIG or control Fc were ineffective. The protective effects of the adapted Fc fragments are partly attributed to interference with complement-mediated oligodendroglia damage. Transcriptome analysis ruled out signatures associated with inflammatory or innate immune responses. Taken together our findings show that recombinant biomimetics can be made that are at least two hundred-fold more effective than IVIG in controlling demyelination by anti-MOG antibodies.

Journal article

Chakraborty A, Perez M, Mohammed NBB, Wells M, Wilmott JS, Thompson JF, Haslam SM, Wang W, Scolyer RA, Murphy GF, Dimitroff CJet al., 2021, Hypoxia-mediated downregulation of GCNT2/I-antigen in metastatic melanoma accelerates disease progression and mortality., Publisher: AMER ASSOC CANCER RESEARCH, ISSN: 0008-5472

Conference paper

Bonnardel F, Haslam SM, Dell A, Feizi T, Liu Y, Tajadura-Ortega V, Akune Y, Sykes L, Bennett PR, MacIntyre DA, Lisacek F, Imberty Aet al., 2021, Proteome-wide prediction of bacterial carbohydrate-binding proteins as a tool for understanding commensal and pathogen colonisation of the vaginal microbiome, npj Biofilms and Microbiomes, Vol: 7, Pages: 1-10, ISSN: 2055-5008

Bacteria use carbohydrate-binding proteins (CBPs), such as lectins and carbohydrate-binding modules (CBMs), to anchor to specific sugars on host surfaces. CBPs in the gut microbiome are well studied, but their roles in the vagina microbiome and involvement in sexually transmitted infections, cervical cancer and preterm birth are largely unknown. We established a classification system for lectins and designed Hidden Markov Model (HMM) profiles for data mining of bacterial genomes, resulting in identification of >100,000 predicted bacterial lectins available at unilectin.eu/bacteria. Genome screening of 90 isolates from 21 vaginal bacterial species shows that those associated with infection and inflammation produce a larger CBPs repertoire, thus enabling them to potentially bind a wider array of glycans in the vagina. Both the number of predicted bacterial CBPs and their specificities correlated with pathogenicity. This study provides new insights into potential mechanisms of colonisation by commensals and potential pathogens of the reproductive tract that underpin health and disease states.

Journal article

Ng BG, Sosicka P, Fenaille F, Harroche A, Vuillaumier-Barrot S, Porterfield M, Xia Z-J, Wagner S, Bamshad MJ, Vergnes-Boiteux M-C, Cholet S, Dalton S, Dell A, Dupre T, Fiore M, Haslam SM, Huguenin Y, Kumagai T, Kulik M, McGoogan K, Michot C, Nickerson DA, Pascreau T, Borgel D, Raymond K, Warad D, Flanagan-Steet H, Steet R, Tiemeyer M, Seta N, Bruneel A, Freeze HHet al., 2021, A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction, AMERICAN JOURNAL OF HUMAN GENETICS, Vol: 108, Pages: 1040-1052, ISSN: 0002-9297

Journal article

Wu G, Murugesan G, Nagala M, McCraw A, Haslam SM, Dell A, Crocker PRet al., 2021, Activation of regulatory T cells triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses [version 1; peer review: 2 approved], Wellcome Open Research, Vol: 6, ISSN: 2398-502X

Background: Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. Its upregulation on macrophages in autoimmune disease was shown previously to promote inflammation through suppressing the expansion of regulatory T cells (Tregs). Here we investigate the molecular basis for Siglec-1 binding to Tregs using in vitro-induced cells as a model system. Methods: Glycosylation changes that affect Siglec‑1 binding were studied by comparing activated and resting Tregs using RNA-Seq, glycomics, proteomics and binding of selected antibodies and lectins. A proximity labelling and proteomics strategy was used to identify Siglec-1 counter-receptors expressed on activated Tregs. Results: Siglec-1 binding was strongly upregulated on activated Tregs, but lost under resting conditions. Glycomics revealed changes in N-glycans and glycolipids following Treg activation and we observed changes in expression of multiple 'glycogenes' that could lead to the observed increase in Siglec-1 binding. Proximity labelling of intact, living cells identified 49 glycoproteins expressed by activated Tregs that may function as Siglec-1 counter-receptors. These represent ~5% of the total membrane protein pool and were mainly related to T cell activation and proliferation. We demonstrate that several of these counter-receptors were upregulated following activation of Tregs and provide initial evidence that their altered glycosylation may also be important for Siglec-1 binding. Conclusions: We provide the first comprehensive analysis of glycan changes that occur in activated Tregs, leading to recognition by the macrophage lectin, Siglec-1 and suppression of Treg expansion. We furthermore provide insights into glycoprotein counter-receptors for Siglec-1 expressed by activated Tregs that are likely to be important for suppressing Treg expansion.

Journal article

Wang S-S, Solar VD, Yu X, Antonopoulos A, Friedman AE, Agarwal K, Garg M, Ahmed SM, Addhya A, Nasirikenari M, Lau JT, Dell A, Haslam SM, Sampathkumar S-G, Neelamegham Set al., 2021, Efficient inhibition of O-glycan biosynthesis using the hexosamine analog Ac(5)GalNTGc, CELL CHEMICAL BIOLOGY, Vol: 28, Pages: 699-+, ISSN: 2451-9448

Journal article

Wang Y, Khan A, Antonopoulos A, Bouche L, Buckley CD, Filer A, Raza K, Li K-P, Tolusso B, Gremese E, Kurowska-Stolarska M, Alivernini S, Dell A, Haslam SM, Pineda MAet al., 2021, Loss of alpha 2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis, NATURE COMMUNICATIONS, Vol: 12, ISSN: 2041-1723

Journal article

Donini R, Haslam S, Kontoravdi K, 2021, Glycoengineering Chinese hamster ovary cells: a short history, Biochemical Society Transactions, Vol: 49, Pages: 915-931, ISSN: 0300-5127

Biotherapeutic glycoproteins have revolutionised the field of pharmaceuticals, with new discoveries and continuous improvements underpinning the rapid growth of this industry. N-glycosylation is a critical quality attribute of biotherapeutic glycoproteins that influences the efficacy, half-life and immunogenicity of these drugs. This review will focus on the advances and future directions of remodelling N-glycosylation in Chinese hamster ovary (CHO) cells, which are the workhorse of recombinant biotherapeutic production, with particular emphasis on antibody products, using strategies such as cell line and protein backbone engineering.

Journal article

Cao H, Antonopoulos A, Henderson S, Wassall H, Brewin J, Masson A, Shepherd J, Konieczny G, Patel B, Williams M-L, Davie A, Forrester MA, Hall L, Minter B, Tampakis D, Moss M, Lennon C, Pickford W, Erwig L, Robertson B, Dell A, Brown GD, Wilson HM, Rees DC, Haslam SM, Rowe JA, Barker RN, Vickers MAet al., 2021, Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance, Nature Communications, Vol: 12, Pages: 1-13, ISSN: 2041-1723

In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man5-9GlcNAc2), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum-infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.

Journal article

Antonopoulos A, Broome S, Sharov V, Ziegenfuss C, Easton RL, Panico M, Morris HR, Haslam Set al., 2021, Site-specific characterisation of SARS-CoV-2 spike glycoprotein receptor binding domain, Glycobiology, Vol: 31, Pages: 181-187, ISSN: 0959-6658

The novel coronavirus SARS-CoV-2, the infective agent causing COVID-19, is having a global impact both in terms of human disease as well as socially and economically. Its heavily glycosylated spike glycoprotein is fundamental for the infection process, via its receptor binding domains interaction with the glycoprotein angiotensin converting enzyme 2 on human cell surfaces. We therefore utilized an integrated glycomic and glycoproteomic analytical strategy to characterise both N- and O- glycan site specific glycosylation within the receptor binding domain. We demonstrate the presence of complex type N-glycans with unusual fucosylated LacdiNAc at both sites N331 and N343 and a single site of O-glycosylation on T323.

Journal article

Cao H, Bagchi A, Tampakis D, Laidvee I, Williams M, Minter B, Wright S, Antonopoulos A, Haslam SM, Barker RN, Vickers MAet al., 2021, Human erythrocyte surface fucose expression increases with age and hyperglycemia, Wellcome Open Research, Vol: 6, Pages: 1-14, ISSN: 2398-502X

Background: Reactive oxygen species and other free radicals, together with glucose and its metabolites are believed to play important roles in the aging process. The carbohydrate components of glycosylated proteins are important in mediating cell-cell interactions and a role has been suggested for them in the aging process. Erythrocytes are critical cells in the human body, heavily glycosylated and relatively easily available and so are good candidates to yield insights into how patterns of glycosylation change with age and disease. It has been claimed, based on a periodic acid Schiff assay, that human aging is associated with a decline of erythrocyte surface sialic acids. Plant lectins allow for more specific assays for glycans, including determining the linkage of sialic acids and analysis of single cells by flow cytometry.Methods: Plant lectins, including Maackia amurensis lectin II (MAL), binding to α-2,3 linked sialic acids and Sambucus nigra (SNA), α-2,6 sialic acids, were used in flow cytometry and western blot of erythrocyte surface membrane. N-glycomics mass spectrometry determines glycan structures. Donors varying in age and hyperglycemia, as indicated by HbA1c were analysed.Results: Erythrocyte surface sialic acids have no significant associations with donor age. A combination of storage and cellular aging produces a specific loss of α-2,6 sialic acids. By contrast, erythrocyte surface terminal fucoses increase significantly with donor age. In order to determine which aspects of aging are important in determining this change, we investigated whether this novel human aging biomarker is associated with higher plasma glucose values, assessed by glycated hemoglobin (HbA1c) and reactive oxygen species (ROS) generation. Fucose levels were associated with HbA1c levels, but not ROS generation.Conclusion: Our study identifies novel glycan-based biomarkers for human aging and disease. The simplicity of lectin-based assays provide an attractive cel

Journal article

Mendoza M, Lu D, Ballesteros A, Blois SM, Abernathy K, Feng C, Dimitroff CJ, Zmuda J, Panico M, Dell A, Vasta GR, Haslam SM, Dveksler Get al., 2020, Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand, Glycobiology, Vol: 30, Pages: 895-909, ISSN: 0959-6658

Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 μM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands.

Journal article

Loxley GM, Hooks DO, Antonopoulos A, Dell A, Haslam SM, Linklater WL, Hurst JL, Beynon RJet al., 2020, Vulpeculin: a novel and abundant lipocalin in the urine of the common brushtail possum,Trichosurus vulpecula, OPEN BIOLOGY, Vol: 10

Journal article

Murphy N, Rooney B, Bhattacharyya T, Triana-Chavez O, Krueger A, Haslam SM, O'Rourke V, Panczuk M, Tsang J, Bickford-Smith J, Gilman RH, Tetteh K, Drakeley C, Smales CM, Miles MAet al., 2020, Glycosylation of trypanosoma cruzi TcI antigen reveals recognition by chagasic sera, Scientific Reports, Vol: 10, ISSN: 2045-2322

Chagas disease is considered the most important parasitic disease in Latin America. The protozoan agent, Trypanosoma cruzi, comprises six genetic lineages, TcI-TcVI. Genotyping to link lineage(s) to severity of cardiomyopathy and gastrointestinal pathology is impeded by the sequestration and replication of T. cruzi in host tissues. We describe serology specific for TcI, the predominant lineage north of the Amazon, based on expression of recombinant trypomastigote small surface antigen (gTSSA-I) in the eukaryote Leishmania tarentolae, to allow realistic glycosylation and structure of the antigen. Sera from TcI-endemic regions recognised gTSSA-I (74/146; 50.7%), with no cross reaction with common components of gTSSA-II/V/VI recombinant antigen. Antigenicity was abolished by chemical (periodate) oxidation of gTSSA-I glycosylation but retained after heat-denaturation of conformation. Conversely, non-specific recognition of gTSSA-I by non-endemic malaria sera was abolished by heat-denaturation. TcI-specific serology facilitates investigation between lineage and diverse clinical presentations. Glycosylation cannot be ignored in the search for immunogenic antigens.

Journal article

Debets MF, Tastan OY, Wisnovsky SP, Malaker SA, Angelis N, Moeckl LKR, Choi J, Flynn H, Wagner LJS, Bineva-Todd G, Antonopoulos A, Cioce A, Browne WM, Li Z, Briggs DC, Douglas HL, Hess GT, Agbay AJ, Roustan C, Kjaer S, Haslam S, Snijders AP, Bassik MC, Moerner WE, Li VSW, Bertozzi CR, Schumann Bet al., 2020, Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation, Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 25293-25301, ISSN: 0027-8424

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe GalNAzMe that is specific for cancer-relevant Ser/Thr-N-acetylgalactosamine (O-GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase GALE like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor UDP-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR knock-out (KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, BH-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

Journal article

Li H, Marceau M, Yang T, Liao T, Tang X, Hu R, Xie Y, Tang H, Tay A, Shi Y, Shen Y, Yang T, Pi X, Lamichhane B, Luo Y, Debowski AW, Nilsson H, Haslam SM, Mulloy B, Dell A, Stubbs KA, Marshall BJ, Benghezal Met al., 2020, East-Asian Helicobacter pylori strains synthesize heptan-deficient lipopolysaccharide, Publisher: WILEY, Pages: 75-75, ISSN: 1083-4389

Conference paper

Sela I, Goss V, Becker-Cohen M, Dell A, Haslam SM, Mitrani-Rosenbaum Set al., 2020, The glycomic sialylation profile of GNE Myopathy muscle cells does not point to consistent hyposialylation of individual glycoconjugates, NEUROMUSCULAR DISORDERS, Vol: 30, Pages: 621-630, ISSN: 0960-8966

Journal article

Hautala LC, Pang P-C, Antonopoulos A, Pasanen A, Lee C-L, Chiu PCN, Yeung WSB, Loukovaara M, Butzow R, Haslam SM, Dell A, Koistinen Het al., 2020, Altered glycosylation of glycodelin in endometrial carcinoma, Laboratory Investigation, Vol: 100, Pages: 1014-1025, ISSN: 0023-6837

Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galβ1–4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not

Journal article

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