1) Stacy A. Malaker1,2, Jusal Quanico1, Antonella Raffo Romero1, Firas Kobeissy3, Soulaimane Aboulouard1, Dominique Tierny4, Carolyn R. Bertozzi2,5, Isabelle Fournier1*, Michel Salzet1*
1Université de Lille 1, INSERM, U1192 - Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse (PRISM), F-59000 Lille, France
2Department of Chemistry and ChEM-H, Stanford University, California, 94035 United States
3Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, Lebanon
4OCR (Oncovet Clinical Research), Parc Eurasanté Lille Métropole, 80 rue du Dr Yersin, F-59120 Loos, France
5Howard Hughes Medical Institute, Stanford University, Stanford, California, 94305 United States
E-mail: smalaker@stanford.edu
Here we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of MALDI mass spectrometry N-glycan imaging and spatially-resolved glycoproteomic strategies. We subjected formalin-fixed, paraffin-embedded glioma biopsies to on-tissue PNGaseF digestion and MALDI imaging and found that the glycan HexNAc4-Hex5-NeuAc2 was found to be predominantly expressed in necrotic regions of high-grade canine gliomas, whereas high mannose HexNAc2-Hex5 was predominantly found in benign regions. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and the extracts were analyzed by LC-MS/MS without further glycopeptide enrichment. Results identified haptoglobin, which is involved in iron scavenging that presents aberrant fucosylation/sialylation in various cancers, as the protein associated with HexNAc4-Hex5-NeuAc2. Additionally, we identified several high-mannose (Hex2-HexNAc5) glycopeptides enriched in benign regions. To the best of our knowledge, this is the first report that directly links glycan imaging with intact glycopeptide identification. In total, our spatially-resolved glycoproteomics technique identified over 400 N-glycosylated, O-GalNAcylated, O-mannosylated, and S- and O- GlcNAcylated glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slides and the sensitivity of our technique. While N-glycosylation and O- mannosylation were similar between benign and tumor/necrotic sections, S- and O-GlcNAc glycopeptides were significantly deceased in tumor/necrotic sections, whereas sialylated O-GalNAc glycopeptides were significantly upregulated. Ultimately, this proof-of-principle work demonstrates the capability of spatially-resolved glycoproteomics to complement MALDI-imaging technologies in understanding dysregulated glycosylation in cancer.
2) “Use of hydroxylamines, hydroxamic acids, oximes and amines as nucleophiles in the Zbiral oxidative deamination of N-acetyl neuraminic acid. Isolation and characterization of novel mono- and disubstitution products”
Mohammed Hawsawia,b*, Michael G. Pirroneb,c, Anura Wickramasingheb,c, David Crichb,c,d,e
aDepartment of Chemistry, Umm AlQura University, Makkah, Saudi Arabia.
bDepartment of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI, 48202, USA.
cDepartment of Pharmaceutical and Biomedical Sciences, University of Georgia, 250 West Green Street, Athens, GA, 30602, USA.
dDepartment of Chemistry, University of Georgia, 140 Cedar Street, Athens, GA, 30602, USA.
eComplex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA, 30602, USA.
E-mail: Mbhawsawi@uqu.edu.sa
We extended the range of nucleophiles for the oxidative deamination of N-acetyl neuraminic acid studies by the use of hydroxylamines and weakly basic amines nucleophiles. Alkenediazonium intermediate gave access to deamination products of sialic acid derivatives. Aniline as nucleophile provided a novel aziridine moiety attaching to C4 and C5 of neuraminic acid derivative.
3) “Synthesis of the complete O-antigen of Vibrio cholerae O1, serotype Inaba”
Mana M. Mukherjee, John A. Hanover and Pavol Kováč
NIDDK, National Institutes of Health, Bethesda, MD 20892-0815
E-mail: mana.mukherjee@nih.gov
The O-SPs of the two strains of V. cholerae O1, Ogawa and Inaba, consist of a chain of 15-20 (1®2)-a-linked repeats of 4-amino-4,6-dideoxy-D-mannopyranose (D-perosamine), {1} where the amino group is acylated with 3-deoxy-L-glycero-tetronic acid.{2} The two serotypes differ in that the terminal, upstream residue is methylated at O-2 in the Ogawa O-SP. Here we report the first synthesis of the 18-mer of the monosaccharide repeat, which is, essentially, the complete O-antigen of serotype Inaba. On the way towards the octadecasaccharide, we used a mono-, tri- and hexasaccharide glycosyl donors, containing 4-azido group as a latent amino group. These were coupled with suitable glycosyl acceptors. The dodecasaccharide was built via coupling of a hexasaccharide donor and a hexasaccharide acceptor. The corresponding octadecasaccharide was built via coupling of a hexasaccharide donor and a dodecasaccharide acceptor. D-Perosamine being a rare sugar, we had to make its immediate precursor on a few hundred-gram scale from the commercially available methyl a-D-mannopyranoside. Many reaction protocols involved have been substantially improved, compared to the conversions developed earlier, and gave the desired products in excellent yields and with high stereoselectivity. Most intermediates were obtained in the analytically pure state and were fully characterized for the first time.
References:
1. a) Manning, P.A.; Stroeher, U. H.; Morona, R.; Ed. Wachsmuth, I. K.; Blake, P. A.; Olsvik, O. Vibri cholerae and cholera, American socity for microbiology, Washington DC, 1984, p. 77.; b) Dick Jr., W. E.; Beuret, M.; Ed. Cruse, J. M.; Lewis, R. E. Conjugate Vaccines, 1989, 10, p. 48.
2. Kenne, L.; Lindberg, B.; Unger, P.; Gustafsson, B.; Holme, T. Carbohydr. Res., 1982, 100, 341.
4) Adam Kositzke
University of Wisconsin-Madison
E-mail: akositzke@wisc.edu
The essential human O-linked a-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme responsible for modifying thousands of intracellular proteins with the monosaccharide O-GlcNAc. This unique modification plays crucial roles in human health and disease, but the substrate recognition of OGT remains poorly understood. Intriguingly, the only human enzyme reported to remove this modification, O-GlcNAcase (OGA), is itself O-GlcNAc modified. Here, we exploited a GlcNAc electrophilic probe (GEP1A) to rapidly screen OGT mutants in a fluorescence assay that can discriminate between altered OGT-sugar and -protein substrate binding to help elucidate the binding mode of OGT toward OGA protein substrate. By revealing how OGT interacts with OGA, we have advanced our understanding of OGT-protein substrate recognition and our findings will facilitate the development of new strategies to investigate the role of substrate-specific O-GlcNAcylation.
5) “Desialyaltion Profile of THP-1 Macrophage upon LPS Stimulation”
Yu Zhao1, Xue-Long Sun*1
1Department of Chemistry, Chemical and Biomedical Engineering and Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, Cleveland, OH, 44115, USA
E-mail: y.zhao26@vikes.csuohio.edu
Macrophages are versatile cells that take part in many physiological and pathological processes. Macrophage cell surface expresses a dense layer of glycans often terminated with sialic acids (Sias), which are involved in many biological processes of macrophages, such as host-pathogen recognition, migration, and antigen presentation. In addition, the remove of Sisa from cell surface receptors of macrophages is involved in signal transduction of either physiological or pathological pathways as well. It was reported that LPS induces endogenous sialidase Neu1 expression, which causes desialylation and enhances NF-κB activation and pro-inflammatory cytokine production. In this study, we investigated the desialylation profile of THP-1 macrophage upon LPS stimulation, by measuring the Sia amount changes in the cells, free Sia release in the cell culture medium by LC-MS/MS method. We also examined sialidase NEU1 and NEU3 expression and activity of THP-1 macrophage upon LPS stimulation by western blotting and sialidase specific substrates. As a result, LPS causes the total Sias decreased in cells and free Sias increased in the cell culture medium of THP-1 macrophage upon LPS stimulation. In addition, cell membrane Neu1 activity increased, while Neu3 activity decreased, but both were not significant. Interestingly, the Neu1 and Neu3 activity in the cell culture medium increased significantly. To quantify the desialyaltion profile of macrophage upon LPS Stimulation will be useful for understanding of the desialylation mechanism in signal transduction of macrophages related to either physiological or pathological processes.
6) “Computational modelling of sugar C-H bond dissociation energies and reactivities towards hydrogen atom transfer (HAT)”
Timur Adrianov1, Julia Turner1, Mia Zakaria1, and Prof. Mark S. Taylor1
1Department of Chemistry, University of Toronto, Toronto, Canada
E-mail: timur.adrianov@mail.utoronto.ca
Recently, several methods have been developed to achieve site-selective C-H functionalization of carbohydrates, which includes photoredox C-C bond formation by Minnaard et al., epimerization by Wendlandt et al., as well as C-H alkylation and redox isomerization reactions reported by our group.1–4 Due to the structural complexity of sugars, however, controlling and predicting the selectivity of such transformations remains a challenge. Herein, in an attempt to address this issue, we employed computational modelling based on density functional theory (DFT) to gain insight into the trends in the strengths (bond dissociation enthalpies) and stabilities (reactivity towards hydrogen atom transfer) of C–H bonds in various sugars, as a function of configuration and substitution pattern. The results of our study can be potentially used to develop novel site-selective radical reactions of sugars and to understand the radical scavenging capability of carbohydrates.
References:
[1] Minnaard, Chem. Commun., 2017, 53, 4926-4929
[2] Wendlandt, Nature, 2020, 578, 403-408
[3] Taylor, J. Am. Chem. Soc., 2019, 141, 5149-5153
[4] Taylor, Chem. Sci., 2020, 11, 1531-1537
7) Vinod Kumar Gattoji
University of Toledo
E-mail: vinodkumar.gattoji@rockets.utoledo.edu
Disialoganglioside GD2 is a tumor associated carbohydrate antigen (TACA) expressed on neuroblastomas (NB), a cancer which starts in early nerve cells i.e., neuroblasts of the sympathetic nervous system. Approximately 700 children under the age of five, are diagnosed with NB in the United States every year and accounts for 6% of all childhood cancers. 99% of the NB cells have GD2 expressed on their cell surface and approximately 5-10 million molecules of GD2/cell. In an attempt to validate our entirely carbohydrate-based immunogens we envisage a strategy to chemically prepare a linker free construct by synthesizing a aminooxy derivative of the GD2 and then conjugating it to PS A1, isolated from B.fragilis ATCC 25285/NCTC 9343 to get GD2 specific mABs.
8) Ka Keung Chan
Cleveland State University
E-mail: k.chan81@vikes.csuohio.edu
Many biological processes depend on carbohydrate recognitions to relay critical information and trigger crucial signaling events. Therefore, glycoscience research specifically concerning the carbohydrate-protein interactions can provide an abundant opportunity to discover molecular mechanisms of biological processes, potential therapeutic targets, and diagnostic mechanisms for various diseases. Developing useful tools to comprehensively elucidate the specificity of the carbohydrate-binding proteins (CBPs) and their functions. To achieve the goal of identifying the specificity of CBPs for particular glycan structures, a novel CBPs probing tool involving affinity photo-crosslinking was designed and its function was demonstrated. The aryl azide chain-end functionalized N-glycan polymer was first synthesized from free glycan via glycosylamine intermediate followed by acrylation and polymerization via cyanoxyl-mediated free radical polymerization (CMFRP) in one-pot fashion. Affinity-assisted photo-labeling capability of the aryl azide N-glycan polymers was demonstrated using beta-galactose-specific lectin from Arachis hypogaea (PNA) and glucose-specific lectin from Concanavalin A (ConA) followed by UV irradiation and confirmed by SDS-PAGE with silver staining. Overall, out of the synthesized aryl azide chain-end functionalized polymeric probes, the probes expressing lactose exhibited PNA specificity while those expressing glucose exhibited ConA specificity, which was consistent with expectation as they processed the carbohydrate moieties specifically known for respective interactions. The results demonstrated our novel probes’ specific capability for binding and labeling lectins based on the lectin’s affinity for particular glycol groups on the glycopolymer, whose synthesis can be tailored to test the specificity of a lectin for different glycans. The aryl azide chain-end functionalized glycopolymers will be useful and effective biomimetic probes for specific protein labeling, functionality study and biomarker identification application both in vitro and in vivo.
9) “Glyco-engineering of natural killer cells with CD22 ligands for enhanced anti-cancer immunotherapy”
Shuyao Lang1, Xianwu Wang2, Yunpeng Tian3, Jianghong Zhang3, Xu Yan4, Zhihong Fang5, Jian Weng1, Na Lu6, Xuanjun Wu1,6, Tianlu Li1,6, Hongzhi Cao6, Zhu Li*3, Xuefei Huang*1
1Department of Chemistry and Biomedical Engineering, Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, Michigan 48824, United States.
2Department of Biomaterials, College of Materials, Xiamen University, 422 Siming Nan Road, Xiamen 361005, P. R. China.
3Xiamen Nuokangde Biological Technology Co., Ltd. Xiamen 361006, China.
4School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China.
5Department of Hematology, The First Affiliated Hospital of Xiamen University, Xiamen 361003, China.
6National Glycoengineering Research Center, Shandong University, Qingdao, Shandong 266237, China.
E-mail: langshuy@msu.edu; lizhu@nkdbio.com; huangxu2@msu.edu
Adoptive transfer of immune cells is being actively pursued for cancer treatment. Natural killer (NK) cells, a class of cytotoxic immune cells, generally lack inherent selectivities towards cancer. To bestow tumor targeting abilities and enhance anti-cancer efficacy, a new strategy is established to glyco-engineer NK cells. Carbohydrate based ligands for CD22, a marker for B cell lymphoma, are introduced onto NK cells through either metabolic engineering or glyco-polymer insertion. Such NK cells exhibited much enhanced cytotoxicities towards CD22+ lymphoma cells in a CD22 dependent manner. Importantly, both CD22+ lymphoma cell lines and primary lymphoma cells from human cancer patients can be effectively killed by the engineered NK cells. Furthermore, glyco-engineered NK cells provided significant protection to tumor bearing mice. Thus, NK cell glyco-engineering is an exciting new approach for cancer treatment complementing the current immune cell genetic engineering strategy.
10) Roger Diehl
Massachusetts Institute of Technology
E-mail: rcdiehl@mit.edu
Protein-carbohydrate interactions play crucial roles in biological processes from embryonic development to immune responses, but an understanding of the intermolecular forces that contribute to them is limited. Previously, it has been shown that aromatic residues, especially Trp are heavily overrepresented in carbohydrate binding sites, particularly those involving β-galactose, due to a CH-π interaction between the carbohydrate and the aromatic system. Our goal is to measure the strength and importance of this interaction using the example
of human galectin-3.
Galectin-3 binds lactose-containing glycans using a CH-π interaction between Trp181 and the a-galactoside residue of the ligand. To investigate the contribution of this interaction to binding affinity, we replaced Trp181 with Phe and Tyr to provide a smaller aromatic system, His to provide an electron-poor aromatic system, and Arg and Met to determine the impact of removing the CH-π interaction entirely. All variants were successfully expressed in E. coli, and all were stable at 25°C as measured by differential scanning fluorimetry. Binding affinities of
wild type galectin-3 and each variant were determined by isothermal titration calorimetry.
Replacement of Trp181 with Phe or Tyr caused a modest loss in binding affinity, whereas replacement with His, Arg, or Met allowed only trace binding. These results indicate that electron-rich aromatic systems are crucial for CH-π interactions in carbohydrate binding sites, and that such interactions can provide the majority of the binding energy for some lectins.