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- PDB-7t62: GPC2 HEP CT3 complex -

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Basic information

Entry
Database: PDB / ID: 7t62
TitleGPC2 HEP CT3 complex
Components
  • CT3
  • Glypican-2
KeywordsIMMUNE SYSTEM / Glypican-3 complex
Function / homology
Function and homology information


regulation of protein localization to membrane / Defective B3GALT6 causes EDSP2 and SEMDJL1 / Defective B4GALT7 causes EDS, progeroid type / Defective B3GAT3 causes JDSSDHD / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / A tetrasaccharide linker sequence is required for GAG synthesis / HS-GAG biosynthesis / HS-GAG degradation / smoothened signaling pathway ...regulation of protein localization to membrane / Defective B3GALT6 causes EDSP2 and SEMDJL1 / Defective B4GALT7 causes EDS, progeroid type / Defective B3GAT3 causes JDSSDHD / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / A tetrasaccharide linker sequence is required for GAG synthesis / HS-GAG biosynthesis / HS-GAG degradation / smoothened signaling pathway / regulation of signal transduction / Retinoid metabolism and transport / lysosomal lumen / positive regulation of neuron projection development / neuron differentiation / Golgi lumen / cell migration / collagen-containing extracellular matrix / Attachment and Entry / synapse / cell surface / endoplasmic reticulum / extracellular space / plasma membrane
Similarity search - Function
Glypican-2 / Glypican, conserved site / Glypicans signature. / Glypican / Glypican
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 21 Å
AuthorsZhu, J. / Cachau, R. / De Val Alda, N. / Li, N. / Ho, M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)Z01 BC010891 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)Cancer Moonshot Program United States
CitationJournal: Cell Rep Med / Year: 2021
Title: CAR T cells targeting tumor-associated exons of glypican 2 regress neuroblastoma in mice.
Authors: Nan Li / Madeline B Torres / Madeline R Spetz / Ruixue Wang / Luyi Peng / Meijie Tian / Christopher M Dower / Rosa Nguyen / Ming Sun / Chin-Hsien Tai / Natalia de Val / Raul Cachau / Xiaolin ...Authors: Nan Li / Madeline B Torres / Madeline R Spetz / Ruixue Wang / Luyi Peng / Meijie Tian / Christopher M Dower / Rosa Nguyen / Ming Sun / Chin-Hsien Tai / Natalia de Val / Raul Cachau / Xiaolin Wu / Stephen M Hewitt / Rosandra N Kaplan / Javed Khan / Brad St Croix / Carol J Thiele / Mitchell Ho /
Abstract: Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. ...Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. Glypican 2 (GPC2) is overexpressed in neuroblastoma. Using RNA sequencing (RNA-seq) analysis, we show that exon 3 and exons 7-10 of GPC2 are expressed in cancer but are minimally expressed in normal tissues. Accordingly, we discover a monoclonal antibody (CT3) that binds exons 3 and 10 and visualize the complex structure of CT3 and GPC2 by electron microscopy. The potential of this approach is exemplified by designing CT3-derived chimeric antigen receptor (CAR) T cells that regress neuroblastoma in mice. Genomic sequencing of T cells recovered from mice reveals the CAR integration sites that may contribute to CAR T cell proliferation and persistence. These studies demonstrate how RNA-seq data can be exploited to help identify tumor-associated exons that can be targeted by CAR T cell therapies.
History
DepositionDec 13, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 22, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Glypican-2
B: CT3


Theoretical massNumber of molelcules
Total (without water)108,3622
Polymers108,3622
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Glypican-2 /


Mass: 61002.066 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GPC2 / Production host: Homo sapiens (human) / References: UniProt: Q8N158
#2: Antibody CT3


Mass: 47359.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: GPC2 HET CT3 complex / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Buffer solutionpH: 7.5
SpecimenConc.: 0.01 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE
Details: A 3 uL aliquot containing ~0.01 mg/mL of the samples was applied for 20 s onto a carbon-coated 200 Cu mesh grid (Electron Microscopy Sciences, Protochips, Inc.) that had been glow discharged ...Details: A 3 uL aliquot containing ~0.01 mg/mL of the samples was applied for 20 s onto a carbon-coated 200 Cu mesh grid (Electron Microscopy Sciences, Protochips, Inc.) that had been glow discharged at 30 mA for 30 s (Pelco easiGlow, Ted Pella, Inc.), then negatively stained with 0.7% (w/v) uranyl formate for 40 sec.
Material: uranyl formate
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 20
Electron gunElectron source: LAB6 / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 100000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 2000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI EAGLE (2k x 2k) / Num. of real images: 200
Image scansWidth: 2048 / Height: 2048

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
10RELION3.08initial Euler assignment
11RELION3.08final Euler assignment
12RELION3.08classification
13RELION3.083D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 21000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21000 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT

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