Proteoglycans: Methods and Protocols

Preface
Contents
Contributors
Part I: Glycosaminoglycans: Strategies for Detection and Structure Characterization
Chapter 1: Detection of Glycosaminoglycans in Biological Specimens
1 Introduction
2 Materials
2.1 Enzyme-Linked Immunosorbent Assay (ELISA)
2.2 Materials for ELISA
2.3 Liquid Chromatography Tandem Mass Spectrometry Materials
2.3.1 Standards and Enzymes
2.3.2 Extraction of GAGs from Tissue
2.3.3 Extraction of GAGs from Dried Blood Spot (DBS)
2.3.4 Apparatus
2.4 Non-reducing End Disaccharide Materials
2.4.1 Cell Lines and Culture
2.4.2 Urine Samples from Mice and Human
2.4.3 Apparatus
2.5 Methanolysis Materials
2.5.1 Reagents
2.5.2 Urine Collection
2.5.3 Apparatus
2.6 Automated High-Throughput Mass Spectrometry Materials
2.6.1 Standards and Enzymes
2.6.2 Preparation of DBS for Extracting GAGs
2.6.3 Establishing Interface Between Mass Spectrometer and RapidFire
2.6.4 Apparatus
3 Methods
3.1 ELISA
3.1.1 Sandwich ELISA Assay to Quantify KS
3.1.2 Sandwich ELISA Assay to Quantify HS
3.2 Liquid Chromatography Tandem Mass Spectrometry
3.2.1 Enzyme-Mediated Degradation of Polysaccharides to Disaccharides and Detection by LC-MS/MS
3.2.2 Non-reducing Ends Disaccharides Measurement by LC-MS/MS
3.2.3 Methanolysis
3.2.4 Automated High-Throughput Mass Spectrometry (HT-MS/MS)
4 Notes
References
Chapter 2: Immunochemical Detection and Glycosaminoglycan Disaccharide-Based Characterization of Chondroitin Sulfate Proteogly...
1 Introduction
2 Materials
2.1 CS-Degrading Enzyme
2.2 Immunochemical Detection of CSPGs
2.3 Analysis of CS Disaccharide Composition
3 Methods
3.1 Immunocytochemistry
3.2 Immunohistochemistry (e.g., for Skeletal Muscle Tissue Sections)
3.3 Analysis of CS Disaccharide Composition (Small-Scale Version Using 1.5 mL Tubes)
4 Notes
References
Chapter 3: Proteoglycans: A Tool for Detecting Hyaluronan by ELISA-Like Methods
1 Introduction
2 Materials
2.1 Materials and Solutions for HA Sandwich ELISA
2.2 Materials and Solutions for HA-Competitive ELISA
2.3 Instruments
3 Methods
3.1 Samples
3.1.1 Cell Supernatant
3.1.2 Serum
3.1.3 Plasma
3.1.4 Alternative Samples
3.2 HA Sandwich ELISA
3.2.1 Plate Preparation
3.2.2 Assay Procedure
3.2.3 Analysis of Results
3.3 HA-Competitive ELISA
3.3.1 Plate Preparation
3.3.2 Pre-incubation Step of Free HA with Biotinylated-HABP
3.3.3 Assay Procedure
3.3.4 Analysis of Results
4 Notes
References
Chapter 4: Particle Exclusion Assay: A Tool for Measuring Hyaluronan Pericellular Matrix
1 Introduction
2 Materials
2.1 Red Blood Cells Fixation
2.2 Cell Culture
2.3 Particle Exclusion Assay
3 Method
3.1 Red Blood Cells Fixation
3.2 Particle Exclusion Assay
4 Notes
References
Chapter 5: Urinary Glycosaminoglycans: Characterization and Quantification
1 Introduction
2 Materials
2.1 Semiquantitative Urinary Glycosaminoglycan Determination
2.2 Quantitative Urinary Glycosaminoglycan Determination
2.3 Urinary GAG Isolation
2.4 Thin Layer Chromatography GAG Characterization
3 Methods
3.1 Dimethylmethylene Blue Solution
3.2 Chondroitin 6-Sulfate Solution
3.3 Semiquantitative Urinary Glycosaminoglycan Determination
3.4 Urinary GAG Isolation
3.5 Thin Layer Chromatography GAG Characterization
4 Notes
References
Chapter 6: HPLC-MS Characterization of Tissue-Derived Heparan Sulfate and Chondroitin Sulfate
1 Introduction
2 Materials
2.1 Tissue Preparation for Digestion
2.2 Chondroitin Sulfate Digestion
2.3 Heparan Sulfate Digestion
2.4 Cotton-HILIC (CT) SPE Purification
2.5 Graphite-Based (GP) SPE Purification
2.6 HPLC Separation and Column Packing
2.7 Calibration Standards
3 Methods
3.1 Tissue Preparation for Digestion
3.2 On-Tissue CS Digestion
3.3 On-Tissue HS Digestion
3.4 Cotton-HILIC Solid-Phase Extraction
3.5 Graphite-Based Solid-Phase Extraction
3.6 Preparation of Capillary Columns
3.7 HPLC-MS Measurements
3.8 Data Evaluation
4 Notes
References
Chapter 7: Evaluation of Glycosaminoglycans Biological Age in Cells and Tissues
1 Introduction
2 Materials
2.1 Sample Preparation of Biological Materials
2.2 Extraction of Extracellular Matrix Components
2.3 Accelerator Mass Spectrometry Analysis
3 Methods
3.1 Sample Preparation of Biological Materials
3.2 Extraction of Extracellular Matrix Components
3.3 Accelerator Mass Spectrometry Analysis
4 Notes
References
Chapter 8: Studying the Effects of Glycosaminoglycans in Cell Morphological Aspect with Scanning Electron Microscopy
1 Introduction
2 Materials
2.1 Cell Culture
2.2 GAG Solution Preparation
2.3 Cell Substrates Preparation
2.4 Cell Fixation
2.5 Scanning Electron Microscopy
3 Methods
3.1 Cell Culture
3.2 Harvesting and Seeding of the Cells
3.3 Treatment of Cells with GAGs
3.4 Preparation of Different Cell Substrates
3.5 Cell Seeding on Boyden Chambers
3.6 Cell Fixation Procedure for Scanning Electron Microscopy
3.7 Observing Cell Morphology with Scanning Electron Microscopy
4 Notes
References
Part II: Proteoglycans: Separation, Analysis, and Interactions
Chapter 9: Evaluation of the In Vitro and In Vivo Effects of Biglycan in Innate Immunity
1 Introduction
1.1 In Vitro Studies of Biglycan in Macrophages
1.2 In Vivo Studies of Biglycan in a Transgenic Mouse Model
2 Materials
2.1 Isolation and Stimulation of Murine Peritoneal Macrophages
2.2 In Vivo Studies of Biglycan in a Transgenic Mouse Model
2.2.1 Construction and Retro-Orbital Intravenous (i.v.) Injection of pLIVE-hBGN Plasmid
2.2.2 Liver-Specific Expression of Transgene and Extraction of Proteoglycans (PGs)
2.2.3 IHC
3 Methods
3.1 In Vitro Studies of Biglycan in Murine Peritoneal Macrophages
3.1.1 Isolation of Murine Peritoneal Macrophage
3.1.2 Macrophage Stimulation
3.2 In Vivo Studies of Biglycan in a Transgenic Mouse Model
3.2.1 Plasmid Construction and Retro-Orbital Intravenous Injection in Mice (See Note 9)
3.2.2 RT-PCR-RFLP Method to Identify hBGN Transgene in the Mouse Liver
3.2.3 Extraction of PGs/Detection of Transgenic Biglycan in Mouse Plasma
3.2.4 IHC Staining for Human Biglycan and F4/80+ Macrophages in the Renal Tissue of Transgenic Mice
4 Notes
References
Chapter 10: Analysis of Extracellular Vesicle-Associated Proteoglycans
1 Introduction
2 Materials
2.1 EV Isolation
2.2 Sample Preparation for MS/MS Analysis
2.3 Glycosaminoglycan Digestion
2.4 [35S]-Sulfate PG Labeling, DEAE Anion-Exchange Chromatography, and Gel Electrophoresis
2.5 Immunoblot Analysis of HSPG Core Protein Profile
3 Methods
3.1 EV Isolation from Conditioned Medium
3.1.1 Differential Ultracentrifugation (dUC)
3.1.2 Optiprep Density Gradient (ODG)
3.1.3 Fraction Collection
3.2 EV Isolation from Plasma
3.2.1 Size Exclusion Chromatography (SEC) Using qEV Columns
3.2.2 Sample Preparation for MS/MS Analysis
3.2.3 Proteomic Profile: Long Gradient, Tandem Mass Tag (TMT), and HiRIEF Quantitative LC-MS/MS or Other MS/MS Method of Choice
3.3 Bioinformatics Tools for Posterior Analysis
3.4 [35S]-Sulfate PG Labeling, DEAE Purification, and Autoradiographic Visualization of EV-Associated PGs
3.5 Immunoblot Analysis of EV-Associated HSPG Core Protein Profile
4 Notes
References
Chapter 11: Analysis of Aggrecan Glycanation by Western Blot in Cell Culture
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Layer and Medium Processing
2.3 Western Blot Analysis
3 Methods
3.1 Cell Culture and Sample Harvest
3.2 Cell Layer Processing
3.3 Medium Processing
3.4 Chondroitinase Digestion and Sample Preparation
3.5 Western Blot Analysis
4 Notes
References
Chapter 12: Repulsive Scaling Replica Exchange Molecular Dynamics in Modeling Protein-Glycosaminoglycan Complexes
1 Introduction
2 Methods
3 Notes
References
Chapter 13: In Vitro Investigation of Hyaluronan/CD44 Network
1 Introduction
2 Materials
2.1 Determination of Secreted Hyaluronan Concentration
2.2 Determination of Hyaluronan Size
2.3 Determination of Hyaluronan Binding by Live Cells
2.4 Determination of Cell Surface and Intracellular Hyaluronan with Fluorescent Staining
2.5 HAS2 Silencing with siRNAs
2.6 Inhibition of Hyaluronan Synthesis by 4-MU
2.7 CD44 Silencing with siRNAs
2.8 Blocking Hyaluronan/CD44 Interactions with Anti-CD44 Monoclonal Antibody
3 Methods
3.1 Determination of Secreted Hyaluronan Concentration
3.2 Determination of Hyaluronan Size
3.3 Determination of Hyaluronan Binding by Live Cells
3.4 Determination of Cell Surface and Intracellular Hyaluronan with Fluorescent Staining
3.5 Inhibition of Hyaluronan Biosynthesis by HAS2 Silencing
3.6 Inhibition of Hyaluronan Biosynthesis by 4-MU
3.7 CD44 Silencing
3.8 Blocking Hyaluronan/CD44 Interactions Using Anti-CD44 Monoclonal Antibodies
4 Notes
References
Chapter 14: Separation and Identification of Native Proteoglycans by Composite Agarose-Polyacrylamide Gel Electrophoresis and ...
1 Introduction
1.1 Discontinuous SDS-Polyacrylamide Gel Electrophoresis
1.2 Native Gel Electrophoresis
2 Materials
2.1 Equipment
2.2 Stock Reagents
2.3 Working Reagents
3 Methods
3.1 Sample Preparation and Proteoglycan Loadings
3.1.1 Proteoglycan Extraction
3.1.2 Application of Samples
3.1.3 Suggested Sample Loadings
3.2 Casting a 0.6% Agarose 1.2% Acrylamide 10 x 16 Cm CAPAGE Slab Gel
3.3 Electrophoretic Conditions
3.4 Staining of Gels
3.5 Semi-Dry Immunoblotting Using the Bio-Rad Trans-Blot Semi-Dry Transfer Cell and Bio-Rad 200/2.0 Constant Power Supply
3.5.1 Development of Nitrocellulose Blots
4 Notes
References
Chapter 15: Building, Visualizing, and Analyzing Glycosaminoglycan-Protein Interaction Networks
1 Introduction
2 Materials
3 Methods
3.1 The Collection of Glycosaminoglycan Interaction Data
3.1.1 The Collection of Glycosaminoglycan Interaction Data
3.1.2 Published heparin/heparan sulfate and GAG interactomes
3.1.3 Large Interaction Datasets Generated by Affinity Chromatography
3.1.4 Manual Curation of Publications
3.2 The Visualization of GAG Interaction Networks
3.2.1 Tools Available in Molecular Interaction Databases
3.2.2 Visualization of Interaction Networks Using Cytoscape
3.3 The Enrichment Analyses of Glycosaminoglycan Interaction Networks
4 Notes
References
Part III: Proteoglycans Related Molecules (Enzymes, Inhibitors, and Epigenetics)
Chapter 16: An Enzymatic Activity Assay for Heparanase That Is Useful for Evaluating Clinically Relevant Inhibitors and Studyi...
1 Introduction
2 Materials
3 Methods
3.1 Preparing Blocked Microplates
3.2 Conducting Heparanase Assays
3.3 Assay Run-in Procedure
4 Notes
References
Chapter 17: A Method for Urinary Trypsin Inhibitor (UTI) Purification Combining Anion-Exchange Chromatography Enrichment and P...
1 Introduction
2 Materials
2.1 Anion-Exchange Chromatography
2.2 Sample Concentration, Dialysis, and Quantitation
2.3 SDS-Polyacrylamide Gel Casting and Electrophoresis
2.4 Gel Staining and Acquisition
2.5 Protein Elution from Gel
2.6 Chondroitin Sulfate Depolymerization
3 Methods
3.1 Anion-Exchange Chromatography
3.2 Sample Concentration, Dialysis, and Quantitation
3.3 SDS-Polyacrylamide Gel Casting and Electrophoresis (SDS-PAGE)
3.4 Protein Elution from Gel
3.5 Purity Assessment of UTI
4 Notes
References
Chapter 18: Enzymatic Sequencing of Heparin Oligosaccharides Using Exolyase
1 Introduction
2 Materials
2.1 Preparation of BIexoHep (See Note 1)
2.2 Enzymatic Digest Reaction
2.3 Collection of UDP6 and UDP4 Products
2.4 O3 Treatment of UDP4
2.5 Enzyme Digestion of UDP8, UDP6, UDP4, and O3-Treated UDP4
2.6 Disaccharide Analysis by Anion Exchange HPLC
3 Methods
3.1 Expression and Purification of BIexoHep
3.2 Enzymatic Digest Reaction (See Note 4)
3.3 Collection of UDP6 and UDP4 Products
3.4 O3 Treatment of UDP4
3.5 Enzymatic Digestion of UDP8, UDP6, UDP4, and O3-Treated UDP4
3.6 Disaccharide Composition Analysis by Anion Exchange HPLC
4 Notes
References
Chapter 19: Evaluating the Effects of MicroRNAs on Proteoglycans and Matrix Constituents´ Expression and Functional Properties
1 Introduction
2 Materials
2.1 Cell Cultures
2.2 Cell Transfections with miRNA Precursors or Inhibitors
2.3 Cell Harvesting, Extraction, and Supernatant Processing
2.4 RNA Extraction, miRNA Isolation, Reverse Transcription, and Real-Time PCR
2.5 Immunofluorescence
2.6 SDS Polyacrylamide Gel Casting and Electrophoresis for Gelatin Zymography
2.7 Gel Staining and Acquisition
2.8 Western Blot
3 Methods
3.1 Cell Cultures
3.2 Cell Transfections with miRNA Precursors or Inhibitors
3.3 Cell Harvesting and Supernatant Processing
3.4 RNA Extraction, miRNA Isolation, Reverse Transcription, and Real-Time PCR
3.5 RNA Reverse Transcription and Real-Time PCR
3.6 Immunofluorescence
3.7 SDS Polyacrylamide Gel Casting and Electrophoresis
3.8 Gel Staining and Acquisition
3.9 Western Blot
4 Notes
References
Chapter 20: Proteoglycan Expression Studied by MicroRNAs
1 Introduction
2 Materials
2.1 Common Requirements for All Protocols
2.2 Cell Culture
2.3 Transfection
2.4 RNA Isolation
2.5 cDNA Reverse Transcription
2.6 Reverse Transcription of the miRNA in cDNA
2.7 Real-Time PCR for miRNA Targets and miR-10b
2.8 Flow Cytometry
2.9 Invasion Assay
3 Methods
3.1 Target Prediction Analysis
3.2 miRNA Transfection of Cell Lines
3.3 Luciferase Reporter Assay
3.4 Functional Analysis and Complementation Assay
3.4.1 Functional Assay: Invasion Assay
3.4.2 Complementation Assay (Co-Transfection)
4 Notes
References
Chapter 21: Modelling and Simulations of Extracellular Glycoproteins
1 Introduction
2 Materials
3 Methods
3.1 Construction of Glycoprotein/Glycoconjugate Model
3.1.1 CHARMM-GUI
3.1.2 doGlycans
3.1.3 Glycosciences.de
3.2 Analysis of Carbohydrate Dynamics
3.2.1 RMSD-Based Clustering of Glycan Conformations
3.2.2 Calculation of Dihedral Angles
3.3 Analysis of Protein Dynamics
3.4 Analysis of Carbohydrate-Protein Interactions
3.4.1 Gmx Select
3.4.2 Conan
3.4.3 Analysis of Protein Solvent Accessibility as a Result of Glycosylations
3.4.4 Gmx Sasa
4 Notes
References
Index