Proteoglycans: Methods and Protocols

321 Pages·2023·10.951 MB·English

by  Nikos K. Karamanos

Methods in
Molecular Biology 2619
Nikos K. Karamanos Editor
Proteoglycans
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
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Proteoglycans
Methods and Protocols
Edited by
Nikos K. Karamanos
Biochemistry, Biochemical Analysis and Matrix Pathobiology Research Group, Laboratory of Biochemistry,
Department of Chemistry, University of Patras, Patras, Greece
Editor
NikosK.Karamanos
Biochemistry,BiochemicalAnalysis
andMatrixPathobiologyResearch
Group,LaboratoryofBiochemistry,
DepartmentofChemistry
UniversityofPatras
Patras,Greece
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-2945-1 ISBN978-1-0716-2946-8 (eBook)
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Preface
Extracellular matrix (ECM) is a non-cellular three-dimensional dynamic macromolecular
network composed of several macromolecules with proteoglycans (PGs) and glycosamino-
glycans (GAGs) among the major ones [1]. Deregulation of ECM composition and struc-
ture is associated with the development and progression of several pathologic conditions
[2]. The field of PGs/GAGs research and applications has expanded rapidly. Due to their
high structural complexity and heterogeneity, PGs mediate several physio(patho)logical
processes. PGs are ubiquitously expressed in all cell types and composed of a protein core
containingcovalentlyattachedGAGschains.PGsarehighlycomplexheterogeneousmacro-
molecules expressed at all cellular levels, extracellularly, pericellularly, at cell surface, and
evenintracellularly,andplaykeyregulatoryrolesincellsignaling,functionalcellproperties,
andmorphology.PGsorchestratephysiologicalprocesses,suchascelladhesion,migration,
proliferation,andangiogenesis,andpathobiologicalconditions,amongtheminflammatory
responses,fibrosis,osteoarthritis,atherosclerosis,andcancer[3–4].
Proteoglycans – Methods and Protocols is a comprehensive and up-to-date collection of
strategies, reproducible methods, and protocols for the in-depth analysis of PGs and their
glycan part, the GAGs. The step-to-step protocols and detailed techniques included will
enablebothyoungandexperiencedresearcherstoisolate,detect,andstructurallycharacter-
izebothatGAGandPGlevels;tostudytheircellexpression,applyimagingtechniquesand
modeling, and identifytheinteractions networkswith matrix effectors; and to evaluatethe
enzymesinvolvedintheir metabolismandstructuremodification.
This volume, dedicated to proteoglycans and their methods and protocols, will offer
biochemists; matrix, molecular, and cellular biologists; pathologists; and geneticists a
detailed reference and practical guide to the current methodologies used for the isolation,
expression, analysis and structural characterization, modeling and imaging, and modifica-
tionapproachesofPGsandGAGsfromtissuesandcells.
The presented methods and protocols are provided and written by internationally
recognized experts in the field, applying different approaches to the studies of PGs/GAGs
andrelatedmolecules.I’mgratefullyindebtedtoallauthorsforsharingtheirprotocolsand
willing to contribute to this new endeavor following the first successful one published in
2001under theleadershipofProf.Renatov.Iozzoandin2012byFranc¸oiseRe´dini.
The volume is organized into three parts. The first part, Glycosaminoglycans: Strategies
for Detection and Structure Characterization, provides state-of-the-art approaches for
detection of GAGs in biological specimens (Shaukat Khan et al., Chap. 1), the immuno-
chemical detection of chondroitin sulfate PGs based on GAG disaccharides (Tadahisa
Mikami and Hiroshi Kitagava, Chap. 2), tools for detecting hyaluronan using ELISA-like
methods (Daiana Vitale et al., Chap. 3), and measuring hyaluronan pericellular matrix by
particle exclusion assay (Arianna Parnigoni et al., Chap. 4). Moreover, a method for the
detectionandcharacterizationofurinaryGAGs(NaimaFdilandEs-SaidSabir,Chap.5),an
HPLC-MSapproachfortissue-derivedheparansulfateandchondroitinsulfate(GaborToth
andLillaTuriak,Chap.6),andaprotocoltoevaluatetheGAGsbiologicalage(Chrysostomi
Gialeli and Isabel Gonc¸alves, Chap. 7) are included. Closing the first part, a protocol to
evaluatetheeffectsofexogenousGAGsinthemorphologicalfeaturesofcellswithscanning
electronmicroscopyispresented(ChristosKoutsakisetal.,Chap.8).
v
vi Preface
The second part, Proteoglycans: Separation, Analysis and Interactions, is dedicated to
protocolsfortheevaluationoftheinvitroandinvivoeffectsofbiglycanininnateimmunity
(Jinyang Zeng-Brouwers et al. Chap. 9), the analysis of extracellular vesicle-associated
proteoglycans (Juliana Poc¸as and Mattias Belting, Chap. 10), the analysis of aggrecan
glycanation by western blot in cell culture (Chiara Gramegna Tota et al., Chap. 11), as
well as the repulsive scaling replica exchange molecular dynamics in modeling protein-
glycosaminoglycan complexes (Mateusz Marcisz et al., Chap. 12). Two chapters provide a
detailed pipeline for the in vitro investigation of hyaluronan/CD44 network (Theodore
Karalis et al., Chap. 13) and the separation and identification of native proteoglycans by
composite agarose-polyacrylamide gel electrophoresis and immunoblotting (James Mel-
rose, Chap. 14). Finally, the last chapter of this part presents the tools to build, visualize,
andanalyzetheGAG-proteininteractomes(SylvieRicard-Blum,Chap.15).
The third part, Proteoglycans-Related Molecules (Enzymes, Inhibitors, Epigenetics),
involvesmethodsandprotocolsforcompoundsrelatedwiththemetabolicenzymes,epige-
neticregulation,andPGs/GAGs-basedinhibitors.Itstartswithanenzymaticactivityassay
forheparanasethatisusefulforevaluatingclinicallyrelevantinhibitorsandstudyingkinetics
(EdwardHammondandVitoFerro,Chap.16).Thispartisalsodedicatedtoamethodfor
urinary trypsin inhibitor (uti) purification combining anion-exchange chromatography
enrichmentandpreparativeSDS-PAGE(GabrieleNiedduetal.,Chap.17)andanapproach
fortheenzymaticsequencingofheparinoligosaccharidesusingexolyase(QingdongZhang
etal.,Chap.18).Thispartprovidesalsotwochaptersrelatedwiththestrategiestoevaluate
the effects of microRNAs on the matrix molecules and matrix functions properties (Zoi
Piperigkou and Nikos Karamanos, Chap. 19) and tools to evaluate the proteoglycans
expression studied by microRNAs (Nancy Andriana Espinoza-Sanchez et al., Chap. 20).
Glycosylationhasgarneredconsiderableinterestinrecentpast,fromtherecentknowledge
gained on their role in physiology and disease. The last chapter (Rajas M Rao et al.,
Chap.21)describesprotocolsforconstructionofaglycoproteinmodelaswellasmethods
toanalyzeglycoprotein/carbohydrateMDsimulations,whichenablesuncoveringinterest-
inginsightsaboutcarbohydrates.
Proteoglycans – Methods and Protocols contains 21 chapters and has been developed
through the efforts of 65 scientists representing 14 countries and 5 continents. I thank all
the authors for their outstanding work and their willingness to write and share the lab
protocols and strategies used for elucidating the structural and functional aspects of the
complexmatrixmacromoleculessuchastheproteoglycansandglycosaminoglycans.
Iwishtothanktheserieseditor,JohnWalker,forhiskindinvitationandguidanceinall
stepsofthearticlescollection,andallpersonsinvolvedinproductionatSpringerNaturefor
theircontributiontoasuccessfulpublicationofthismultiauthor volume.
We hope that this book provides a useful guide on current protocols and methods for
thestudyofproteoglycans,glycosaminoglycans,andcompoundsrelatedwiththemetabolic
enzymes, epigenetic regulation, and PGs/GAGs-based inhibitors. Personally, I was
rewarder by reading and editing the published articles. I gained new knowledge, inspired
andlearnedalotduringthisendeavor.
Patras,Greece NikosK.Karamanos
Preface vii
References
1. TheocharisAD,SkandalisSS,GialeliC,KaramanosNK(2016)Extracellularmatrixstructure.AdvDrug
DelivRev97:4–27
2. KaramanosNK,TheocharisAD,PiperigkouZetal(2021)Aguidetothecompositionandfunctionsof
theextracellularmatrix.FEBSJ288(24):6850–6912
3. Karamanos NK, Piperigkou Z, Theocharis AD et al (2018) Proteoglycan chemical diversity drives
multifunctionalcellregulationandtherapeutics.ChemRev118(18):9152–9232
4. Iozzo RV, Schaefer L (2015) Proteoglycan form and function: a comprehensive nomenclature of
proteoglycans.MatrixBiol42:11–55
Contents
Preface ..................................................................... v
Contributors................................................................. xi
PART I GLYCOSAMINOGLYCANS: STRATEGIES FOR DETECTION
AND STRUCTURE CHARACTERIZATION
1 DetectionofGlycosaminoglycansinBiologicalSpecimens .... ....... ........ 3
ShaukatA.Khan,F.N.U.Nidhi,PaigeC.Amendum,
andShunjiTomatsu
2 ImmunochemicalDetectionandGlycosaminoglycanDisaccharide-Based
CharacterizationofChondroitinSulfateProteoglycans ...................... 25
TadahisaMikamiandHiroshiKitagawa
3 Proteoglycans:AToolforDetectingHyaluronanby
ELISA-LikeMethods....... ........ ....... ....... ........ ....... ........ 39
DaianaL.Vitale,Rosalı´aI.Cordo-Russo,andLauraAlaniz
4 ParticleExclusionAssay:AToolforMeasuringHyaluronan
PericellularMatrix ......... ........ ....... ...... .... ..... ....... ........ 53
AriannaParnigoni,PaolaMoretto,SimonaRovera,ManuelaViola,
EvgeniaKarousou,AlbertoPassi,andDavideVigetti
5 UrinaryGlycosaminoglycans:CharacterizationandQuantification.... ........ 61
NaimaFdilandEs-SaidSabir
6 HPLC-MSCharacterizationofTissue-DerivedHeparanSulfate
andChondroitinSulfate .... ........ ....... ....... ........ ....... ........ 71
Ga´borT(cid:1)othandLillaTuria´k
7 EvaluationofGlycosaminoglycansBiologicalAgeinCellsandTissues......... 91
ChrysostomiGialeliandIsabelGonc¸alves
8 StudyingtheEffectsofGlycosaminoglycansinCellMorphological
AspectwithScanningElectronMicroscopy...... .... ... ..... ....... ........ 99
ChristosKoutsakis,MarcoFranchi,Anastasia-GerasimoulaTavianatou,
ValentinaMasola,andNikosK.Karamanos
PART II PROTEOGLYCANS: SEPARATION, ANALYSIS, AND INTERACTIONS
9 EvaluationoftheInVitroandInVivoEffectsofBiglycan
inInnateImmunity ........ ........ ....... ....... ........ ....... ........ 109
JinyangZeng-Brouwers,LisaSophieHuber,RosettaMerline,
JonelTrebicka,MalgorzataWygrecka,andLilianaSchaefer
10 AnalysisofExtracellularVesicle-AssociatedProteoglycans..... ....... ........ 125
JulianaPoc¸asandMattiasBelting
ix
x Contents
11 AnalysisofAggrecanGlycanationbyWestern BlotinCellCulture.... ........ 141
ChiaraGramegnaTota,AlessandraLeone,AntonioRossi,
andChiaraPaganini
12 RepulsiveScalingReplicaExchangeMolecularDynamicsin
ModelingProtein-GlycosaminoglycanComplexes ........... ....... ........ 153
MateuszMarcisz,MartynaMaszota-Zieleniak,andSergeyA.Samsonov
13 InVitroInvestigationofHyaluronan/CD44Network ....... ....... ........ 169
Theodore Karalis,ParaskeviHeldin,andSpyrosS.Skandalis
14 SeparationandIdentificationofNativeProteoglycansbyComposite
Agarose-PolyacrylamideGelElectrophoresisandImmunoblotting.... ........ 187
JamesMelrose
15 Building,Visualizing,andAnalyzingGlycosaminoglycan–Protein
InteractionNetworks....... ........ ....... ....... ....... ....... ....... .. 211
SylvieRicard-Blum
PART III PROTEOGLYCANS RELATED MOLECULES (ENZYMES,
INHIBITORS, AND EPIGENETICS)
16 AnEnzymaticActivityAssayforHeparanaseThatIsUsefulfor
EvaluatingClinicallyRelevantInhibitorsandStudyingKinetics....... ........ 227
EdwardHammondandVitoFerro
17 AMethodforUrinaryTrypsinInhibitor(UTI)Purification
CombiningAnion-ExchangeChromatographyEnrichment
andPreparativeSDS-PAGE ......... ....... ....... ........ ....... ........ 239
GabrieleNieddu,MarilenaFormato,andAntonioJuniorLepedda
18 EnzymaticSequencingofHeparinOligosaccharidesUsingExolyase .......... 249
QingdongZhang,DanrongLu,andFuchuanLi
19 EvaluatingtheEffectsofMicroRNAsonProteoglycansand
MatrixConstituents’ExpressionandFunctionalProperties .......... ........ 257
ZoiPiperigkouandNikosK.Karamanos
20 ProteoglycanExpressionStudiedbyMicroRNAs .... ........ ....... ........ 273
NancyAdrianaEspinoza-Sanchez,FabianTroschel,
BurkhardGreve,andMartinG¨otte
21 ModellingandSimulationsofExtracellularGlycoproteins .... ....... ........ 293
RajasM.Rao,NicolasBelloy,Jean-MarcCrowet,ManuelDauchez,
andSte´phanieBaud
Index .......... ........ ....... ........ ....... ....... ........ ....... ........ 315