Fragmentation characteristics of glycopeptides

Mass spectrometric analysis of glycopeptides is an emerging strategy for analysis of glycosylation patterns. Here we present an approach using energy resolved collision induced decomposition (CID) spectra to determine structural features of glycopeptides. Fragmentation of multiply protonated glycopeptides proceeds by a series of competing charge separation processes by cleavage of a glycosidic bond, each producing two charged products: a singly charged, “B” type sugar (oxonium) ion, and a complementary high mass fragment. Energy requirements (activation energies) of these processes are similar to each other, and are far less, than that required for peptide fragmentation. At higher collision energies these first generation products fragment further, yielding a complex fragmentation pattern. Analysis of low energy spectra (those corresponding to ca. 50% survival yield) are straightforward; the ions observed correspond to structural features present in the oligosaccharide, and are not complicated by consecutive reactions. This makes it feasible to identify and distinguish antenna- and core-fucosylated isomers; antenna fucosylation usually suggests presence of the Lewis-X antigen. In general, analysis of the triply protonated molecules are most advantageous, where neutral losses and monosaccharide oxonium ion formation are less abundant

Lewis X antigen mediates adhesion of human breast carcinoma cells to activated endothelium. Possible involvement of the endothelial scavenger receptor C-Type lectin

Lewis x (Lex, CD15), also known as SSEA-1 (stage specific embryonic antigen-1), is a trisaccharide with the structure Galβ(1–4)Fucα(1–3)GlcNAc, which is expressed on glycoconjugates in human polymorphonuclear granulocytes and various tumors such as colon and breast carcinoma. We have investigated the role of Lex in the adhesion of MCF-7 human breast cancer cells and PMN to human umbilical endothelial cells (HUVEC) and the effects of two different anti-Lex mAbs (FC-2.15 and MCS-1) on this adhesion. We also analyzed the cytolysis of Lex+-cells induced by anti-Lex mAbs and complement when cells were adhered to the endothelium, and the effect of these antibodies on HUVEC. The results indicate that MCF-7 cells can bind to HUVEC, and that MCS-1 but not FC-2.15 mAb inhibit this interaction. Both mAbs can efficiently lyse MCF-7 cells bound to HUVEC in the presence of complement without damaging endothelial cells. We also found a Lex-dependent PMN interaction with HUVEC. Although both anti-Lex mAbs lysed PMN in suspension and adhered to HUVEC, PMN aggregation was only induced by mAb FC-2.15. Blotting studies revealed that the endothelial scavenger receptor C-type lectin (SRCL), which binds Lex-trisaccharide, interacts with specific glycoproteins of Mr␣∼␣28 kD and 10 kD from MCF-7 cells. The interaction between Lex+-cancer cells and vascular endothelium is a potential target for cancer treatment.Fil: Elola, Maria Teresa. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Capurro, Mariana Isabel. University of Toronto; CanadáFil: Barrio, Maria Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Investigación, Docencia y Prevención del Cáncer; ArgentinaFil: Coombs, Peter J.. Imperial College London; Reino UnidoFil: Taylor, Maureen E.. Imperial College London; Reino UnidoFil: Drickamer, Kurt. Imperial College London; Reino UnidoFil: Mordoh, Jose. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Investigación, Docencia y Prevención del Cáncer; Argentin

Lectin-gated and glycan functionalized mesoporous silica nanocontainers for targeting cancer cells overexpressing Lewis X antigen

[EN] Gated mesoporous silica nanoparticles can deliver payload upon the application of a predefined stimulus, and therefore are promising drug delivery systems. Despite their important role, relatively low emphasis has been placed on the design of gating systems that actively target carbohydrate tumor cell membrane receptors. We describe herein a new Lewis X (Le(x)) antigen-targeted delivery system comprising mesoporous silica nanoparticles (MSNs) loaded with ATTO 430LS dye, functionalized with a Le(x) derivative (1) and capped with a fucose-specific carbohydrate-binding protein (Aleuria aurantia lectin (AAL)). This design takes advantage of the affinity of AAL for Le(x) overexpressed receptors in certain cancer cells. In the proximity of the cells, AAL is detached from MSNs to bind Le(x), and selectins in the cells bind Le(x) in the gated MSNs, thereby inducing cargo delivery. Gated MSNs are nontoxic to colon cancer DLD-1 cells, and ATTO 430LS dye delivered correlated with the amount of Le(x) antigen overexpressed at the DLD-1 cell surface. This is one of the few examples of MSNs using biologically relevant glycans for both capping (via interaction with AAL) and targeting (via interaction with overexpressed Le(x) at the cell membrane).The authors thank the Spanish Government (Projects MAT2015-64139-C4-1-R and MAT2013-46101-R (MINECO/ FEDER)), Fondo de Investigacion Sanitaria (PI15/00480) and Generalitat Valenciana (Project PROMETEOII/2014/047 and project GVA/2014/13) for support. R. B. is thankful to Svagata. Eu (Erasmus Mundus Action-II program) for his fellowship. The authors also thank the Electron Microscopy Service at the UPV for support.Bhat, R.; García, I.; Aznar, E.; Arnáiz, B.; Martínez-Bisbal, M.; Liz-Marzán, L.; Penadés, S.... (2018). Lectin-gated and glycan functionalized mesoporous silica nanocontainers for targeting cancer cells overexpressing Lewis X antigen. Nanoscale. 10(1):239-249. https://doi.org/10.1039/c7nr06415bS239249101Argyo, C., Weiss, V., Bräuchle, C., & Bein, T. (2013). Multifunctional Mesoporous Silica Nanoparticles as a Universal Platform for Drug Delivery. Chemistry of Materials, 26(1), 435-451. doi:10.1021/cm402592tAznar, E., Martínez-Máñez, R., & Sancenón, F. (2009). Controlled release using mesoporous materials containing gate-like scaffoldings. Expert Opinion on Drug Delivery, 6(6), 643-655. doi:10.1517/17425240902895980Aznar, E., Oroval, M., Pascual, L., Murguía, J. R., Martínez-Máñez, R., & Sancenón, F. (2016). Gated Materials for On-Command Release of Guest Molecules. Chemical Reviews, 116(2), 561-718. doi:10.1021/acs.chemrev.5b00456Wang, X., Tan, L.-L., Li, X., Song, N., Li, Z., Hu, J.-N., … Yang, Y.-W. (2016). 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Advanced Functional Materials, 25(9), 1404-1417. doi:10.1002/adfm.201403636Burchell, J., Poulsom, R., Hanby, A., Whitehouse, C., Cooper, L., Clausen, H., … Taylor-Papadimitriou, J. (1999). An  2,3 sialyltransferase (ST3Gal I) is elevated in primary breast carcinomas. Glycobiology, 9(12), 1307-1311. doi:10.1093/glycob/9.12.1307Pinho, S. S., Reis, C. A., Paredes, J., Magalhaes, A. M., Ferreira, A. C., Figueiredo, J., … Seruca, R. (2009). The role of N-acetylglucosaminyltransferase III and V in the post-transcriptional modifications of E-cadherin. Human Molecular Genetics, 18(14), 2599-2608. doi:10.1093/hmg/ddp194Takahashi, M., Kuroki, Y., Ohtsubo, K., & Taniguchi, N. (2009). Core fucose and bisecting GlcNAc, the direct modifiers of the N-glycan core: their functions and target proteins. Carbohydrate Research, 344(12), 1387-1390. doi:10.1016/j.carres.2009.04.031Li, M., Song, L., & Qin, X. (2010). Glycan changes: cancer metastasis and anti-cancer vaccines. 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Epigenetic Silencing of the Sulfate Transporter Gene DTDST Induces Sialyl Lewisx Expression and Accelerates Proliferation of Colon Cancer Cells. Cancer Research, 70(10), 4064-4073. doi:10.1158/0008-5472.can-09-2383Golijanin, D., Sherman, Y., Shapiro, A., & Pode, D. (1995). Detection of bladder tumors by immunostaininc of the lewis x antigen in cells from voided urine. Urology, 46(2), 173-177. doi:10.1016/s0090-4295(99)80189-7Hittelet, A., Camby, I., Nagy, N., Legendre, H., Bronckart, Y., Decaestecker, C., … Yeaton, P. (2003). Binding Sites for Lewis Antigens Are Expressed by Human Colon Cancer Cells and Negatively Affect Their Migration. Laboratory Investigation, 83(6), 777-787. doi:10.1097/01.lab.0000073129.62433.39De la Torre, C., Casanova, I., Acosta, G., Coll, C., Moreno, M. J., Albericio, F., … Martínez-Máñez, R. (2014). Gated Mesoporous Silica Nanoparticles Using a Double-Role Circular Peptide for the Controlled and Target-Preferential Release of Doxorubicin in CXCR4-Expresing Lymphoma Cells. Advanced Functional Materials, 25(5), 687-695. doi:10.1002/adfm.201403822De la Fuente, J. M., & Penadés, S. (2002). Synthesis of Lex-neoglycoconjugate to study carbohydrate–carbohydrate associations and its intramolecular interaction. Tetrahedron: Asymmetry, 13(17), 1879-1888. doi:10.1016/s0957-4166(02)00480-9Zhu, T., & Boons, G.-J. (2000). A Novel and Efficient Synthesis of a Dimeric LexOligosaccharide on Polymeric Support. Journal of the American Chemical Society, 122(41), 10222-10223. doi:10.1021/ja001930lMartínez-Ávila, O., Hijazi, K., Marradi, M., Clavel, C., Campion, C., Kelly, C., & Penadés, S. (2009). GoldManno-Glyconanoparticles: Multivalent Systems to Block HIV-1 gp120 Binding to the Lectin DC-SIGN. Chemistry - A European Journal, 15(38), 9874-9888. doi:10.1002/chem.200900923Cory, A. H., Owen, T. 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Studies of the role of sialyl Lewis X antigen and E selectin ligands in colorectal cancer

Glycosylation alterations dysregulate multiple biological processes and are a hallmark of cancer, linked to tumorigenesis and tumour progression. The present study focuses on altered glycosylation in colorectal cancer (CRC), the third most common cancer worldwide. Increased sialylation and fucosylation are reported in CRC and associated with malignant tumour features. This increase is translated by upregulation of the sialofucosylated sialyl Lewis X (sLeX) antigen, a ligand of the endothelial E-selectin, having a potential role in metastasis. Thus, overexpressing sLeX antigen may affect the expression of E-selectin ligands and the invasion capacity of CRC cell lines. Moreover, the effect of increased sLeX antigen expression on tumour cells immunosuppressive strategies has not been clearly examined so far. To address these hypotheses, we first characterised the impact on the biology and the glycan profile of sLeX overexpression in CRC cells. The results showed improvement of cell migration and reactivity with E-selectin, upon increased sLeX expression. Then, we identified the glycoproteins immunoprecipitated with E-selectin by mass spectrometry, and our results revealed neural cell adhesion molecule L1 (L1CAM). Furthermore, we showed that the sLeX antigen overexpression by CRC cells reduces the maturation profile of dendritic cells (DCs), as inferred by a decreased expression of the antigen presenting molecule, MHC-II, and the co-stimulatory molecule, CD86. This thesis is the first to report the L1CAM ability to interact with E-selectin. Since L1CAM is known to be elevated in cancer and associated with metastasis and progression, this should contribute to better understand its action mechanism. Also, the reduced DCs maturation induced by sLeX expressing CRC cells, may diminished the capacity to appropriately engage immune response against tumour cells. Overall, these findings contribute to elucidate the role of sLeX antigen and E-selectin ligands on CRC progression, metastasis and in the tumour immune system escape strategy, proposing potential novel targets for therapeutic treatments of CRC.As alterações da glicosilação desregulam múltiplos processos biológicos e são características do cancro, relacionadas com a tumorigénese e progressão tumoral. Este estudo foca-se na glicosilação alterada em cancro colorretal (CRC), o terceiro cancro mais comum no mundo. Um aumento da sialilação e fucosilação foram reportados em CRC e associados com características de tumores malignos. Estas alterações resultaram num incremento da expressão do antigénio sialofucosilado sialil Lewis X (sLeX), um ligando da E-selectina endotelial, tendo potencial na formação de metástases. Assim, a sobreexpressão do antigénio sLeX pode afectar a expressão dos ligandos da E-selectina e a capacidade de invasão de linhas celulares de CRC. Além disso, o efeito da expressão aumentada do antigénio sLeX em estratégias imunossupressoras de células tumorais não foram claramente discutidas até à data. Para responder a estas hipóteses, primeiro caracterizámos o impacto na biologia e no perfil de glicanos da sobreexpressão de sLeX em células de CRC. Os resultados mostraram melhoria na migração celular e na reatividade com a E-selectina quando a expressão de sLeX foi aumentada. Em seguida, identificámos as glicoproteínas imunoprecipitadas com E-selectina por espectrometria de massa, e os nossos resultados revelaram a molécula de adesão das células neurais L1 (L1CAM). Para além disso, mostrámos que a sobreexpressão do antigénio sLeX nas células de CRC reduz o perfil de maturação das células dendríticas (DCs), conforme indicado pela diminuição na expressão da molécula de apresentação de antigénios, MHC-II, e a molécula co-estimuladora, CD86. Esta tese é a primeira a relatar a capacidade de interação da L1CAM com a E-selectina. Como é conhecido que a L1CAM possui uma elevada expressão em cancro e que está associada com metástases e progressão tumoral, estes resultados devem contribuir para melhor compreender o seu mecanismo de ação. Também a redução na maturação das DCs induzida por células de CRC que expressam sLeX pode diminuir a capacidade de iniciar adequadamente a resposta imunológica contra células tumorais. Em geral, estes resultados contribuem para elucidar o papel do antigénio sLeX e dos ligandos de E-selectina na progressão do CRC, na formação de metástases e na estratégia tumoral de evasão do sistema imunológico, propondo novos potenciais alvos para tratamentos terapêuticos para o CRC

Study of glycosylation of prostate-specific antigen secreted by cancer tissue-originated spheroids reveals new candidates for prostate cancer detection

Prostate-specific antigen (PSA) is the most frequently used biomarker for the screening of prostate cancer. Understanding the structure of cancer-specific glycans can help us improve PSA assay. In the present study, we analysed the glycans of PSA obtained from culture medium containing cancer tissue-originated spheroids (CTOS) which have similar characteristics as that of the parent tumour to explore the new candidates for cancer-related glycoforms of PSA. The glycan profile of PSA from CTOS was determined by comparing with PSA from normal seminal plasma and cancer cell lines (LNCaP and 22Rv1) using lectin chromatography and mass spectrometry. PSA from CTOS was mostly sialylated and the content of Wisteria floribunda agglutinin reactive glycan (LacdiNAc) was similar to that of PSA derived from seminal plasma and 22Rv1. Conversely, concanavalin A (Con A)-unbound PSA was definitely detected from the three cancer origins but was almost negligible in seminal PSA. Two novel types of PSA were elucidated in the Con A-unbound fraction: one is a high molecular weight PSA with highly branched N-glycans, and the other is a low molecular weight PSA without N-glycans. Furthermore, the existence of Lewis X antigen group on PSA was indicated. These PSAs will be candidates for new cancer-related markers

Targeting Human Dendritic Cells with Lewis X Modified Liposomes

In this short report, we demonstrate that liposomes bearing the Lewis X trisaccharide on the surface (“Awesosomes”) efficiently target human dendritic cells. We chose a glycolipid with Lewis X trisaccharide headgroup to facilitate the targeted liposome uptake via the DC-SIGN internalization pathway. While no uptake of Awesosomes was detected with wild-type human HEK293 cells, HEK293 cells transfected with human DC-SIGN internalized Awesosomes extensively. In samples of human blood-derived leukocytes, the extent of uptake of Awesosomes correlated with the expression of DC-SIGN, which is a dendritic cells marker. There was a marked difference in the uptake of Awesosomes and plain liposomes by DC-SIGN expressing dendritic cells. There was no difference in uptake of Awesosomes and plain liposomes by wt HEK293 cells or macrophages. These results indicate that Lewis X trisaccharide can “sweet-talk” dendritic cells into internalizing a delivery vehicle, and that Awesosomes are promising as “magic bullets” for specific delivery of drugs, antigens, or immunostimulatory molecules to human dendritic cells without influencing other cell types

Glycosylation pattern of brush border-associated glycoproteins in enterocyte-like cells: involvement of complex-type N-glycans in apical trafficking

We have previously reported that galectin-4, a tandem repeat-type galectin, regulates the raft-dependent delivery of glycoproteins to the apical brush border membrane of enterocyte-like HT-29 cells. N-Acetyllactosamine-containing glycans, known as galectin ligands, were found enriched in detergent-resistant membranes. Here, we analyzed the potential contribution of N-and/ or O-glycans in this mechanism. Structural studies were carried out on the brush border membrane-enriched fraction using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and nano-ESI-QTOF-MS/MS. The pattern of N-glycans was very heterogeneous, with the presence of high mannose- and hybrid-type glycans as well as a multitude of complex-type glycans. In contrast, the pattern of O-glycans was very simple with the presence of two major core type 1 O-glycans, sialylated and bisialylated T-antigen structures {[}Neu5Ac alpha 2-3Gal beta 1-3GalNAc-ol and Neu5Ac alpha 2-3Gal beta 1 -3(Neu5Ac alpha 2-6)GalNAc-ol]. Thus, N-glycans rather than O-glycans contain the N-acetyllactosamine recognition signals for the lipid raft-based galectin-4-dependent apical delivery. In the presence of 1-deoxymannojirimycin, a drug which inhibits the generation of hybrid-type or complex type N-glycans, the extensively O-glycosylated mucin-like MUC1 glycoprotein was not delivered to the apical brush border but accumulated inside the cells. Altogether, our data demonstrate the crucial role of complex N-glycans in the galectin-4-dependent delivery of glycoproteins to the apical brush border membrane of enterocytic HT-29 cells

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression

Small-cell lung cancer with a rare epidermal growth factor receptor gene mutation showing “wax-and-wane” transformation

BACKGROUND: Small-cell lung cancer with epidermal growth factor receptor (EGFR) gene mutation typically manifests as a transformation occurring after EGFR tyrosine kinase inhibitor therapy for adenocarcinoma with EGFR mutation, whereas primary small-cell lung cancer showing EGFR mutation is extremely rare. Second biopsy of EGFR-mutated tumor has been broadly recognized as necessary, but is not always performed in daily practice, mainly due to the imbalance between the potential risk of the diagnostic procedure and the therapeutic impact of the biopsy result. CASE PRESENTATION: A 70-year-old woman who had never smoked was referred to our hospital with chief complaints of cough and back pain. Transbronchial lung biopsy from the primary tumor of the left upper lobe revealed combined small-cell lung cancer and adenocarcinoma, a subtype of small-cell lung cancer. EGFR L861Q mutation was detected in both small-cell lung cancer and adenocarcinoma components. Given the staging of cT2aN3M1b (Stage IV) and histological diagnosis, first-line chemotherapy with cisplatin plus irinotecan was initiated, and partial response was achieved. Seven months after initial diagnosis, the primary tumor enlarged again, and a second biopsy from the enlarged lesion detected only adenocarcinoma with the L861Q mutation. Erlotinib was started, but multiple brain metastases and enlarged mediastinal lymph nodes subsequently appeared. Whole-brain radiation therapy was performed, and endobronchial ultrasonography-guided transbronchial biopsy from the lymph node revealed reverse transformation to small-cell lung cancer with the L861Q mutation. Amrubicin therapy achieved partial response after two cycles, with the shrinkage lasting for eight months. Serum sialyl Lewis X antigen level increased when the adenocarcinoma component was dominant, whereas plasma pro-gastrin-releasing peptide level increased when the small-cell lung cancer component became dominant. CONCLUSIONS: Transformation of the tumor correlates with the difference between small-cell lung cancer and adenocarcinoma in sensitivity to therapies, so repeated biopsies are beneficial for choosing appropriate treatments. Noninvasively obtainable parameters such as tumor markers can support the need for biopsy

Cell surface-specific N-glycan profiling in breast cancer

Aberrant changes in specific glycans have been shown to be associated with immunosurveillance, tumorigenesis, tumor progression and metastasis. In this study, the N-glycan profiling of membrane proteins from human breast cancer cell lines and tissues was detected using modified DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). The N-glycan profiles of membrane proteins were analyzed from 7 breast cancer cell lines and MCF 10A, as well as from 100 pairs of breast cancer and corresponding adjacent tissues. The results showed that, compared with the matched adjacent normal tissue samples, two biantennary N-glycans (NA2 and NA2FB) were significantly decreased (p <0.0001) in the breast cancer tissue samples, while the triantennary glycan (NA3FB) and a high-mannose glycan (M8) were dramatically increased (p = 0.001 and p <0.0001, respectively). Moreover, the alterations in these specific N-glycans occurred through the oncogenesis and progression of breast cancer. These results suggested that the modified method based on DSA-FACE is a high-throughput detection technology that is suited for analyzing cell surface N-glycans. These cell surface-specific N-glycans may be helpful in recognizing the mechanisms of tumor cell immunologic escape and could be potential targets for new breast cancer drugs