TriCEPS to visualize binding to the cells with
Flow cytometry and microscopy
Flow-TriCEPS (for flow cytometry) and TAMRA-TriCEPS technology (for microscopy) are tools to perform pretests prior your target identification studies on the living cells for drug candidates/ligands such as peptides, antibodies, ADC’s, proteins and small molecules. The TriCEPS variants we use (Flow-TriCEPS and TAMRA-TriCEPS) are very similar to the TriCEPS/HATRIC molecules used for the identification experiment. The only difference is that we have a visualization function instead of a pull out function. Further, since cells are not oxidized the glycans of the cell surface do not react with TRICEPS thus the observed binding is due to the interaction of the ligand of interest with the unknown targets. Pretest TriCEPS molecules are used to
- Determine the best cell type to use in your target identification experiment
- Define the optimal binding conditions on the living cells (time, temperature, pH)
- Identify co-factors needed for binding to the cells of your drug candidates
Perform functional assays with Flow-TriCEPS coupled drug candidates/ligands.
Flow-TriCEPS with flow cytometry
Utilize Flow-TriCEPS to visualize the binding of TriCEPS coupled ligands to the unknown target(s) at the cell membrane by flow cytometry. Optimize the binding conditions by varying different parameters (pH, temperature, time, co-factors etc.) to define the best conditions for the identification studies using the LRC platforms.
TAMRA-TriCEPS for microscopy
Use TAMRA-TriCEPS to visualize the binding of TriCEPS coupled ligand to the unknown target(s) at the cell membrane by microscopy. Test different binding conditions (pH, temperature, time, co-factors etc.) to find the optimal parameters for the identification experiments with the LRC platforms.
Identify the mode of action
using the TriCEPS platforms
Customers Testimonials – LRC-TriCEPS Service
Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.
OncoLille Cancer Institute
Best,
Silvia Gaggero, PhD
Mitra Lab, Inserm
OncoLille Cancer Institute
Lille, France
AstraZeneca
James Dodgson
AstraZeneca
Cambridge, UK.
UCF College of Medicine
Justine Tigno-Aranjuez, Ph.D.
Assistant Professor of Medicine
UCF College of Medicine
Cohbar
Dr. Lindsay Stark
Drug Discovery Scientist at CohBar
Technical University of Munich
Using LRC-TriCEPS, we aimed to identify novel direct cell surface receptors of our ligand of interest.
At any time, we experienced great support of Dualsystems Biotech. They kindly helped to find optimal conditions for our purposes and provided help with any kind of question before, during and after the experiment. LRC-TriCEPS allowed us to identify novel cell surface receptors of our ligand, which we could successfully validate in different cell types and with different biochemical assays. We can fully recommend Dualsystems Biotech and are looking forward to perform further analyses using LRC-TriCEPS.
Prof. Dr. rer. nat. Achim Krüger
Institute of Experimental Oncology and Therapy Research
Klinikum rechts der Isar, Technical University of Munich
University of Miyazaki
Hideyuki Sakoda, MD, PhD
Associate professor
Department of Biological Sciences, Faculty of Medicine, University of Miyazaki, Japan.
Lund University Diabetes Centre
Dr. Claire L. Lyons,
Associate Researcher
Unit of Medical Protein Science
Lund University Diabetes Centre
Sweden
Australian National University
The Australian National University
Co-Director, Centre for Personalised Immunology, NHMRC Centre of Research Excellence
College of Health & Medicine
The Australian National University
Harvard Medical School, Brigham and Women’s Hospital
Maximillian Rogers, PhD
Research Scientist
Harvard Medical School, Brigham and Women's Hospital
Department of Medicine, Cardiovascular Division
Boston, MA
Center for Biomolecular & Cellular Structure, Institute for Basic Science
Associate Professor
Graduate School of Medical Science and Engineering, KAIST
Chief Investigator
Center for Biomolecular & Cellular Structure, Institute for Basic Science (IBS)
Department of Internal Medicine Erasmus MC
Dr Patric Delhanty
Laboratory of Metabolism and Reproduction
Department of Internal Medicine
Erasmus MC
Rotterdam, The Netherlands
Seoul National University
Chung Hwan Cho, Ph. D. candidate
Environmental Health Microbiology Laboratory
Department of Environmental Public Health
Seoul National University
Immuno-Oncology Discovery from Bristol-Myers Squibb published in Nature
Identification of a new immune-oncology drug target using the LRC-TriCEPS platform on primary human T-cells.
The University of Oklahoma – Health Sciences Center
Anne Kasus-Jacobi, PhD
Associate Professor of Research
University of Oklahoma Health Sciences Center
Department of Pharmaceutical Sciences
Oklahoma City, Oklahoma, USA
CuroNZ Ltd
Frank Sieg, PhD
CSO
CuroNZ Ltd
Mangawhai in New Zealand
University of Pittsburgh
Maliha Zahid, M.D., Ph.D.
Assistant Professor
Departement of Developmental Biology
University of Pittsburgh
University of Oklahoma Health Sciences Center
Anne Kasus-Jacobi, PhD
Assistant Professor of Research
University of Oklahoma Health Sciences Center
Department of Pharmaceutical Sciences
Oklahoma City, Oklahoma, USA
Biomedical Research Institute
The identification of a T cell co-receptor for staphylococcal superantigens had been challenging due to the structural features of the interaction and its kinetics. However, working with Dualstystems Biotech AG, and with Dr. Paul Helbling in particular, and using the LRC-TriCEPS technology, we were able to identify a candidate that was subsequently corroborated by biochemical and functional assays. We are very happy with this collaboration , and sincerely recommend it for the identification of novel receptor or co-receptor candidates.(Quim) Madrenas, MD, PhD, FCAHS
Chief Scientific Officer
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center
Torrance, USA
QIMR Berghofer Medical Research Institute
Hepatic Fibrosis Group
QIMR Berghofer Medical Research Institute, Australia
University of Miami, Miller School of Medicine
I would like to thank once again the company and, particularly, Dr Helbling for his attention and collaboration.
Dr Karina Galoian
Research associate professor
University of Miami, Miller School of Medicine
Department of Orthopedic surgery
Miami, Florida, USA
Münster University Hospital (UKM)
Working group from Prof. Dr. med. Luisa Klotz
Münster University Hospital (UKM), Germany
University of Manitoba
Sari S. Hannila, PhD
Associate Professor, Department of Human Anatomy and Cell Science
Associate Member, Spinal Cord Research Centre
Max Rady College of Medicine, Rady Faculty of Health Sciences
University of Manitoba
The Rockefeller University
Assistant Professor of Clinical Investigation
The Rockefeller University
Medizinische Hochschule Hannover
East Tennessee State University
Assistant Professor
East Tennessee State University
Igenica Biotherapeutics
Senior Director, Preclinical Development
Igenica Biotherapeutics
Centro de Estudos de Doenças Crónicas
« The fruitful collaboration with Dualsystems Biotech using the LRC-TriCEPS (CaptiRec) technology showed that even on insect cells receptors could be identified »
Alisson M. Gontijo,
Principal Investigator at CEDOC
Centro de Estudos de Doenças Crónicas
Washington University School of Medicine
University of California San Francisco
Assistant Professor in Residence
University of California San Francisco (UCSF)
LRC-TriCEPS / HATRIC-LRC Publications
Concerning the LRC-TriCEPS or HATRIC-LRC platforms.
The LRC-TriCEPS platform identified 2 new receptors for ApoA-1, potentially new drug targets for Diabetes Type 2
Apolipoprotein A-I priming via SR-BI and ABCA1 receptor binding upregulates mitochondrial metabolism to promote insulin secretion in INS-1E cells
Abstract : Apolipoprotein A-I (ApoA-I), the primary component of high-density lipoprotein (HDL) cholesterol primes β-cells to increase>INS secretion, however, the mechanisms involved are not fully defined. Here, we aimed to confirm ApoA-I receptors in β-cells and delineate ApoA-I-receptor pathways in β-cell>INS output. An LRC-TriCEPS experiment was performed using the INS-1E rat β-cell model and ApoA-I for unbiased identification of ApoA-I receptors. We confirm that SR-BI and ABCA1 are the primary β-cell ApoA-I receptors and demonstrate that ApoA-I priming enhances β-cell>INS secretion via the upregulation of mitochondrial metabolism through ApoA-I-SR-BI and ApoA-I-ABCA1 pathways. We propose that SR-BI relies on mitochondrial and exocytotic pathways, while ABCA1 depends solely on mitochondrial pathways. Our findings uncover new targets in ApoA-I β-cell mechanism for type 2 diabetes therapies. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0311039
Identification of a receptor for house dust mite allergens
Development of therapies with the potential to change the allergic asthmatic disease course will require the discovery of targets that play a central role during the initiation of an allergic response, such as those involved in the process of allergen recognition. We use a receptor glycocapture technique to screen for house dust mite (HDM) receptors and identify LMAN1 as a candidate. We verify the ability of LMAN1 to directly bind HDM allergens and demonstrate that LMAN1 is expressed on the surface of dendritic cells (DCs) and airway epithelial cells (AECs) in vivo. Overexpression of LMAN1 downregulates NF-κB signaling in response to inflammatory cytokines or HDM. HDM promotes binding of LMAN1 to the FcRγ and recruitment of SHP1. Last, peripheral DCs of asthmatic individuals show a significant reduction in the expression of LMAN1 compared with healthy controls. These findings have potential implications for the development of therapeutic interventions for atopic disease.
LRC-TriCEPS identified a completely new mechanism of monocyte activation that might be clinically relevant in Alzheimers’ and inflammation diseases. Feb 2023
The emerging cytokine tissue inhibitor of metalloproteinases-1 (TIMP-1) correlates with the progression of inflammatory diseases, including cancer. However, the effects of TIMP-1 on immune cell activation and underlying molecular mechanisms are largely unknown. The LRC-TriCEPS platform revealed TIMP-1-interaction with Amyloid Precursor Protein (APP) family members. We found that TIMP-1 triggered glucose uptake and flammatory cytokine expression in human monocytes. In cancer patients, TIMP-1 expression positively correlated with flammatory cytokine expression and processes associated with monocyte activation. This novel monocyte activation pathway might be clinically relevant for inflammation diseases such as pancreatic cancer but might also be involved in Alzheimers’. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9837626/
Sensei Biotherapeutics Presents Preclinical Data at the 37th Society for Immunotherapy of Cancer (SITC) Annual Meeting
Akkermansia muciniphila secretes a gluc.-like peptide-1-inducing protein that improves glucose homeostasis and ameliorates metabolic disease in mice
Nature Microbiol (2021) https://doi.org/10.1038/s41564-021-00880-5
Here, we report that A. muciniphila increases thermogenesis and gluc.-like peptide-1 (GLP-1) secretion in high-fat-diet (HFD)-induced C57BL/6J mice by induction of uncoupling protein 1 in brown adipose tissue and systemic GLP-1 secretion. We apply fast protein liquid chromatography and liquid chromatography coupled to mass spectrophotometry analysis to identify an 84 kDa protein, named P9, that is secreted by A. muciniphila. Using L cells and mice fed on an HFD, we show that purified P9 alone is sufficient to induce GLP-1 secretion and brown adipose tissue thermogenesis. Using ligand–receptor capture analysis, we find that P9 interacts with intercellular adhesion molecule 2 (ICAM-2). Interleukin-6 deficiency abrogates the effects of P9 in glucose homeostasis and downregulates ICAM-2 expression. Our results show that the interactions between P9 and ICAM-2 could be targeted by therapeutics for metabolic diseases. Hyo Shin Yoon, Chung Hwan Cho, Myeong Sik Yun, Sung Jae Jang, Hyun Ju You, Jun-hyeong Kim, Dohyun Han, Kwang Hyun Cha, Sung Hyun Moon, Kiuk Lee, Yeon-Ji Kim, Sung-Joon Lee, Tae-Wook Nam & GwangPyo Ko
Neuroplastin Modulates Anti-inflammatory Effects of MANF
iScience Volume 23, ISSUE 12, 101810, December 18, 2020, DOI:https://doi.org/10.1016/j.isci.2020.101810
Identification of Neuroplastin (NPTN) as receptor of MANF polypeptide by LRC-TriCEPS. Endoplasmic reticulum (ER) stress is known to induce pro-inflammatory response and ultimately leads to cell death. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER-localized protein whose expression and secretion is induced by ER stress and a crucial survival factor. However, the underlying mechanism of how MANF exerts its cytoprotective activity remains unclear due to the lack of knowledge of its receptor. Here we show that Neuroplastin (NPTN) is such a receptor for MANF. Biochemical analysis shows the physiological interaction between MANF and NPTN on the cell surface. Binding of MANF to NPTN mitigates the inflammatory response and apoptosis via suppression of NF-kβ signaling. Our results demonstrate that NPTN is a cell surface receptor for MANF, which modulates inflammatory responses and cell death, and that the MANF-NPTN survival signaling described here provides potential therapeutic targets for the treatment of ER stress-related disorders, including diabetes mellitus, neurodegeneration, retinal degeneration, and Wolfram syndrome. Takuya Yagi, Rie Asada, Kohsuke Kanekura, Ave Eesmaa, Maria Lindahl, Mart Saarma, Fumihiko Urano
NKG2A/CD94 Is a New Immune Receptor for HLA-G and Distinguishes Amino Acid Differences in the HLA-G Heavy Chain
International Journal of Molecular Sciences. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352787/
The immune checkpoint molecule human leukocyte antigen (HLA)-G is upregulated on malignant cells but not on healthy surrounding cells, the requirement of understanding the basis of receptor mediated events at the HLA-G/NK cell interface becomes obvious. The NK cell receptors ILT2 and KIR2DL4 have been described to bind to HLA-G; however, their differential function and expression levels on NK cell subsets suggest the existence of an unreported receptor. Here, we performed a ligand-based receptor capture on living cells utilizing sHLA-G*01:01 molecules coupled to TriCEPS and bound to NK cells followed by mass spectrometric analyses. We could define NKG2A/CD94 as a cognate receptor of HLA-G. Gia-Gia T. Hò, Alexander A. Celik, Trevor Huyton, Wiebke Hiemisch, Rainer Blasczyk, Gwendolin S. Simper, and Christina Bade-Doeding
Acidity changes immunology: a new VISTA pathway
Kathleen M. Mahoney and Gordon J. Freeman
Nature Article VISTA interaction with PSGL-1 identified by LRC-TriCEPS
https://www.nature.com/articles/s41586-019-1674-5 Nature volume 574, pages565–570 (2019)
Summary We then performed ligand-based receptor capture with VISTA–Fc chimeric protein and human CD4 + Tcells at acidic pH 19 . PSGL-1 was one of few proteins that were enriched relative to controls (Fig.3a). In addition to its well-characterized role facilitating adhesion interactions between leukocytes, platelets and endothelial cells 20,21 , PSGL-1 has been identified as a negative regulator of T cell responses in contexts of chronic viral infection, cancer, and some autoimmune diseases 22–28 . Robert J. Johnston, Linhui Julie Su, Jason Pinckney, David Critton, Eric Boyer, Arathi Krishnakumar, Martin Corbett, Andrew L. Rankin, Rose Dibella, Lynne Campbell, Gaelle H. Martin, Hadia Lemar, Thomas Cayton, Richard Y.-C. Huang, Xiaodi Deng, Akbar Nayeem, Haibin Chen, Burce Ergel, Joseph M. Rizzo, Aaron P. Yamniuk, Sanjib Dutta, Justine Ngo, Andrea Olga Shorts, Radha Ramakrishnan, Alexander Kozhich, Jim Holloway, Hua Fang, Ying-Kai Wang, Zheng Yang, Kader Thiam, Ginger Rakestraw, Arvind Rajpal, Paul Sheppard, Michael Quigley, Keith S. Bahjat & Alan J. Korman
Anti-VISTA antibody that inhibits Vista function and blocks interaction with PSGL-1 and VSIG3 proteins slows tumor growth
https://www.nature.com/articles/s41598-020-71519-4 Mehta, N., Maddineni, S., Kelly, R.L. et al. An engineered antibody binds a distinct epitope and is a potent inhibitor of murine and human VISTA. Sci Rep 10, 15171 (2020). https://doi.org/10.1038/s41598-020-71519-4 PDF
Anti-VISTA antibody that inhibits Vista function and blocks interaction with PSGL-1 and VSIG3 proteins slows tumor growth. Mehta, N., Maddineni, S., Kelly, R.L. et al.
Phage resistance at the cost of virulence
Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlBmediated invasion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008032 October 7, 2019
PDF
L. monocytogenes is a Gram-positive, food-borne, intracellular pathogen that causes severe infection in susceptible individuals. Interestingly, almost all infections are caused by a subset of strains belonging to certain serovars featuring a complex glycosylation pattern on their cell surface. Using an engineered bacteriophage that specifically recognizes these modifications we selected for mutants that lost these sugars. We found that the resulting strains are severely deficient in invading host cells as we observed that a major virulence factor mediating host cell entry requires galactose decoration of the cell surface for its function. Without this galactose decoration, the strain represents a serovar not associated with disease. Altogether, we show a complex interplay between bacteriophages, bacteria, and the host, demonstrating that cellular invasiveness is dependent upon a serovar-defining structure, which also serves as a phage receptor. Eric T. Sumrall, Yang Shen, Anja P. Keller, Jeanine Rismondo, Maria Pavlou, Marcel R. Eugster, Samy Boulos, Olivier Disson, Pierre Thouvenot, Samuel Kilcher, Bernd Wollscheid, Didier Cabanes, Marc Lecuit, Angelika Gründling, Martin J. Loessner
TFF3 interacts with LINGO2 to regulate EGFR activation for protection against colitis and gastrointestinal helminths
Nature Communications volume 10, Article number: 4408 (2019) https://doi.org/10.1038/s41467-019-12315-1
Intestinal epithelial cells (IEC) have important functions in nutrient absorption, barrier integrity, regeneration, pathogen-sensing, and mucus secretion. Goblet cells are a specialized cell type of IEC that secrete Trefoil factor 3 (TFF3) to regulate mucus viscosity and wound healing, but whether TFF3-responsiveness requires a receptor is unclear. We postulated that TFF3 interacted with its receptor through low-affinity interactions reliant upon carbohydrates because TFF3 glycosylation has been shown critical for biological activity34,35, therefore U937 cells were subjected to the TRICEPSTM protocol as a biochemical screening strategy to identify glycosylated transmembrane protein(s) on the cell surface32,36. As bait, rhTFF3 was covalently linked to the TRICEPS probe and incubated with PMA-treated U937 that had been treated with sodium periodate. Cell pellets were subjected to glycosidase digestion and peptides generated using mass spectrometry. As a positive control to account for enrichment efficiency, recombinant human>INS was used as bait for the>INS receptor. Whereas the TFF3-probe induced an 8-fold enrichment of LINGO2 (LIGO2) peptide, the>INS-probe induced a 7.76-fold-enrichment in INSR peptide (Fig. 1b). No other enriched peptides in this screen met this level of enrichment or were derived from proteins that satisfied our selection criterion (e.g., extracellular domain, transmembrane region, cytoplasmic tail).Here, we show that leucine rich repeat receptor and nogo-interacting protein 2 (LINGO2) is essential for TFF3-mediated functions. LINGO2 immunoprecipitates with TFF3, co-localizes with TFF3 on the cell membrane of IEC, and allows TFF3 to block apoptosis. We further show that TFF3-LINGO2 interactions disrupt EGFR-LINGO2 complexes resulting in enhanced EGFR signaling. Excessive basal EGFR activation in Lingo2 deficient mice increases disease severity during colitis and augments immunity against helminth infection. Conversely, TFF3 deficiency reduces helminth immunity. Thus, TFF3-LINGO2 interactions de-repress inhibitory LINGO2-EGFR complexes, allowing TFF3 to drive wound healing and immunity. Nicole Maloney Belle 1, Yingbiao Ji 1,5, Karl Herbine 1,5, Yun Wei 2, 3,5, JoonHyung Park 1,5, Kelly Zullo1, Li-Yin Hung 1,2, Sriram Srivatsa 1, Tanner Young 1, Taylor Oniskey 2, Christopher Pastore 1, Wildaliz Nieves 4, Ma Somsouk 4 & De’Broski R. Herbert 1,2 1 Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19140, USA. 2 Division of Experimental Medicine, University of California, San Francisco, San Francisco, CA 94110, USA. 3 Department of Inflammation and Oncology, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA. 4 Division of Gastroenterology at ZSFG, University of California, San Francisco, San Francisco, CA 94110, USA. 5These authors contributed equally: Yingbiao Ji, Karl Herbine, Yun Wei, JoonHyung Park. Email: debroski@vet.upenn.edu
White Paper Ligand-Receptor Identification Methodologies Details Matter
European Biopharmaceutical Review April 2019, pages 16-20 / Maria P. Pavlou and Paul Helbling / White -Paper PDF
Comparison of different technologies to identify Ligand-Receptor interactions. Maria P. Pavlou and Paul Helbling
Validation of extracellular ligand–receptor interactions by Flow‑TriCEPS
BMC Research Notes 2018 11:863 https://doi.org/10.1186/s13104-018-3974-5 Laura A. Lopez‑Garcia, Levent Demiray, Sandra Ruch‑Marder, Ann‑Katrin Hopp, Michael O. Hottiger, Paul M. Helbling and Maria P. Pavlou Received: 28 October 2018 – Accepted: 30 November 2018- Published: 5 December 2018 PDF
The advent of ligand based receptor capture methodologies, allows the identification of unknown receptor candidates for orphan extracellular ligands. However, further target validation can be tedious, laborious and time consuming. Here, we present a methodology that provides a fast and costefficient alternative for candidate target verification on living cells for a peptide, a protein and an antibody. Laura A. Lopez‑Garcia, Levent Demiray, Sandra Ruch‑Marder, Ann‑Katrin Hopp, Michael O. Hottiger, Paul M. Helbling and Maria P. Pavlou
Cardiac Targeting Peptide, a Novel Cardiac Vector: Studies in Bio-Distribution, Imaging Application, and Mechanism of Transduction
Maliha Zahid, Kyle S. Feldman, Gabriel Garcia-Borrero, Timothy N. Feinstein, Nicholas Pogodzinski, Xinxiu Xu, Raymond Yurko, Michael Czachowski , Yijen L. Wu, Neale S. Mason and CeciliaW. Lo Biomolecules 2018, 8, 147; doi:10.3390/biom8040147 Received: 24 September 2018 / Accepted: 8 November 2018 / Published: 14 November 2018
Our previous work identified a 12-amino acid peptide that targets the heart, termed cardiac targeting peptide (CTP).We now quantitatively assess the bio-distribution of CTP, show a clinical application with the imaging of the murine heart, and study its mechanisms of transduction. Bio-distribution studies of cyanine5.5-N-Hydroxysuccinimide (Cy5.5) labeled CTP were undertaken in wild-type mice. Cardiac targeting peptide was labeled with Tc. 99m (99mTc) using the chelator hydrazino-nicotinamide (HYNIC), and imaging performed using micro-single photon emission computerized tomography/computerized tomography (SPECT/CT). Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMCs) were incubated with dual-labeled CTP, and imaged using confocal microscopy. TriCEPs technology was utilized to study the mechanism of transduction. Bio-distribution studies showed peak uptake of CTP at 15 min. 99mTc-HYNIC-CTP showed heart-specific uptake. Robust transduction of beating human iPSC-derived CMCs was seen. TriCEPs experiments revealed five candidate binding partners for CTP, with Kcnh5 being felt to be the most likely candidate as it showed a trend towards being competed out by siRNA knockdown. Transduction efficiency was enhanced by increasing extracellular potassium concentration, and with Quin., a Kcnh5 inhibitor, that blocks the channel in an open position. We demonstrate that CTP transduces the normal heart as early as 15 min. 99mTc-HYNIC-CTP targets the normal murine heart with substantially improved targeting compared with 99mTc Sestamibi. Cardiac targeting peptide’s transduction ability is not species limited and has human applicability. Cardiac targeting peptide appears to utilize Kcnh5 to gain cell entry, a phenomenon that is affected by pre-treatment with Quin. and changes in potassium levels. Maliha Zahid 1,*, Kyle S. Feldman 1, Gabriel Garcia-Borrero 1, Timothy N. Feinstein 1, Nicholas Pogodzinski 1, Xinxiu Xu 1, Raymond Yurko 2, Michael Czachowski 3, Yijen L. Wu 1, Neale S. Mason 3 and CeciliaW. Lo 1 1 Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15201, USA; ksf23@pitt.edu (K.S.F.); gag44@pitt.edu (G.G.-B.); tnf8@pitt.edu (T.N.F.); nrp30@pitt.edu (N.P.);xux@pitt.edu (X.X.); yijenwu@pitt.edu (Y.L.W.); cel36@pitt.edu (C.W.L.) 2 Peptide Synthesis Facility, University of Pittsburgh, Pittsburgh, PA 15201, USA; yurko@pitt.edu 3 Department of Radiology, University of Pittsburgh, Pittsburgh, PA 15201, USA; michael.czachowski@chp.edu (M.C.); masonns@upmc.edu (N.S.M.)
Leukocyte differentiation by histidine-rich glycoprotein/stanniocalcin-2 complex regulates murine glioma growth through modulation of anti-tumor immunity
Francis P Roche, Ilkka Pietilä, Hiroshi Kaito, Elisabet O Sjöström, Nadine Sobotzki, Oriol Noguer, Tor Persson Skare, Magnus Essand, Bernd Wollscheid, Michael Welsh and Lena Claesson-Welsh DOI: 10.1158/1535-7163.MCT-18-0097 Received January 27, 2018, Revision received April 21, 2018, Accepted June 19, 2018, Copyright ©2018, American Association for Cancer Research. PDF
The plasma-protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and anti-tumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45+ leukocyte and CD8+ T cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45+ and CD8+ infiltration. Using a novel protein interaction-decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells in vitro and co-localization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14+ differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG treated gliomas displayed decreased numbers of Interleukin-35+ Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control anti-tumor immunity. Francis P Roche1, Ilkka Pietilä2, Hiroshi Kaito3, Elisabet O Sjöström1, Nadine Sobotzki4, Oriol Noguer1, Tor Persson Skare1, Magnus Essand1, Bernd Wollscheid5, Michael Welsh2, and Lena Claesson-Welsh1,*
1Department of Immunology, Uppsala University
2Department of Medical Cell Biology, Uppsala University
3Department of Immunology, Uppsala university
4Department of Health Sciences, ETH Zurich
5Department of Health Sciences, ETH Zürich
Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions
Lisa Lasswitz, Naresh Chandra, Niklas Arnberg, Gisa Gerold jmb Journal of Molecular Biology, doi.org/10.1016/j.jmb.2018.04.039 Received 15 February 2018, Revised 24 April 2018, Accepted 30 April 2018, Available online 7 May 2018.
Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells.
HATRIC-based identification of receptors for orphan ligands
Nadine Sobotzki, Michael A. Schafroth, Alina Rudnicka, Anika Koetemann, Florian Marty, Sandra Goetze, Yohei Yamauchi, Erick M. Carreira & Bernd Wollscheid Nature Communications, volume 9, Article number: 1519 (2018) doi:10.1038/s41467-018-03936-z Published online: 17 April 2018
Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors. The sensitivity of these interactions to non-physiological conditions, however, makes them challenging to study using in vitro assays. Here we present HATRIC-based ligand receptor capture (HATRIC-LRC), a chemoproteomic technology that successfully identifies target receptors for orphan ligands on living cells ranging from small molecules to intact viruses. HATRIC-LRC combines a click chemistry-based, protein-centric workflow with a water-soluble catalyst to capture ligand-receptor interactions at physiological pH from as few as 1 million cells. We show HATRIC-LRC utility for general antibody target validation within the native nanoscale organization of the surfaceome, as well as receptor identification for a small molecule ligand. HATRIC-LRC further enables the identification of complex extracellular interactomes, such as the host receptor panel for influenza A virus (IAV), the causative agent of the common flu. Nadine Sobotzki, Michael A. Schafroth, Alina Rudnicka, Anika Koetemann, Florian Marty, Sandra Goetze, Yohei Yamauchi, Erick M. Carreira and Bernd Wollscheid
Staphylococcal Superantigens Use LAMA2 as a Coreceptor GPCT signaling To Activate T Cells
Zhigang Li, Joseph J. Zeppa, Mark A. Hancock, John K. McCormick, Terence M. Doherty, Geoffrey N. Hendy and Joaquín Madrenas J Immunol January 15, 2018, ji1701212; DOI: https://doi.org/10.4049/jimmunol.1701212 (Published online February 5, 2018) This work was supported by the Canadian Institutes for Health Research. J.M. holds a tier I Canada Research Chair in Human Immunology. The Department of Microbiology and Immunology Flow Cytometry and Cell Sorting Facility and McGill Surface Plasmon Resonance–Mass Spectrometry Facility are supported by the Canada Foundation for Innovation.
Canonical Ag-dependent TCR signaling relies on activation of the src-family tyrosine kinase LCK. However, staphylococcal superantigens can trigger TCR signaling by activating an alternative pathway that is independent of LCK and utilizes a Gα11-containing G protein–coupled receptor (GPCR) leading to PLCβ activation. The molecules linking the superantigen to GPCR signaling are unknown. Using the ligand-receptor capture technology LRC-TriCEPS, we identified LAMA2, the α2 subunit of the extracellular matrix protein laminin, as the coreceptor for staphylococcal superantigens. Complementary binding assays (ELISA, pull-downs, and surface plasmon resonance) provided direct evidence of the interaction between staphylococcal enterotoxin E and LAMA2. Through its G4 domain, LAMA2 mediated the LCK-independent T cell activation by these toxins. Such a coreceptor role of LAMA2 involved a GPCR of the calcium-sensing receptor type because the selective antagonist NPS 2143 inhibited superantigen-induced T cell activation in vitro and delayed the effects of toxic shock syndrome in vivo. Collectively, our data identify LAMA2 as a target of antagonists of staphylococcal superantigens to treat toxic shock syndrome. Zhigang Li, Joseph J. Zeppa, Mark A. Hancock, John K. McCormick, Terence M. Doherty, Geoffrey N. Hendy and Joaquín Madrenas
Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma
International Journal of Oncology, published online on: November 9, 2017 doi.org/10.3892/ijo.2017.4199 Authors: Karina Galoian, Silva Abrahamyan, Gor Chailyan, Amir Qureshi, Parthik Patel, Gil Metser, Alexandra Moran, Inesa Sahakyan, Narine Tumasyan, Albert Lee, Tigran Davtyan, Samvel Chailyan and Armen Galoyan Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed.
Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function. Karina Galoian, Silva Abrahamyan, Gor Chailyan, Amir Qureshi, Parthik Patel, Gil Metser, Alexandra Moran, Inesa Sahakyan, Narine Tumasyan, Albert Lee, Tigran Davtyan, Samvel Chailyan and Armen Galoyan
Phenotypic screening—the fast track to novel antibody discovery
ScienceDirekt, doi.org/10.1016/j.ddtec.2017.03.004 Department of Antibody Discovery and Protein Engineering, MedImmune, Milstein Building, Granta Park, Cambridge CB21 6GH, UK Available online 25 April 2017
The majority of antibody therapeutics have been isolated from target-led drug discovery, where many years of target research preceded drug program initiation. However, as the search for validated targets becomes more challenging and target space becomes increasingly competitive, alternative strategies, such as phenotypic drug discovery, are gaining favour. This review highlights successful examples of antibody phenotypic screens that have led to clinical drug candidates. We also review the requirements for performing an effective antibody phenotypic screen, including antibody enrichment and target identification strategies. Finally, the future impact of phenotypic drug discovery on antibody drug pipelines will be discussed. Section editors: Neil O Carragher – Institute of Genetics and Molecular Medicine, Cancer Research UK Edinburgh Centre, University of Edinburgh, Edinburgh, United Kingdom. Jonathan A. Lee – Quantitative Biology, Eli Lilly and Company, Indianapolis, Indiana. Ellen L. Berg – BioMAP Systems Division of DiscoverX, DiscoverX Corporation.