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LRC-TriCEPS™ Publications


Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology


ABSTRACT

PLoS One. 2016 Oct 11;11(10):e0164593. doi: 10.1371/journal.pone.0164593. eCollection 2016.Logo East Tennessee State University

Li Y1, Ozment T2, Wright GL1, Peterson JM1,3.

We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and insulin as a control ligand was performed on the H4IIE rat hepatoma cell line.

RESULTS

Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and Insulin-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the insulin 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.

AUTHORS

Li Y1, Ozment T2, Wright GL1, Peterson JM1,3.

  • 1Quillen College of Medicine, Department of Biomedical Sciences, East Tennessee State University, Johnson City, Tennessee, United States of America.
  • 2Quillen College of Medicine, Department of Internal Medicine, East Tennessee State University, Johnson City, Tennessee, United States of America.
  • 3College of Public Health, Department of Health Sciences, East Tennessee State University, Johnson City, Tennessee, United States of America.

Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5


ABSTRACT

Current Issue – vol. 113 no. 45 – Manish P. Ponda,  E7059–E7068, doi: 10.1073/pnas.1615671113rockefeller-university-logo

Contributed by Jan L. Breslow, September 23, 2016 (sent for review August 1, 2016; reviewed by Myron Cybulsky and Carl F. Nathan)

Manish P. Pondaa and Jan L. Breslowa,1

Chemokines 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.

To identify potential receptors in an unbiased manner, we used ligand–receptor capture (LRC-TriCEPS) technology as a tool for detecting T-lymphocyte surface proteins that physically interact with H497–K520 (Fig. 5A) (20). Transferrin was used as a control ligand to eliminate nonspecific interactions with the TriCEPS reagent.

METHODE

TriCEPS Ligand-Receptor Capture.

The TriCEPS reagent is a proprietary trifunctional molecule with three key moieties: (i) an amine-reactive group capable of binding a peptide ligand of interest, (ii) a cross-linking group capable of bonding to oxidized glycans, and (iii) an affinity tag for downstream extraction (20, 50). These studies were performed by DualSystems Biotech. Briefly, either human H497–K520 or transferrin was coupled to the TriCEPS reagent. The TriCEPS–ligand complex was added to CCRF-CEM cells in complete medium in the presence or absence of zinc (100 μM), after treatment with an oxidizing reagent to oxidize surface glycoproteins. Cells were then lysed and processed as described, including LC-MS/MS analysis of captured peptides (20). Comparisons between conditions were made in triplicate to determine the relative fold enrichment of a given protein. P values were determined for pairwise comparisons and adjusted for multiple comparisons.

AUTHORS

Manish P. Ponda

aLaboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY 10065

Jan L. Breslow

aLaboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY 10065


Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization


ABSTRACT

Current Issue – vol. 113 no. 45- Heather L. Glasgow,  12774–12779, doi: 10.1073/pnas.161164211

Contributed by Roger Y. Tsien, August 3, 2016 (sent for review November 16, 2015; reviewed by Joshua E. Elias and Jeff W. Lichtman)

Heather L. Glasgowa,1, Michael A. Whitneya, Larry A. Grossb, Beth Friedmana, Stephen R. Adamsa, Jessica L. Crispb, Timon Hussainc, Andreas P. Freid, Karel Novyd, Bernd Wollscheidd, Quyen T. Nguyenc, and Roger Y. Tsiena,b,e,2

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 biotin 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.

AUTHORS

Heather L. Glasgow

aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Michael A. Whitney

aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Larry A. Gross

bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;

Beth Friedman

aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Stephen R. Adams

aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Jessica L. Crisp

bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;

Timon Hussain

cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;

Andreas P. Frei

dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;

Karel Novy

dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;

Bernd Wollscheid

dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;

Quyen T. Nguyen

cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;

Roger Y. Tsien

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


ABSTRACT

Logo East Tennessee State UniversityApril 2016, The FASEB Journal, vol. 30 no. 1 Supplement 1249.2

Jonathan M Peterson

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. .

METHODS

We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and Insulin as the control ligand was performed on H4IIE rat hepatoma cell line. Additional analysis using siRNA induced knockdown of the identified receptors was used to validate the findings.

RESULTS

Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and Insulin-TriCEPS to the cell surface glycans. In total, an enrichment of glycopeptides of 11% (261 peptides) was observed. Under these conditions, INSR (Insulin receptor) could be identified and quantified in the control dataset. For the ligand sample two unique receptors were identified. Flow cytometry analysis with siRNA induced knockdown of these proteins confirmed that the presence of the protein is needed for CTRP3 binding to occur.

CONCLUSION

The LRC-TriCEPS methodology was successful in identifying the receptor 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.

AUTHOR

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


ABSTRACT

Cedoc-logoNature Communications 6, Article number: 8732 (2015), doi:10.1038/ncomms9732

Received:
Accepted:
Published online:

Andres Garelli, Fabiana Heredia, Andreia P. Casimiro, Andre Macedo, Catarina Nunes, Marcia Garcez, Angela R. Mantas Dias, Yanel A. Volonte, Thomas Uhlmann, Esther Caparros, Takashi Koyama & Alisson M. Gontijo

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 insulin-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.

AUTHORS

Phenotypic screening—the fast track to novel antibody discovery


ABSTRACT

ScienceDirektdoi.org/10.1016/j.ddtec.2017.03.004

Department of Antibody Discovery and Protein Engineering, MedImmune, Milstein Building, Granta Park, Cambridge CB21 6GH, UK

Available online 25 April 2017

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.

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.


A Mass Spectrometric-Derived Cell Surface Protein Atlas


ABSTRACT

Published: April 20, 2015 – http://dx.doi.org/10.1371/journal.pone.0121314

Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa).

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.

AUTHORS

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

Competing Interests

The authors have declared that no competing interests exist.

Author Contributions

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.


Direct identification of ligand-receptor interactions on living cells and tissues


AFFILIATIONS

Nature Biotechnology 30, 997–1001 doi:10.1038/nbt.2354 – ReceivedAcceptedPublished online

Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties—one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. In some cases, we could also determine the approximate ligand-binding sites on the receptors. Using TRICEPS to label intact mature vaccinia viruses, we identified the cell surface proteins AXL, M6PR, DAG1, CSPG4 and CDH13 as binding factors on human cells. This technology enables the identification of receptors for many types of ligands under near-physiological conditions and without the need for genetic manipulations.

AUTHORS

Ligand-based receptor identification on living cells and tissues using TRICEPS


ABSTRACT

Affiliations ¦ Contributions ¦Corresponding authors

Nature Protocols 8, 1321–1336 (2013) doi:10.1038/nprot.2013.072

Published online 13 June 2013

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 biotin 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., insulin) 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.

AUTHORS

Discovering ligand-receptor interactions


ABSTRACT

Affiliations ¦ Corresponding authors

Nature Biotechnology 30, 959–961 (2012) doi:10.1038/nbt.2373

Published online 10 October 2012
A new probe identifies cell-surface receptors bound by known ligands.
AUTHORS

The power of TRICEPS


ABSTRACT

Nature Methods 9, 1044–1045 (2012) doi:10.1038/nmeth.2237

Published online 06 November 2012

AUTHORS

Flex your TRICEPS


ABSTRACT

Nature Chemical Biology 8, 950 (2012) doi:10.1038/nchembio.1126
Published online 26 November 2012
Identifying ligands for receptors in live cells can provide valuable information, providing insight into signaling pathways as well as drug targets, but these interactions can be difficult to detect and quantify. Frei et al. now report a trifunctional chemoproteomic reagent called TRICEPS that binds glycosylated receptors on live cells and allows for the purification of the ligand-receptor complex and identification of receptor-derived peptides by MS. TRICEPS contains three functional groups: an N-hydroxysuccinimide ester for ligand conjugation, a protein….

AUTHORS

Protter


ABSTRACT

Protter — the open-source tool for visualization of proteoforms and interactive integration of annotated and predicted sequence features together with experimental proteomic evidence.

The ability to integrate and visualize experimental proteomic evidence in the context of rich protein feature annotations represents an unmet need of the proteomics community. Protter, a web-based tool that supports interactive protein data analysis and hypothesis generation by visualizing both annotated sequence features and experimental proteomic data in the context of protein topology. Protter supports numerous proteomic file formats and automatically integrates a variety of reference protein annotation sources, which can be readily extended via modular plug-ins. A built-in export function produces publication-quality customized protein illustrations, also for large datasets. Visualizations of surfaceome datasets show the specific utility of Protter both for the integrated visual analysis of membrane proteins and peptide selection for targeted proteomics.

AUTHORS

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