Dualsystems Biotech AG is testing the limits of the LRC-TriCEPSTM (CaptiRec) technology using antibodies with different affinities against the same target and cells expressing the target receptor with different expression levels.
Limits-of-LRC-TriCEPS
Details of the assesment see here
Dualsystems Biotech AG and Biaffin GmbH & Co KG perform together an example of an experiment.
In the case study Dualsystems Biotech AG determined the targets of the Anti EGFR antibodies and Biaffin GmbH& Co KG measured the binding characteristics of the ligand receptor interaction using Surface Plasmon Resonance (SPR).
Dualsystems Biotech AG Press Release
Zürich Switzerland – June 23, 2014 – Dualsystems Biotech AG offers the most advanced technology for identifying receptor(s) for orphan ligands. The ligand-receptor capture technology (LRC-TriCEPS™) is an innovative and conceptually new approach for the unbiased discovery of cell surface interactions of ligands ranging from peptides, proteins, antibodies to viruses. Dualsystems offers the LRC-TriCEPS™ technology in our service line CaptiRec. We offer a variety of formats which will fit your needs: From CaptiRec Kits for your own in-house experiments to an all-inclusive CaptiRec Service.
Dualsystems Biotech AG is looking forward to support your project with the newest invention in receptor/target discovery.
Please watch the video where we explain how the LRC-TriCEPS™ technology works: LRC-TriCEPS™-Video
Watch the video to see how the LRC-TriCEPS™ technology works
LRC-TriCEPS™ is a novel approach which enables the identification of cell surface receptors and off-targets on the living cells for a wide range of orphan ligands, such as:
Please fill out all mandatory (*) fields.
If you want to be best service for you unkown target do not hesitate and write us a mail!
Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.
De'Broski R. Herbert Ph.D.
Fumihiko Urano, MD, PhD
Alisson M. Gontijo,
Dr. Edward van der Horst,
Jonathan M Peterson
Dr. rer. nat. Inga Sörensen-Zender
Manish Ponda, M.D., M.S.
Sari S. Hannila, PhD
Dr. rer. nat. Martin Herold
Dr Karina Galoian
Anita Burgess | PhD
(Quim) Madrenas, MD, PhD, FCAHS
Over 200 satisfied customers from 28 countries.
Years working with LRC-TriCEPS
LRC-TriCEPS clients served
Cell types used for LRC-TriCEPS
Years of experience
Concerning the LRC-TriCEPS or HATRIC-LRC platforms.
Cardiac Targeting Peptide, a Novel Cardiac Vector: Studies in Bio-Distribution, Imaging Application, and Mechanism of Transduction
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 Technetium 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 Quinidine, 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 Quinidine 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
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,*
Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions
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
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
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
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
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.
Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology
Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and INS-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the>INS receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells.
Conclusion
The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3.
Relevance
The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.
Li Y1, Ozment T2, Wright GL1, Peterson JM1,3.
Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5
MChemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulation factor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity.
Manish P. Pondaa and Jan L. Breslowa,1
a Laboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY 10065
Contributed by Jan L. Breslow, September 23, 2016 (sent for review August 1, 2016; reviewed by Myron Cybulsky and Carl F. Nathan)
Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization
Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41’s binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue–NP41-bound nerves, followed by hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10−7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve “ghosts” months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
eDepartment of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093
Identification of cell surface receptors for the novel adipokine CTRP3
C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects throughout the body. Our initial work shows promise in the development of CTRP3-induced cellular processes as a means to combat nonalcoholic fatty liver disease (NAFLD). Clinically, NAFLD is defined as the excessive accumulation of fat in the liver, usually due to obesity, and NAFLD effects 1 in 10 Americans. Our previous data show that when high fat fed mice are treated with CTRP3 they are protected from developing NAFLD. However, the mechanism for this effect remains unclear. The purpose of this project was to identify the unknown receptor that mediates the hepatic actions of CTRP3.
Jonathan M Peterson, Health Sciences, East Tennessee State University, Johnson City, TN
This abstract is from the Experimental Biology 2016 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing
How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in INS-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.
A Mass Spectrometric-Derived Cell Surface Protein Atlas
The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.
Damaris Bausch-Fluck
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
Andreas Hofmann
Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Thomas Bock
Current Address: European Molecular Biology Laboratory, Heidelberg, Germany
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Andreas P. Frei
Current Address: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, United States of America
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Ferdinando Cerciello
Current Address: James Thoracic Center, James Cancer Center, The Ohio State University Medical Center, Columbus, Ohio, United States of America
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Laboratory of Molecular Oncology, University Hospital Zurich, Zurich, Switzerland
Andrea Jacobs
Current Address: Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Hansjoerg Moest
Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Ulrich Omasits
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
Rebekah L. Gundry
Affiliation Department of Biochemistry, Medical College of Wisconsin, Wisconsin, Milwaukee, United States of America
Charles Yoon
Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada
Ralph Schiess
Current Address: ProteoMediX AG, Schlieren, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Alexander Schmidt
Current Address: Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Paulina Mirkowska
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland
Anetta Härtlová
Current Address: College of Life Sciences, University of Dundee, Dundee, United Kingdom
Affiliation Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech Republic
Jennifer E. Van Eyk
Current Address: Cedars-Sinai, Clinical Biosystem Research Institute, Los Angeles, California, United States of America
Affiliation Department of Medicine, Biological Chemistry and Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Jean-Pierre Bourquin
Affiliation Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland
Ruedi Aebersold
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland, Faculty of Science, University of Zurich, Zurich, Switzerland
Kenneth R. Boheler
Affiliations SCRMC, LKS Faculty of Medicine, Hong Kong University, Hong Kong, Hong Kong SAR, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Peter Zandstra
Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada
Bernd Wollscheid
* E-mail: wbernd@ethz.ch
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
The authors have declared that no competing interests exist.
Conceived and designed the experiments: DBF BW KRB PZ RA. Performed the experiments: DBF AH TB APF FC AJ HM RLG CY RS AS PM AH. Analyzed the data: DBF UO. Contributed reagents/materials/analysis tools: AS APF FC TB BW. Wrote the paper: DBF BW. Supervised experiments: JVE JPB.
Protter
Ligand-based receptor identification on living cells and tissues using TRICEPS
Physiological responses to ligands such as peptides, proteins, pharmaceutical drugs or whole pathogens are generally mediated through interactions with specific cell surface protein receptors. Here we describe the application of TRICEPS, a specifically designed chemoproteomic reagent that can be coupled to a ligand of interest for the subsequent ligand-based capture of corresponding receptors on living cells and tissues. This is achieved by three orthogonal functionalities in TRICEPS—one that enables conjugation to an amino group containing ligands, a second for the ligand-based capture of glycosylated receptors on gently oxidized living cells and a tag for purifying receptor peptides for analysis by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., INS) or no binding preferences (e.g., TRICEPS quenched with glycine). In combination with powerful statistical models, this ligand-based receptor capture (LRC) technology enables the unbiased and sensitive identification of one or several specific receptors for a given ligand under near-physiological conditions and without the need for genetic manipulations. LRC has been designed for applications with proteins but can easily be adapted for ligands ranging from peptides to intact viruses. In experiments with small ligands that bind to receptors with comparatively large extracellular domains, LRC can also reveal approximate ligand-binding sites owing to the defined spacer length of TRICEPS. Provided that sufficient quantities of the ligand and target cells are available, LRC can be carried out within 1 week.
Dualsystems Biotech AG Press Release
Schlieren, Switzerland, January 2014: – Dualsystems Biotech AG has announced it will collaborate with another Swiss laboratory, Institute for Biopharmaceutical Research (IBR Inc.) to validate the CaptiRec Triceps/Ligand Receptor Capture Technology. This technology is used to identify the Cell Membrane Receptors on Living Cells.
The partnership will combine the respective strengths of Dualsystems, a leading service provider for protein interaction at the cell membrane and IBR Inc., a GLP-compliant contract research organization specializing in cell-based assays
for Dualsystems Biotech AG
Following two financing rounds in 2000 and 2007, Dualsystems Biotech has built a solid investor base. This includes both private individuals as well as institutional investors to support our technology driven growth strategy.
Should you be interested in supporting us and becoming an investor in Dualsystems Biotech, please contact Dr. Paul Helbling, at paul.helbling@dualsystems.com
If you want to get support for your project
Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.
De'Broski R. Herbert Ph.D.Fumihiko Urano, MD, PhDAlisson M. Gontijo,Dr. Edward van der Horst,Jonathan M PetersonDr. rer. nat. Inga Sörensen-ZenderManish Ponda, M.D., M.S.Sari S. Hannila, PhDDr. rer. nat. Martin HeroldDr Karina GaloianAnita Burgess | PhD(Quim) Madrenas, MD, PhD, FCAHS
Over 200 satisfied customers from 28 countries.
Schlieren – Switzerland
Dualsystems Biotech is a provider of proteomics services for industry and academia. The LRC TriCEPS™ technology platform is designed to identify targets and off-targets in the cell membrane of living cells.
Please fill out all mandatory (*) fields.
Concerning the LRC-TriCEPS or HATRIC-LRC platforms.
Cardiac Targeting Peptide, a Novel Cardiac Vector: Studies in Bio-Distribution, Imaging Application, and Mechanism of Transduction
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 Technetium 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 Quinidine, 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 Quinidine 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
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,*
Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions
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
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
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
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
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.
Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology
Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and INS-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the>INS receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells.
Conclusion
The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3.
Relevance
The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.
Li Y1, Ozment T2, Wright GL1, Peterson JM1,3.
Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5
MChemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulation factor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity.
Manish P. Pondaa and Jan L. Breslowa,1
a Laboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY 10065
Contributed by Jan L. Breslow, September 23, 2016 (sent for review August 1, 2016; reviewed by Myron Cybulsky and Carl F. Nathan)
Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization
Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41’s binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue–NP41-bound nerves, followed by hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10−7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve “ghosts” months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
eDepartment of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093
Identification of cell surface receptors for the novel adipokine CTRP3
C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects throughout the body. Our initial work shows promise in the development of CTRP3-induced cellular processes as a means to combat nonalcoholic fatty liver disease (NAFLD). Clinically, NAFLD is defined as the excessive accumulation of fat in the liver, usually due to obesity, and NAFLD effects 1 in 10 Americans. Our previous data show that when high fat fed mice are treated with CTRP3 they are protected from developing NAFLD. However, the mechanism for this effect remains unclear. The purpose of this project was to identify the unknown receptor that mediates the hepatic actions of CTRP3.
Jonathan M Peterson, Health Sciences, East Tennessee State University, Johnson City, TN
This abstract is from the Experimental Biology 2016 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing
How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in INS-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.
A Mass Spectrometric-Derived Cell Surface Protein Atlas
The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.
Damaris Bausch-Fluck
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
Andreas Hofmann
Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Thomas Bock
Current Address: European Molecular Biology Laboratory, Heidelberg, Germany
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Andreas P. Frei
Current Address: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, United States of America
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Ferdinando Cerciello
Current Address: James Thoracic Center, James Cancer Center, The Ohio State University Medical Center, Columbus, Ohio, United States of America
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Laboratory of Molecular Oncology, University Hospital Zurich, Zurich, Switzerland
Andrea Jacobs
Current Address: Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Hansjoerg Moest
Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Ulrich Omasits
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
Rebekah L. Gundry
Affiliation Department of Biochemistry, Medical College of Wisconsin, Wisconsin, Milwaukee, United States of America
Charles Yoon
Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada
Ralph Schiess
Current Address: ProteoMediX AG, Schlieren, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Alexander Schmidt
Current Address: Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Paulina Mirkowska
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland
Anetta Härtlová
Current Address: College of Life Sciences, University of Dundee, Dundee, United Kingdom
Affiliation Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech Republic
Jennifer E. Van Eyk
Current Address: Cedars-Sinai, Clinical Biosystem Research Institute, Los Angeles, California, United States of America
Affiliation Department of Medicine, Biological Chemistry and Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Jean-Pierre Bourquin
Affiliation Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland
Ruedi Aebersold
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland, Faculty of Science, University of Zurich, Zurich, Switzerland
Kenneth R. Boheler
Affiliations SCRMC, LKS Faculty of Medicine, Hong Kong University, Hong Kong, Hong Kong SAR, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Peter Zandstra
Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada
Bernd Wollscheid
* E-mail: wbernd@ethz.ch
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
The authors have declared that no competing interests exist.
Conceived and designed the experiments: DBF BW KRB PZ RA. Performed the experiments: DBF AH TB APF FC AJ HM RLG CY RS AS PM AH. Analyzed the data: DBF UO. Contributed reagents/materials/analysis tools: AS APF FC TB BW. Wrote the paper: DBF BW. Supervised experiments: JVE JPB.
Protter
Ligand-based receptor identification on living cells and tissues using TRICEPS
Physiological responses to ligands such as peptides, proteins, pharmaceutical drugs or whole pathogens are generally mediated through interactions with specific cell surface protein receptors. Here we describe the application of TRICEPS, a specifically designed chemoproteomic reagent that can be coupled to a ligand of interest for the subsequent ligand-based capture of corresponding receptors on living cells and tissues. This is achieved by three orthogonal functionalities in TRICEPS—one that enables conjugation to an amino group containing ligands, a second for the ligand-based capture of glycosylated receptors on gently oxidized living cells and a tag for purifying receptor peptides for analysis by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., INS) or no binding preferences (e.g., TRICEPS quenched with glycine). In combination with powerful statistical models, this ligand-based receptor capture (LRC) technology enables the unbiased and sensitive identification of one or several specific receptors for a given ligand under near-physiological conditions and without the need for genetic manipulations. LRC has been designed for applications with proteins but can easily be adapted for ligands ranging from peptides to intact viruses. In experiments with small ligands that bind to receptors with comparatively large extracellular domains, LRC can also reveal approximate ligand-binding sites owing to the defined spacer length of TRICEPS. Provided that sufficient quantities of the ligand and target cells are available, LRC can be carried out within 1 week.
If you want to get support for your project
Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.
De'Broski R. Herbert Ph.D.Fumihiko Urano, MD, PhDAlisson M. Gontijo,Dr. Edward van der Horst,Jonathan M PetersonDr. rer. nat. Inga Sörensen-ZenderManish Ponda, M.D., M.S.Sari S. Hannila, PhDDr. rer. nat. Martin HeroldDr Karina GaloianAnita Burgess | PhD(Quim) Madrenas, MD, PhD, FCAHS
Over 200 satisfied customers from 28 countries.
Dualsystems Biotech AG
Do you want to know the mode-of-action of your ligand – small molecule, peptide, protein, antibody, virus – and use a technology that works with natural, not modified cells? Then call us or fill in the form below and we will get back to you and advise you how the LRC-TriCEPS™ technology or the Hatric-LRC technology can solve your open questions.
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Dualsystems Biotech AG
Dr. Paul Helbling
Grabenstrasse 11a
8952 Schlieren
Switzerland
phone +41 44 738 50 00
fax +41 44 738 50 05
Contact Dr. Paul Helbling to discuss how we can support your projects.
Dualsystems Biotech is located in Schlieren (Zurich), close to the railway station.
If you want to get support for your project
Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.
De'Broski R. Herbert Ph.D.Fumihiko Urano, MD, PhDAlisson M. Gontijo,Dr. Edward van der Horst,Jonathan M PetersonDr. rer. nat. Inga Sörensen-ZenderManish Ponda, M.D., M.S.Sari S. Hannila, PhDDr. rer. nat. Martin HeroldDr Karina GaloianAnita Burgess | PhD(Quim) Madrenas, MD, PhD, FCAHS
Over 200 satisfied customers from 28 countries.
Watch the video to see how the LRC-TriCEPS™ technology works
LRC-TriCEPS™ is a novel approach which enables the identification of cell surface receptors and off-targets on the living cells for a wide range of orphan ligands, such as:
Please fill out all mandatory (*) fields.
If you want to be best service for you unkown target do not hesitate and write us a mail!
Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.
De'Broski R. Herbert Ph.D.Fumihiko Urano, MD, PhDAlisson M. Gontijo,Dr. Edward van der Horst,Jonathan M PetersonDr. rer. nat. Inga Sörensen-ZenderManish Ponda, M.D., M.S.Sari S. Hannila, PhDDr. rer. nat. Martin HeroldDr Karina GaloianAnita Burgess | PhD(Quim) Madrenas, MD, PhD, FCAHS
Over 200 satisfied customers from 28 countries.
Years working with LRC-TriCEPS
LRC-TriCEPS clients served
Cell types used for LRC-TriCEPS
Years of experience
Concerning the LRC-TriCEPS or HATRIC-LRC platforms.
Cardiac Targeting Peptide, a Novel Cardiac Vector: Studies in Bio-Distribution, Imaging Application, and Mechanism of Transduction
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 Technetium 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 Quinidine, 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 Quinidine 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
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,*
Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions
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
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
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
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
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.
Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology
Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and INS-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the>INS receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells.
Conclusion
The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3.
Relevance
The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.
Li Y1, Ozment T2, Wright GL1, Peterson JM1,3.
Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5
MChemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulation factor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity.
Manish P. Pondaa and Jan L. Breslowa,1
a Laboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY 10065
Contributed by Jan L. Breslow, September 23, 2016 (sent for review August 1, 2016; reviewed by Myron Cybulsky and Carl F. Nathan)
Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization
Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41’s binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue–NP41-bound nerves, followed by hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10−7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve “ghosts” months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
eDepartment of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093
Identification of cell surface receptors for the novel adipokine CTRP3
C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects throughout the body. Our initial work shows promise in the development of CTRP3-induced cellular processes as a means to combat nonalcoholic fatty liver disease (NAFLD). Clinically, NAFLD is defined as the excessive accumulation of fat in the liver, usually due to obesity, and NAFLD effects 1 in 10 Americans. Our previous data show that when high fat fed mice are treated with CTRP3 they are protected from developing NAFLD. However, the mechanism for this effect remains unclear. The purpose of this project was to identify the unknown receptor that mediates the hepatic actions of CTRP3.
Jonathan M Peterson, Health Sciences, East Tennessee State University, Johnson City, TN
This abstract is from the Experimental Biology 2016 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing
How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in INS-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.
A Mass Spectrometric-Derived Cell Surface Protein Atlas
The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.
Damaris Bausch-Fluck
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
Andreas Hofmann
Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Thomas Bock
Current Address: European Molecular Biology Laboratory, Heidelberg, Germany
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Andreas P. Frei
Current Address: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, United States of America
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Ferdinando Cerciello
Current Address: James Thoracic Center, James Cancer Center, The Ohio State University Medical Center, Columbus, Ohio, United States of America
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Laboratory of Molecular Oncology, University Hospital Zurich, Zurich, Switzerland
Andrea Jacobs
Current Address: Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Hansjoerg Moest
Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Ulrich Omasits
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
Rebekah L. Gundry
Affiliation Department of Biochemistry, Medical College of Wisconsin, Wisconsin, Milwaukee, United States of America
Charles Yoon
Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada
Ralph Schiess
Current Address: ProteoMediX AG, Schlieren, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Alexander Schmidt
Current Address: Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Paulina Mirkowska
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland
Anetta Härtlová
Current Address: College of Life Sciences, University of Dundee, Dundee, United Kingdom
Affiliation Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech Republic
Jennifer E. Van Eyk
Current Address: Cedars-Sinai, Clinical Biosystem Research Institute, Los Angeles, California, United States of America
Affiliation Department of Medicine, Biological Chemistry and Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Jean-Pierre Bourquin
Affiliation Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland
Ruedi Aebersold
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland, Faculty of Science, University of Zurich, Zurich, Switzerland
Kenneth R. Boheler
Affiliations SCRMC, LKS Faculty of Medicine, Hong Kong University, Hong Kong, Hong Kong SAR, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Peter Zandstra
Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada
Bernd Wollscheid
* E-mail: wbernd@ethz.ch
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
The authors have declared that no competing interests exist.
Conceived and designed the experiments: DBF BW KRB PZ RA. Performed the experiments: DBF AH TB APF FC AJ HM RLG CY RS AS PM AH. Analyzed the data: DBF UO. Contributed reagents/materials/analysis tools: AS APF FC TB BW. Wrote the paper: DBF BW. Supervised experiments: JVE JPB.
Protter
Ligand-based receptor identification on living cells and tissues using TRICEPS
Physiological responses to ligands such as peptides, proteins, pharmaceutical drugs or whole pathogens are generally mediated through interactions with specific cell surface protein receptors. Here we describe the application of TRICEPS, a specifically designed chemoproteomic reagent that can be coupled to a ligand of interest for the subsequent ligand-based capture of corresponding receptors on living cells and tissues. This is achieved by three orthogonal functionalities in TRICEPS—one that enables conjugation to an amino group containing ligands, a second for the ligand-based capture of glycosylated receptors on gently oxidized living cells and a tag for purifying receptor peptides for analysis by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., INS) or no binding preferences (e.g., TRICEPS quenched with glycine). In combination with powerful statistical models, this ligand-based receptor capture (LRC) technology enables the unbiased and sensitive identification of one or several specific receptors for a given ligand under near-physiological conditions and without the need for genetic manipulations. LRC has been designed for applications with proteins but can easily be adapted for ligands ranging from peptides to intact viruses. In experiments with small ligands that bind to receptors with comparatively large extracellular domains, LRC can also reveal approximate ligand-binding sites owing to the defined spacer length of TRICEPS. Provided that sufficient quantities of the ligand and target cells are available, LRC can be carried out within 1 week.
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Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.
De'Broski R. Herbert Ph.D.Fumihiko Urano, MD, PhDAlisson M. Gontijo,Dr. Edward van der Horst,Jonathan M PetersonDr. rer. nat. Inga Sörensen-ZenderManish Ponda, M.D., M.S.Sari S. Hannila, PhDDr. rer. nat. Martin HeroldDr Karina GaloianAnita Burgess | PhD(Quim) Madrenas, MD, PhD, FCAHS
Over 200 satisfied customers from 28 countries.
Schlieren – Switzerland
Dualsystems Biotech is a provider of proteomics services for industry and academia. The LRC TriCEPS™ technology platform is designed to identify targets and off-targets in the cell membrane of living cells.
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Cardiac Targeting Peptide, a Novel Cardiac Vector: Studies in Bio-Distribution, Imaging Application, and Mechanism of Transduction
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 Technetium 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 Quinidine, 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 Quinidine 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
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,*
Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions
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
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
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
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
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.
Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology
Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and INS-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the>INS receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells.
Conclusion
The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3.
Relevance
The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.
Li Y1, Ozment T2, Wright GL1, Peterson JM1,3.
Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5
MChemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulation factor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity.
Manish P. Pondaa and Jan L. Breslowa,1
a Laboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY 10065
Contributed by Jan L. Breslow, September 23, 2016 (sent for review August 1, 2016; reviewed by Myron Cybulsky and Carl F. Nathan)
Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization
Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41’s binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue–NP41-bound nerves, followed by hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10−7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve “ghosts” months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;
cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;
aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;
bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;
eDepartment of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093
Identification of cell surface receptors for the novel adipokine CTRP3
C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects throughout the body. Our initial work shows promise in the development of CTRP3-induced cellular processes as a means to combat nonalcoholic fatty liver disease (NAFLD). Clinically, NAFLD is defined as the excessive accumulation of fat in the liver, usually due to obesity, and NAFLD effects 1 in 10 Americans. Our previous data show that when high fat fed mice are treated with CTRP3 they are protected from developing NAFLD. However, the mechanism for this effect remains unclear. The purpose of this project was to identify the unknown receptor that mediates the hepatic actions of CTRP3.
Jonathan M Peterson, Health Sciences, East Tennessee State University, Johnson City, TN
This abstract is from the Experimental Biology 2016 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing
How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in INS-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.
A Mass Spectrometric-Derived Cell Surface Protein Atlas
The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.
Damaris Bausch-Fluck
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
Andreas Hofmann
Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Thomas Bock
Current Address: European Molecular Biology Laboratory, Heidelberg, Germany
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Andreas P. Frei
Current Address: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, United States of America
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Ferdinando Cerciello
Current Address: James Thoracic Center, James Cancer Center, The Ohio State University Medical Center, Columbus, Ohio, United States of America
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Laboratory of Molecular Oncology, University Hospital Zurich, Zurich, Switzerland
Andrea Jacobs
Current Address: Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Hansjoerg Moest
Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Ulrich Omasits
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
Rebekah L. Gundry
Affiliation Department of Biochemistry, Medical College of Wisconsin, Wisconsin, Milwaukee, United States of America
Charles Yoon
Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada
Ralph Schiess
Current Address: ProteoMediX AG, Schlieren, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Alexander Schmidt
Current Address: Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland
Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
Paulina Mirkowska
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland
Anetta Härtlová
Current Address: College of Life Sciences, University of Dundee, Dundee, United Kingdom
Affiliation Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech Republic
Jennifer E. Van Eyk
Current Address: Cedars-Sinai, Clinical Biosystem Research Institute, Los Angeles, California, United States of America
Affiliation Department of Medicine, Biological Chemistry and Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Jean-Pierre Bourquin
Affiliation Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland
Ruedi Aebersold
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland, Faculty of Science, University of Zurich, Zurich, Switzerland
Kenneth R. Boheler
Affiliations SCRMC, LKS Faculty of Medicine, Hong Kong University, Hong Kong, Hong Kong SAR, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Peter Zandstra
Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada
Bernd Wollscheid
* E-mail: wbernd@ethz.ch
Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland
The authors have declared that no competing interests exist.
Conceived and designed the experiments: DBF BW KRB PZ RA. Performed the experiments: DBF AH TB APF FC AJ HM RLG CY RS AS PM AH. Analyzed the data: DBF UO. Contributed reagents/materials/analysis tools: AS APF FC TB BW. Wrote the paper: DBF BW. Supervised experiments: JVE JPB.
Protter
Ligand-based receptor identification on living cells and tissues using TRICEPS
Physiological responses to ligands such as peptides, proteins, pharmaceutical drugs or whole pathogens are generally mediated through interactions with specific cell surface protein receptors. Here we describe the application of TRICEPS, a specifically designed chemoproteomic reagent that can be coupled to a ligand of interest for the subsequent ligand-based capture of corresponding receptors on living cells and tissues. This is achieved by three orthogonal functionalities in TRICEPS—one that enables conjugation to an amino group containing ligands, a second for the ligand-based capture of glycosylated receptors on gently oxidized living cells and a tag for purifying receptor peptides for analysis by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., INS) or no binding preferences (e.g., TRICEPS quenched with glycine). In combination with powerful statistical models, this ligand-based receptor capture (LRC) technology enables the unbiased and sensitive identification of one or several specific receptors for a given ligand under near-physiological conditions and without the need for genetic manipulations. LRC has been designed for applications with proteins but can easily be adapted for ligands ranging from peptides to intact viruses. In experiments with small ligands that bind to receptors with comparatively large extracellular domains, LRC can also reveal approximate ligand-binding sites owing to the defined spacer length of TRICEPS. Provided that sufficient quantities of the ligand and target cells are available, LRC can be carried out within 1 week.
If you want to get support for your project
Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.
De'Broski R. Herbert Ph.D.Fumihiko Urano, MD, PhDAlisson M. Gontijo,Dr. Edward van der Horst,Jonathan M PetersonDr. rer. nat. Inga Sörensen-ZenderManish Ponda, M.D., M.S.Sari S. Hannila, PhDDr. rer. nat. Martin HeroldDr Karina GaloianAnita Burgess | PhD(Quim) Madrenas, MD, PhD, FCAHS
Over 200 satisfied customers from 28 countries.
Dualsystems Biotech AG
Do you want to know the mode-of-action of your ligand – small molecule, peptide, protein, antibody, virus – and use a technology that works with natural, not modified cells? Then call us or fill in the form below and we will get back to you and advise you how the LRC-TriCEPS™ technology or the Hatric-LRC technology can solve your open questions.
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Dualsystems Biotech AG
Dr. Paul Helbling
Grabenstrasse 11a
8952 Schlieren
Switzerland
phone +41 44 738 50 00
fax +41 44 738 50 05
Contact Dr. Paul Helbling to discuss how we can support your projects.
Dualsystems Biotech is located in Schlieren (Zurich), close to the railway station.
If you want to get support for your project
Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.
De'Broski R. Herbert Ph.D.Fumihiko Urano, MD, PhDAlisson M. Gontijo,Dr. Edward van der Horst,Jonathan M PetersonDr. rer. nat. Inga Sörensen-ZenderManish Ponda, M.D., M.S.Sari S. Hannila, PhDDr. rer. nat. Martin HeroldDr Karina GaloianAnita Burgess | PhD(Quim) Madrenas, MD, PhD, FCAHS
Over 200 satisfied customers from 28 countries.
