Chapter 28

Discovery and Classification of Glycan-Binding Proteins

Essentials of Glycobiology 3rd edition

DiscoveryandClassificationofGlycan-BindingProteins

Glycansserveavarietyofbiologicalfunctionsbyvirtueoftheirmass,shape,charge,andotherphysicalproperties.Manyoftheirmorespecificbiologicalrolesaremediatedviarecognitionbycomplementaryglycan-bindingproteins(GBPs).Naturehastakenadvantageofthediversityofglycansexpressedinorganismsbyevolvingproteinmodulestorecognizediscreteglycansthatmediatespecificphysiologicalorpathologicalprocesses.ThischapterprovidesageneralclassificationandoverviewofnaturallyoccurringGBPs,thehistoryoftheirdiscovery,someoftheirbiologicalfunctionsandwaysinwhichnewGBPsareidentified.Chaptersthatfollowdescribetheanalysisofglycan–proteininteractions(Chapter29),thephysicalprinciplesinvolved(Chapter30)andthestructuresandbiologicalfunctionsofseveralGBPssubclasses(Chapters31-38).TWODISTINCTCLASSESOFGBPs

GBPsarefoundinalllivingorganisms,andfallintotwooverarchinggroups–lectinsandsulfatedglycosaminoglycan(GAG)-bindingproteins(onlineAppendix28A).Lectinsarefurtherclassifiedintoevolutionarily-relatedfamiliesidentifiedby“carbohydrate-recognitiondomains”(CRDs)basedonprimaryaminoacidand/orthree-dimensionalstructuralsimilarities(Figure28.1).CRDscanexistasstand-aloneproteinsorasdomainswithinlargermulti-domainproteins.Theytypicallyrecognizeterminalgroupsonglycans,whichfitintoshallowbutwell-definedbindingpockets(Chapters29,30).Incontrast,proteinsthatbindtosulfatedGAGs(heparan,chondroitin,dermatanandkeratansulfates,Chapter17)dosoviaclustersofpositivelychargedaminoacidsthatbindspecificarrangementsofcarboxylicacidandsulfategroupsalongGAGchains(Chapter38).Mostoftheseproteinsareevolutionarilyunrelated.GBPsthatbindtothenon-sulfatedGAGhyaluronicacid(hyaladherins)shareanevolutionarilyconservedfoldthatbindstoshortsegmentsoftheinvarianthyaluronanrepeatingdisaccharide(Chapter16),soarebestclassifiedaslectinsratherthangroupedwithsulfatedGAG-bindingproteins.Therestofthischapterconsiderslectins,differentfamiliesofwhicharedetailedinChapters31-37.DISCOVERYANDHISTORYOFLECTINS

Lectinswerediscoveredinplantsin1888whenextractsofcastorbeanseedswerefoundtoagglutinateanimalredbloodcells.Subsequentlyseedsofmanyplantswerefoundtocontainsuch"agglutinins",laterrenamedlectins(Latinfor“select”)whentheywerefoundtodistinguishhumanABObloodgroups(Chapter14)importantforbloodtransfusions.Lectinsareparticularlycommonintheseedsofleguminousplantsandthese"L-type"lectins,includingconcanavalinAandphytohemagglutinin,havebeenextensivelystudied.Althoughtheirspecificglycan-bindingactivitiesmakeplantlectinsextremelyusefulscientifictools,theirbiologicalfunctionsinplantsremainmostlyunknown.

Thefirstanimallectindiscoveredwastheasialoglycoproteinreceptor(ASGPR)identifiedbyAnatolMorellandGilbertAshwellinthelate1960’sduringtheirinvestigationsoftheturnoverofaserumglycoprotein,ceruloplasmin.Likemostglycoproteinscirculatinginblood,ceruloplasminhascomplexN-glycanswithsialicacidtermini.Toprepareradiolabeledceruloplasmin,theterminalsialicacidswereremoved,leavinganexposedgalactose.Surprisingly,asialoceruloplasminhadacirculationhalf-life(inrabbits)ofminuteswhereasintactceruloplasminremainedinthebloodforhours.GlycoproteinswithexposedGalresidueswererapidlyclearedintolivercellsviaanendocyticcellsurfacereceptorthatspecificallyboundtoterminalβ-linkedGalorGalNAc.ASGPRwaspurifiedbyaffinitychromatographyusingacolumnofimmobilizedasialoglycoprotein.Otherglycan-specificreceptorsinvolvedinglycoproteinclearanceandtargetingweresubsequentlydiscovered,includingmannose6-phosphatereceptorsfortargetinglysosomalenzymestothelysosomesandmannosereceptorsthatclearglycoproteinswithterminalmannoseorGlcNAcresiduesfromtheblood.Smallsolublelectinsspecificforβ-linkedgalactose(nowcalled“galectins”,Chapter36)wereisolatedbyaffinitychromatographyinextractsfrommanybiologicalsourcesrangingfromtheslimemoldDictyosteliumdiscoideumtomammaliantissues.Bythe1980’s,theconceptofvertebratelectinsthatrecognizespecificglycanswaswellestablished.Althoughthefirstanimallectinsidentifiedwerespecificforendogenousglycans,manylectinsspecificforexogenousglycansofmicroorganismswerelaterfound.Lectinsrecognizingexogenousglycansincludesolubleproteinsthatcirculateinthebloodofmanyspeciesaswellasmembrane-boundreceptorsoncellsoftheimmunesystem.Lectinsarealsowidespreadinmicroorganisms,althoughtheytendtobecalledbyothernamessuchashemagglutininsandadhesins.Theinfluenzavirushemagglutinin,whichbindstosialicacidonhostcells(Chapter15)wasthefirstGBPisolatedfromamicroorganism.Theviralhemagglutinins,likemanyplantlectins,canagglutinateredbloodcells.Manybacteriallectinshavebeendescribed.Theyfallintotwogeneralclasses:adhesinsonbacterialsurfacesthatrecognizeglycansonhostcellmembraneglycolipidsorglycoproteinstofacilitatebacterialadhesionandcolonization,andsecretedbacterialtoxins(Chapter37).DISCOVERYOFSULFATEDGAG-BINDINGPROTEINS

AlargegroupofGBPsthatdefyclassificationbasedonsequenceorstructurerecognizesulfatedGAGs(Chapter38).Thebest-studiedexampleistheinteractionofheparinwithantithrombin.Heparinwasdiscoveredin1916byJayMcLean,amedicalstudent,butitwasnotuntil1939thatheparinwasshowntobeananticoagulantinthepresenceof“heparincofactor”,whichwasthenidentifiedasantithrombininthe1950s.ManyothersulfatedGAG-bindingproteinswerelaterdiscoveredbyaffinitychromatographyoncolumnsofimmobilizedheparin.Growthfactorsandcytokinesbearingclustersofpositivelychargedaminoacidsalongtheir

proteinsurfaceinteractwithsulfatedGAGsinalooserfashion—i.e.,theydonotalwaysshowthehighspecificityseenwithantithrombin.However,insomecases,specificGAGsequencesmediatetheformationofhigher-ordercomplexes,actingasatemplateforoligomerizationorpositioningofproteinssuchasFGFanditscellsurfacereceptor.

MAJORBIOLOGICALFUNCTIONSOFGBPs

GBPsfunctionincommunicationbetweencellsinmulticellularorganismsandininteractionsbetweenmicrobesandhostsandcanalsobeinvolvedinbindinggrowthfactorsorcytokines.Theseinteractionscantakevariousforms,resultinginmovementofmolecules,cells,andinformation.Trafficking,targetingandclearanceofproteinsDirectingmovementofglycoproteinswithinandbetweencellsisacommonfunctionforlectinsinmanyorganisms.Ineukaryoticcells,includingyeastaswellashighereukaryotes,severalgroupsoflectinsareimportantinglycoproteinbiosynthesisandintracellularmovement(Chapter39).Intheendoplasmicreticulum(ER),twolectins,calnexinandcalreticulinbindmonoglucosylatedhighmannoseglycanspresentonnewlysynthesizedglycoproteins,formingpartofaqualitycontrolsystemforproteinfolding.BindingtocalnexinorcalreticulinkeepsproteinsintheERuntiltheyarecorrectlyfolded.OthergroupsoflectinsintheER,includingM-typelectinsandproteinscontainingmannose6-phosphatereceptorhomologydomainstakepartintheprocessofER-associatedglycoproteindegradation(ERAD),bindingpartiallyprocessedhighmannoseglycansonterminallymisfoldedglycoproteins,causingthemtoberetrotranslocatedintothecytoplasmfordeglycosylation,followedbydegradationintheproteasome.OneofthebestcharacterizedfunctionsofGBPsisindeliveryofnewlysynthesizedlysosomalenzymesfromthetrans-GolgitolysosomesbyP-typelectins(Chapter33)thatrecognizemannose6-phosphateresiduesthathavebeenaddedtoN-glycansonlysosomalenzymesintheGolgiapparatus,targettingthemtoendosomesforfusionwithlysosomes.Oncereleasedfromcells,glycoproteinscanalsobetakenupfordegradationinlysosomes.Asnotedabove,theASGPRonmammalianhepatocytescontrolsturnoverofmanyserumglycoproteinsbyrecognitionofterminalGalorGalNAcresidues.Similarly,themannosereceptoronmacrophagesandsinusoidalcellsoftheliverbindsandclearsglycoproteinswitholigomannoseN-glycansthatarereleasedfromcellsduringinflammationandtissuedamage.NotallGBP-mediatedtargetingleadstodegradation.Glycan-bindingsubunitsofsecretedbacterialandplanttoxinstargetthemtoglycolipidsoncellsurfacesandfacilitateentryofthetoxinsintocells(Chapter37).Manyenzymescontainglycan-bindingdomainsthatbringanotherdomainwithenzymeactivityintoclose

proximitywithitssubstrates.Onenotablegroupincludesbacterialcellulasesinwhichcellulose-bindingmodulespositiontheenzymaticdomainforoptimaldegradationofcellulosefibers.Usingasimilarprinciple,GalNAc-bindingdomainsinpolypeptide-N-acetylgalactosaminyltransferasesthatinitiateO-linkedglycosylationinanimalspositiontheseenzymestoaddfurtherGalNAcresiduestoregionsofpolypeptidesthatalreadybearO-glycans(Chapter10).CelladhesionDistinctiveglycansonthesurfacesofdifferentcells,botheukaryoticandprokaryotic,makethemtargetsforGBPs.BindingofglycansonthesurfaceofonecellbyGBPsonanothercellcaninducerecognitionandadhesion,whereascrosslinkingglycansondifferentcellsbymultivalentsolubleGBPsprovidesanalternativemechanism.Suchinteractionsareexploitedinspecializedsituationsexemplifiedbytransientcontactsbetweenmovingcells.Theselectins,threereceptorsthatfunctionininteractionsbetweenwhitebloodcells,plateletsandendothelia,providethebestcharacterizedexampleoflectin-glycaninteractionsincell-celladhesion(Chapter34).Forexample,L-selectinonlymphocytesbindsglycansonthespecializedendothelialcellsoflymphnodestoinducelymphocyte-homing,whereincirculatinglymphocytesleavethebloodstreamandenterthelymphnode.OthermammalianGBPsthatmediatebindingofcellstoeachotherorthatcrosslinkligandsonthesamecellsurfaceincludeSiglecs(Chapter35)andgalectins(Chapter36).Lectinsinmulticellularorganismsalsoforminteractionsbetweencellsandtheextracellularmatrixandsupporttheorganizationofmatrixcomponents.Forexample,proteinscontaining“linkmodules”thatbindspecificallytohyaluronanincartilage(andothertissues)areessentialforstructuringtheextracellularmatrix(Chapter38)andotherextracellularproteinsbindtosulfatedGAGstoorganizecell-cellandcell-matrixinteractions(Chapter38).Manybacteriaalsouselectinstoadheretoglycansonhostcellsinsituationsinwhichtheywouldotherwisegetwashedaway.Thelectinsareusuallypresentattheendsoflongstructurescalledpiliorfimbriaethatprojectfromthesurfaceofthebacteria(Chapter37).Adhesioncanbepartoftheinfectionprocess.Forexample,amannose-specificadhesinonpathogenicstrainsofEscherichiacolithatcauseurinaryinfectionsbindstoepithelialcellsoftheurinarytract.Otherglycan-proteininteractionsbetweenhostcellsandbacteriaprovideanormalmechanismofco-existence.Severalbacterialspeciesthatarepartofthenormalgutfloraincludingnon-pathogenE.coliuseadhesinstobindtoglycolipidspresentoncellsliningthelargeintestine.ImmunityandinfectionManylectinsareinvolvedinimmuneresponses,in“lower”vertebratesandinvertebratesaswellasinmammals.Differencesinglycansonhostandmicrobialcellsurfacesarecommonlythebasisforinnateimmuneresponses.Phagocytosisisacommonoutcomeofthebindingofglycan-specificreceptorsonmacrophagestoglycanscommontobacteria,fungiandviruses.Otherlectinscirculatingintheblood,

suchasserummannose-bindingproteinandficolins,bindtopathogencellsurfacesandactivatethecomplementcascade,leadingtocomplement-mediatedkilling.Bindingofglycanstolectinsonimmunecellscanalsotriggerintracellularsignalingthatactivatesorsuppressescellularresponses.Receptorsthatrecognizeself-glycanssuchassialicacid,aswellasseveralthatarespecificforglycanscharacteristicofmicro-organismscaninitiatesuchsignaling.Forexample,bindingofα2-6linkedsialicsacidtoCD22,amemberoftheSiglecfamilyofvertebratelectinsfoundonBlymphocytes,initiatessignalingthatinhibitsactivationtopreventself-reactivity(Chapter35).Incontrast,bindingoftrehalosedimycolate,aglycolipidfoundinthecellwallofMycobacteriumtuberculosistothemacrophageC-typelectinmincle,inducesasignalingpathwaythatcausesthemacrophagetosecreteproinflammatorycytokines.Finally,virusesoftenuseGBPstoattachtohostcellsduringinfection(Chapter37).Proteinsonvirussurfaces,includingthoseoninfluenzavirus,reovirus,Sendaivirus,andpolyomavirus,bindtosialicacids.Inadditiontobringingthevirusintocontactwiththeircelltargets,thesehemagglutininstypicallyinducemembranefusion,facilitatingvirusentryanddeliveryofnucleicacidsintothecytosol.Glycan-bindingreceptorsonvirusesareoftenhighlyspecificforaparticularlinkage;humaninfluenzavirusbindstosialicacidslinkedα2-6toGal,whereasbirdinfluenzavirusbindstoα2-3linkedsialicacid.Otherviruses,suchasherpessimplexvirus,haveGAG-bindingproteinsthatbindtoheparansulfateproteoglycansoncellsurfaces.ORGANIZATIONOFLECTINS

Animportantconceptinidentifying,definingandclassifyinglectinsisthatsugar-bindingactivityisembodiedindiscreteproteinmodulesordomains,referredtoascarbohydrate-recognitiondomains(CRDs).CRDsaretypicallyindependentlyfoldingsegmentsofproteins;oftenonecanseparatethesugar-bindingactivityfromotheractivitiesoftheproteinbyexpressingitsCRDinisolation.Insomecases,theCRDsconstitutetheentireGBP(Figure28.2).WhenalectiniscomprisedsimplyofitsCRD,itsfunctionsoftenaredependentonmultivalency,whichendowslectinswiththeabilitytocross-linksugar-containingstructures.Thisarrangementexplainstheabilityofmanyplantlectinstoagglutinatecellsandtoclusterglycoproteinsoncellsurfaces,whichcaninducemitogenesis.OtherGBPsthatfunctionthiswayincludethegalectins,whichcanbridgeglycansononecellsurfaceorbetweencells.Sometimesotheractivitiesareencodedwithinthestructureofthesamedomainthatbindssugars;somecytokinescomprisedofasinglefoldeddomainmayhavedistinctsitesforbindingglycansandothertargetreceptors.Morecommonly,otheractivitiesoflectinsresideinseparatemodulesinmulti-domainproteins(Figure28.2).SucharrangementsarewidespreadandthedomainsassociatedwithCRDsperformmanydifferentfunctions,includingbindingothertypesofligands,performingenzymaticreactions,anchoringproteinsto

membranesanddirectingoligomerization.GBPsoftencontainmultiplemodules,combiningseveralfunctionsinoneprotein.Membraneanchorsinlectinscantakemultipleforms,buttheyoftenspanthemembrane,linkingextracellularCRDswithcytoplasmicdomains.Thisarrangementfacilitatestheflowofinformationbetweenglycan-bindingsitesontheextracellularsurfaceandthecytoplasm.Simplesequencemotifsinthecytoplasmicdomainsoftransmembranelectinsoftencontroltraffickingofreceptorsandtheirboundglycanligands.Commonfunctionsofsuchintracellularmovementsareinternalizationofcellsurfacereceptors,directingboundligandstoendosomesandlysosomes,andmovementthroughintracellularcompartmentssuchastheendoplasmicreticulumandGolgiapparatustothecellsurface.Flowofinformationintheoppositedirectioncanleadtostimulationofsignalingcomplexesonthecytoplasmicsideofthemembraneinresponsetobindingofglycansatthecellsurface.Clusteringofglycan-bindingsites(multivalency)isoftencriticaltobothrecognitionandbiologicalfunctionsoflectins.Clusteringofsitesisachievedindifferentways,byformationofsimpleoligomersofCRDs,asaresultofthepresenceofmultipleCRDsinasinglereceptorpolypeptideandthroughassociationofCRD-containingpolypeptidesthroughindependentoligomerizationdomains.Someoligomersarestable,whileothers,suchasthoseformedbysomegalectins,areinequilibriumwithmonomers.Thesearrangementsfacilitatemultivalentbindingtoincreaseavidityanddirectthegeometricalarrangementofbindingsites.MultipleCRDsmayfaceinthesamedirectionforsurfacerecognitionorinoppositedirectionstofacilitatecrosslinking.MultivalentCRDsmayhavefixedspacingorflexiblespacingtoaccommodatedifferenttargetglycans.Insomecases,oligomerizationdomainsalsoformstructuralfeatures,servingsasstalksthatprojectCRDsfromthecellsurface.Oligomerizationdomainscanalsoembodyotherfunctions,suchastheprotease-bindingsitesinthecollagen-likedomainsofmannose-bindingprotein.CLASSIFICATIONOFLECTINSBASEDONSTRUCTURALSIMILARITIES

ItisconvenienttoclassifylectinsbasedonthestructuresoftheCRDsthattheycontain(Figure28.3).CRDsarefoundinalargenumberofdifferentstructuralcategories,indicatingthatmanydifferentproteinfoldscanaccommodateglycanbinding(Chapter30).Basedonthisobservation,sugar-recognitionmusthaveevolvedindependentlymanytimesandthediversityofCRDstructuresmusthavearisentoaddressadiversityoffunctions.GBPsappearacrossallkingdomsoflife,butthetypesoflectinsineachkingdomvaryconsiderably.Severalfamiliesappearinbothprokaryotesandeukaryotes,buttheirdistributionssuggestdifferentevolutionaryhistories.Themalectindomain,althoughconservedinstructureandwidelydistributedinprokaryotes,plantsand

animals,isfoundinproteinswithdistinctdomainorganizationanddifferentfunctionsinthethreegroups.Animalmalectinisamembrane-anchoredCRDoftheendoplasmicreticulumthatbindsN-linkedglycansduringglycoproteinbiosynthesis.Inplants,themalectinCRDisexpressedatthecellsurfaceandislinkedtoacytoplasmickinasedomain.BacterialmalectinsconsistofCRDsassociatedwithglycohydrolasedomains.Similarly,R-typeCRDs(Chapter31)inplantsformthecellsurface-bindingcomponentoftoxinssuchasricinandarelinkedtoglycohydrolasegenesinbacteria,butinanimalstheyappearintwodistinctcontexts:inpolypeptide-N-acetylgalactosaminyltransferasesthatinitiateO-GalNAcglycans(Chapter10)andinthemannosereceptorfamily.AlthoughtheseCRDshavebeenadaptedtoservedifferentfunctionsindifferentkingdoms,asugar-bindingfunctionappearstohaveevolvedearlyandbeenpreservedinsubsequentlineages.IncontrasttoCRDswithbroadevolutionarydistribution,twoothergroupsoflectinshavesporadicdistributions.B-lectindomainsarebroadlydistributedinbacteriainassociationwithhydrolasedomains,arefoundasisolatedortandemCRDsinmonocotplantsbutnotinotherplants,inbonyfishesbutnotinotheranimals,andinandsomefungi.TheF-typelectinsappearinbacteriaandinalimitednumberofanimalspecies.Inthesecases,thepresenceofrelateddomainsinevolutionarilydistantspeciesmayreflectlateralgenetransferratherthanthepresenceofaprecursorlectininthedistantcommonancestorthattheyshare.AdifferentpatternofevolutionisobservedforPA14domains,theonlyothertypeofCRDfoundinbothbacteriaandeukaryotes.AlthoughthePA14foldisrelativelywidespread,suggestingthatitoriginatedearlyandwasretainedacrossspecies,onlyasubsethavebeenshowntohavesugar-bindingactivity:CRDsassociatedwithbacterialglycohydrolasesandinadhesinsandflocculationfactorsonthesurfaceofyeast.Theintracellularsortinglectinsmentionedearlier,suchascalnexin,calreticulin,andM-typelectins,arethemostbroadlydistributedlectinsthatevolvedfromacommoneukaryoticancestor.Theirdistributionandtheconservationoftheirfunctionsprobablyreflectanancientandconservedroleinintracellulartraffickingofglycoproteinsineukaryotes.TwoothergroupsofCRDsappeartobefoundinmetazoansbutnotsimplereukaryotes.TheL-typeCRDshavedivergedinfunctionbetweenanimals,wheretheyfunctioninintracellularglycoproteinsortingandtrafficking,andplants,wheretheyserveaprotectivefunction(Chapter32).Chitinase-likesugar-bindingdomainsacrossarangeofspeciesretaintheabilitytobindpolymersofGlcNAc,buttheirbiologicalfunctionsarenotwellunderstood,soitisuncleariftheyhavesharedrolesinplantsandanimals.Inadditiontothewidelydistributedfamilies,certainCRDfamiliesareevolutionarilyrestricted.Inadditiontoanimal-specificandvertebrate-specificlectingroups,therearealsogroupssuchastheI-typelectinsfoundonlyinmammals(Chapter35).Thepatternofevolutionofanimal-specificlectinsvaries.Galectinsseemtobesimilarinorganizationinvertebratesandinvertebratesanditmaybepossibletoidentifyorthologsinquitediversespecies(Chapter36).Incontrast,C-typeCRDshave

undergoneindependentradiationinvertebratesandinvertebrates,andidentifyingorthologsevenbetweenmouseandhumanproteinsinsomecasesisdifficult(Chapter34).Ofthetwelvedifferentproteinfoldsfoundinplantlectins,nineappeartobeuniquetoplants.Itisalsonoteworthythatvirusesseemtohavedevelopedtheirownapproachestobindingglycansratherthanborrowingfromhosts(Chapter37).InadditiontofamiliesofproteinsthatshareevolutionarilyrelatedCRDs,thereareindividualproteinsthatbindsugarsthroughdomainsthatarenotrelatedtoCRDsinotherproteins.Examplesincludeproteinswithdedicatedsugarbindingdomains,suchassomelamininGdomains,whichrecognizeglycansonα-dystroglycan(Chapter45),pentraxins,whichbindmodifiedandphosphorylatedsugars,andmacrophageαMβ2integrin,whichbindsfungalglucansandexposedGlcNAcresiduesonglycoproteins.Otherproteinsbindtosugarsthroughdomainsthatalsohaveotherligands:annexinVbindsbisectingGlcNAcresiduesaswellasphospholipidsandseveralcytokineshavebeenreportedtobindsugarsaswellastheirtargetreceptors.Sulfated-GAGbindingproteinshavealsolargelyevolvedbyconvergentevolution.IDENTIFYINGGBPsBYBIOLOGICALANDBIOCHEMICALFUNCTIONANDSTRUCTURALSIMILARITY

Therearemultiplewaysinwhichglycanrecognitioncanbeimplicatedinspecificbiologicalprocesses.Onecommonapproachistodemonstratetheabilityofsimplemonosaccharidesorsmallglycanstocompetewithaprocess.Informationcanoftenalsobegainedbymodifyingsugarsoncellsandglycoproteinswithenzymesthataddorremovesugars,bygeneticmanipulation,andbychemicalinhibitorsofglycanmetabolism.Thesestrategieshaveprovidedinformationabouttheglycansinvolved,forexamplethoseneededforvirusortoxinbindingorthoserequiredforendocytosisofglycoproteins.Basedonthisinformation,itisthenpossibletolookforGBPsthattargettheseparticularsugarsandwhichcanthenbelinkedtothebiologicalprocess.Theabilitytobindspecificsugars,assessedinvariousbiochemicalassays,hasoftenbeenthebasisfordirectidentificationofnovelGBPswithoutreferencetoaparticularbiologicalfunction.Inadditiontoformingabasisforbindingandcompetitionassays,thebindingactivityiscommonlyusedasameansofisolatingtheseproteinsbyemployingaffinitychromatographyonappropriateimmobilizedglycanligands.Awidevarietyofmethodsforcouplingmonosaccharidesandcomplexglycanstocreateaffinityresinshavebeendeveloped.Asmentionedabove,manysulfated-GAGbindingproteinshavebeendiscoveredbyaffinitychromatographyonimmobilizedGAGchains.Alimitationoftheseapproachesisthatbindingactivitydoesnotdirectlyindicateabiologicalfunctionandtherolesofmanywell-characterizedGBPshavenotbeenfullydetermined.

TheobservationthatmanylectinsfallintostructuralfamiliesprovidesanalternativewaytoidentifynovelGBPsthroughanalysisofproteinsequences.SequencemotifscharacteristicofCRDsareroutinelyusedtoscreensequencesfromwholegenomesequencing.ThesemotifscanalsobeusedtoscreenspecificcDNAandgenesequencesofinterestbecauseoftheirassociationwithbiologicalfunctions.DetectionofanappropriatemotifsuggeststhepresenceofafunctionalCRD,andstructuralknowledgeofknownsugar-bindingsitescansuggestwhetheranovelproteinislikelytoretainglycan-bindingactivity.Insomecases,itcanevensuggestpotentialligands.Suchpredictionsoftenmotivatetestingforsugarbindingactivity,eitherbyspecificallyexaminingbindingtopredictedligandsorbyscreeningmoregenerallyusingglycanarrays.Althoughstructure-basedpredictionsdonotdirectlyyieldinformationaboutbiologicalfunction,theorganizationofCRDsandtheirassociationwithotherdomainsoftenprovideinformationaboutpotentialfunctions.Thistypeoftop-downanalysisislimitedtodiscoveryofGBPsthatcontaindomainsresemblingknownCRDs.Asglycanarrayscreeningbecomesmorewidelyaccessible,morebroad-basedscreeningcanbeenvisioned.NATURALLIGANDSFORGBPs

Monosaccharidesorsmalloligosaccharidesinisolationtendtobelow-affinityligandsforGBPs,oftenwithdissociationconstantsinthemillimolarrange.Theseintrinsicaffinitiesareenhancedinseveralways(Figure28.4).Atthelevelofindividualglycans,affinitycanbeenhancedbylinkingthesugartoothertypesofstructures.TypicalconjugationofglycanstoproteinsandlipidscanleadtoenhancedCRDbinding.Forexample,someGBPssuchasthemacrophagereceptorminclebindtoglycolipidswithmuchhigheraffinitythantheybindtofreeoligosaccharides.Inthiscase,enhancedaffinitycanresultfromthepresenceofanextendedoraccessorybindingsiteinaCRDadjacenttothesugar-bindingsite,whichisabletoaccommodatethehydrophobictailofthelipid.OtherGBPsbindselectivelytoaparticularglycanconjugatedtoaspecificpolypeptidemotif.OptimalbindingofP-selectintotheligandPSGL-1requiresanO-linkedglycanbearingasialylLewisxstructureonapeptidewithadjacentacidicresiduesandsulfatedtyrosines(Chapter34).Inyetothercases,glycanrecognitioniscombinedwithotherbindingdomainsonaprotein.ThemannosereceptorcontainsC-typeCRDsthatbindhighmannoseoligosaccharidesandafibronectintypeIIrepeatthatbindstotriplehelicalpolypeptides.Together,thesetwomodalitiesfacilitatebindingtofragmentsofcollagenreleasedatsitesofinflammation.AmajordeterminantofbindingtonaturalligandsistheinteractionofmultivalentglycanswithclusteredCRDs,resultinginhighaviditybinding.Clusteringofligandscanresultfromthepresenceofmultiplebindingepitopesinasingleoligosaccharideorpolysaccharide,thepresenceofmultipleglycansattachedtoasingleproteinscaffoldorthepresenceofadjacentglycoproteinsorglycolipidsinacellmembrane.Similarly,clusteringofCRDscanreflectthepresenceofmultipleCRDsinasingle

polypeptide,formationofoligomersofpolypeptidethateachcontainsasingleCRDandfromclusteringofCRD-containingproteinsinthecellmembrane.EachoftheselevelsoforganizationofCRDshasthepotentialtoplacegeometricalconstraintsontheoptimalarrangementofligands,dependingonthedegreetowhichCRDsareheldinafixedarrangementorareflexiblylinked.Clusteringofglycansattachedtoasinglepolypeptide,particularlyinheavilyO-glycosylatedproteinssuchasmucins,canalsoaffecttheirabilitytotakeondifferentconformations.SinceGBPstypicallyinteractwithasingleconformation,selectingoneofmultipleaccessibleconformations,thereisanentropicpenaltyassociatedwithbindingwhichmaybereducedwhentheglycanhasfewerpotentialconformations.Invitrobiochemicalassays,includingglycanarrays,reflectonlysomeofthesetypesofclusteringofCRDsandligands,sotheymustbeinterpretedwithsomecaution.Insomecases,bindingofaCRDtoisolatedglycansmaybeessentiallyundetectableeventhoughbindingoftheintactCRD-containingproteintoitsendogenousglycoconjugatemaybehighlyselectiveandquitestrong.Caremustalsobeexercisedinuseofthetermligand,todistinguishtheglycanpartofaligandfromtheentirenaturalglycoconjugateorevencellsurface.TERMINOLOGYFORSPECIFICGBPLIGANDS

Basedontheaboveconsiderations,GBPsmaybindselectivelytoaparticularglycanonlywhenitisconjugatedtoaparticularglycoprotein.TheGBPligandisneithertheglycanitselfnortheproteinitself.ExamplesincludeP-selectinbindingtosialylLewisxonPSGL-1(seeabove)andE-selectinbindingtothesameglycan(sialylLewisx)carriedonavariantformoftheproteinCD44.Thereisatpresentnoconsistentwaytodesignateaglycanonparticularproteinasaligandforaspecificlectin.SayingthatsialylLewisxorthatPSGL-1(protein)isthe“ligand”forP-Selectinisnotaccurate.TheE-selectin-bindingformofCD44wasgivenadifferentname(HCELL,hematopoieticstemcellligandforE-selectin)thatfailstoidentifythepolypeptidecarrier.Thismatterhasyettoberesolved,especiallywhenasingleproteinmightbetherequiredpolypeptidescaffoldthatcarriesglycansfordifferentGBPs.Atthispoint,theconceptthatglycansareoftenligandsforGBPsonlyinthecontextoftheirproteinorlipidcarriershasbeenwellestablished.

FIGURELEGENDS

FIGURE28.1.Representativestructuresfromfourcommonanimallectinfamilies.Theemphasisisontheextracellulardomainstructureandtopology.Thefollowingarethedefinedcarbohydrate-bindingdomains(CRDs)shown:(CL)C-typelectin;(GL)Galectin;(MP)P-typelectin;(IL)I-typelectin.Otherdomainsare(EG)EGF-likedomain;(IG2)immunoglobulinC2-setdomain;(TM)transmembranedomain;and(C3)complementregulatoryrepeat.ThenumberofdomainsaccompanyingtheCRDvariesamongfamilymembers.

FIGURE28.2.Arrangementsofcarbohydrate-recognitiondomains(CRDs)inGBPs.ProteinscontainingjustCRDsorCRDsassociatedwithothertypesoffunctionaldomains,withmembraneanchorsorwitholigomerizationdomainsaredepictedschematically.AsingleGBPcancontainalloftheseadditionaldomains.

agglutinin;EDEM,Endoplasmicreticulum-associateddegradation-enhancingα-mannosidase-likeproteins;GH,glycohydrolase;MRH,mannosereceptorhomology.

FIGURE28.4.SourcesofenhancedbindingofnaturalligandstoGBPs.WithinindividualCRDs,secondaryinteractionsbeyondtheprimarybindingsitecanbewithsugar,proteinorlipidportionsofglycoconjugateligands.MultivalentinteractionscanreflectinteractionofsinglebranchedoligosaccharidesormultipleoligosaccharidesattachedtoaglycoproteinwithmultipleCRDsbroughttogetherwithinreceptoroligomersorinGBPclustersonthecellsurface.

FURTHERREADING

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Table1.Appendix26A

Comparisonoftwomajorclassesofglycan-bindingproteins

LectinsaGlycosaminoglycan-binding

proteinsbSharedevolutionaryorigins yes(withineachgroup) noSharedstructuralfeatures yes(withineachgroup) noDefiningAAresiduesinvolvedinbinding

oftentypicalforeachgroup patchofbasicaminoacidresidues

Typeofglycansrecognized N-glycans,O-glycans,glycosphingolipids(afewalsorecognizesulfatedglycosaminoglycans)

differenttypesofsulfatedglycosaminoglycans

Locationofcognateresidueswithinglycans

typicallyinsequencesatouterendsofglycanchains

typicallyinsequencesinternaltoanextendedsulfatedglycosaminoglycanchain

Specificityforglycansrecognized

stereospecificityhighforspecificglycanstructures

oftenrecognizearangeofrelatedsulfatedglycosaminoglycanstructures

Single-sitebindingaffinity oftenlow;highaviditygeneratedbymultivalency

oftenmoderatetohigh

Valencyofbindingsites multivalencycommon(eitherwithinnativestructureorbyclustering)

oftenmonovalent

Subgroups C-typelectins,galectins,P-typelectins,I-typelectins,L-typelectins,R-typelectinsetc.

heparansulfate–bindingproteins,chondroitinsulfate–bindingproteins,dermatansulfate–bindingproteins

Typesofglycansrecognizedwithineachgroup

canbesimilar(e.g.,galectins)orvariable(e.g.,C-typelectins)

classificationitselfisbasedontypeofglycosaminoglycanchainrecognized

ModifiedfromVarkiA.andAngataT.2006.Glycobiology16:1R–27R.aThereareotheranimalproteinsthatrecognizeglycansinalectin-likemanneranddonotappeartofallintooneofthewell-recognizedclasses(e.g.,variouscytokines).bHyaluronan(HA)-bindingproteins(hyaloadherins)fallinbetweenthesetwoclasses.Ontheonehand,some(butnotall)ofthehyaloadherinshavesharedevolutionaryorigins.Ontheotherhand,recognitioninvolvesinternalregionsofHA,whichisanonsulfatedglycosaminoglycan.

Chapter 38

Proteins that Bind Sulfated Glycosaminoglycans

Essentials of Glycobiology, 3rd edition

ProteinsthatBindSulfatedGlycosaminoglycans

Authors:JeffreyD.Esko,JamesPrestegardandRobertJ.LinhardtGlycosaminoglycansbindtomanydifferentclassesofproteinsmostlythroughelectrostaticinteractionsbetweennegativelychargedsulfategroupsanduronicacidsandpositivelychargedaminoacidsintheprotein.Thischapterfocusesonexamplesofglycosaminoglycan-bindingproteins,methodsformeasuringglycosaminoglycan-proteininteraction,andinformationaboutthree-dimensionalstructuresofthecomplexes.

GLYCOSAMINOGLYCAN-BINDINGPROTEINSARECOMMON

Severalhundredglycosaminoglycan(GAG)-bindingproteinshavebeendiscovered,whichmakeuptheGAG-interactomeandfallintothebroadclassespresentedinTable38.1.Toalargeextent,studiesoftheGAG-interactomehavefocusedonproteininteractionswithheparin,amorehighlysulfated,iduronicacid(IdoA)-richformofheparansulfate(HS;Chapter17).Thisbiasreflectsinpartthecommercialavailabilityofheparinandheparin-Sepharose,whicharefrequentlyusedforfractionationstudies,andtheassumptionthatbindingtoheparinmimicsbindingtoHSpresentoncellsurfacesandintheextracellularmatrix.Incomparison,relativelyfewproteinsareknowntointeractwithchondroitinsulfate(CS)orkeratansulfate(KS)withcomparableavidityandaffinity.Insomecases,CSandtherelatedGAG,dermatansulfate(DS),maybephysiologicallyrelevantbindingpartnersbecausetheseGAGspredominateinmanytissues.Determiningthephysiologicalrelevanceoftheseinteractionsisamajorareaofresearch.Incontrasttolectins,whichtendtofallintoevolutionarilyconservedfamilies(Chapters28-37),GAG-bindingproteinsdonothavecommonfoldsandinsteadappeartohaveevolvedbyconvergentevolution.AsshowninTable38.1,theinteractionbetweenGAGsandproteinscanhaveprofoundphysiologicaleffectsonprocessessuchashemostasis,lipidtransportandabsorption,cellgrowthandmigration,anddevelopment.BindingtoGAGscanresultinimmobilizationofproteinsattheirsitesofproductionorintheextracellularmatrixforfuturemobilization;regulationofenzymeactivity;bindingofligandstotheirreceptors;proteinoligomerization;andprotectionofproteinsagainstdegradation.Insomecases,theinteractionmayreflectcomplementarityofcharge(e.g.,histone-heparininteractions)ratherthananyspecificbiologicallyrelevantinteraction.Inothercases,theinteractionhasbeenshowntodependonrarebutveryspecificsequencesofmodifiedsugarsintheGAGchain(e.g.,antithrombinbinding).

METHODSFORMEASURINGGLYCOSAMINOGLYCAN-PROTEIN

BINDING

NumerousmethodsareavailableforanalyzingGAG-proteininteractions,andsomeprovideadirectmeasurementofKdvalues.AcommonmethodinvolvesaffinityfractionationofproteinsonSepharosecolumnscontainingcovalentlylinkedGAGchains,usuallyheparin.Theboundproteinsareelutedwithdifferentconcentrationsofsodiumchloride,andtheconcentrationrequiredforelutionisgenerallyproportionaltotheKd.High-affinityinteractionsrequireatleast1MNaCltodisplaceboundligand,whichtranslatesintoKdvaluesof10−7–10−9M(determinedunderphysiologicalsaltconcentrationsbyequilibriumbinding).Proteinswithlowaffinity(10−4–10−6M)eitherdonotbindunder“normal”conditions(0.15MNaCl)orrequireonly0.3–0.5MNaCltoelute.ThismethodisbasedontheassumptionthatGAG-proteininteractionisentirelyionic,whichisnotentirelycorrect.Nevertheless,itcanprovideanassessmentofrelativeaffinity,whencomparingdifferentGAG-bindingproteins.Anumberofmoresophisticatedmethodsarenowinusethatprovidedetailedthermodynamicdata(ΔH[changeinenthalpy],ΔS[changeinentropy],ΔCp[changeinmolarheatcapacity],etc.),kineticdata(associationanddissociationrates),andhigh-resolutiondataonatomiccontactsinGAG-proteininteractions(Table38.2).Regardlessofthetechniqueoneuses,itmustbekeptinmindthatinvitrobindingmeasurementsarenotlikelytobethesameasthosewhentheproteinbindstoproteoglycansonthecellsurfaceorintheextracellularmatrix,wherethedensityandvarietyofGAG-bindingproteins,proteoglycansandotherinteractingfactorsvariesgreatly.Todeterminethephysiologicalrelevanceoftheinteraction,oneshouldconsidermeasuringbindingunderconditionsthatcanleadtoabiologicalresponse.Forexample,onecanmeasurebindingtocellswithalteredGAGcomposition(Chapter49)oraftertreatmentwithspecificlyasestoremoveGAGchainsfromthecellsurface(Chapter17)andthendeterminewhetherthesameresponseoccursasobservedinthepresenceofGAGchains.Theinteractioncanthenbestudiedmoreintensivelyusingtheinvitroassaysdescribedabove.

CONFORMATIONALANDSEQUENCECONSIDERATIONS

Asmentionedabove,mostGAG-bindingproteinsinteractwithHSorheparin.Thelikelybasisforthispreferenceisgreatersequenceheterogeneityandmorevariablesulfation,comparedtootherGAGs.Theunusualconformationalflexibilityofiduronicacid,whichisfoundinheparin,HS,andDS,alsohasaroleintheirabilitytobindproteins.GAGsarelinearhelicalstructures,consistingofalternatingresiduesofN-acetylglucosamine(GlcNAc)orN-acetylgalactosamine(GalNAc)withglucuronicacid(GlcA)oriduronicacid(IdoA)(withtheexceptionofkeratan

sulfates,whichconsistofalternatingGlcNAcandgalactoseresidues;Chapter17).Inspectionofheparinoligosaccharidescontaininghighlymodifieddomains([GlcNS6S-IdoA2S]n)showsthattheN-sulfoand6-O-sulfogroupsofeachdisacchariderepeatlieonoppositesidesofthehelixfromthe2-O-sulfoandcarboxylgroups(Figure38.1).AnalysisoftheconformationofindividualsugarsshowsthatGlcNAcandGlcAresiduesassumeapreferredconformationinsolution,designated4C1(indicatingthatcarbon4isabovetheplanedefinedbycarbons2,3,and5andtheringoxygen,andthatcarbon1isbelowtheplane;Chapter2).Incontrast,IdoA2Sassumesthe1C4orthe2SOconformation(Figure38.1),whichreorientsthepositionofthesulfosubstituents,therebycreatingadifferentorientationofchargedgroups.InmanycaseswhenaproteinbindstoanHSchain,itinducesachangeinconformationoftheIdoA2Sresidueresultinginabetterfitandenhancedbinding.IdoA2SresidueshavealwaysbeenfoundindomainsrichinN-sulfoandO-sulfogroups(forbiosyntheticreasons;Chapter17),whichisalsowhereproteinsusuallybind.Thus,thegreaterdegreeofconformationalflexibilityinthesemodifiedregionsmayexplainwhysomanymoreproteinsbindwithhighaffinitytoheparin,HS,andDSthantootherGAGs.ThepresenceofanN-acetylgroupinanGlcNAcresiduechangesthepreferredconformationoftheneighboringIdoA2Sresidue,showingthatevenminormodificationscaninfluenceconformationandchainflexibility.BindingtoGAGsthathavealowdegreeofsulfationmayrequirelargerdomainsintheproteintointeractwithlongerstretchesofanoligosaccharide.Moleculardynamicsimulationsonlargeheparinoligosaccharidesarepossiblewiththeavailabilityofsupercomputers(seeSimulation35.1ontheaccompanyingwebsite).Suchsimulationscanbeusedtopredicttheconformationalflexibilityofdifferentdomainswithinthechainandwhencombinedwithrecentadvancesinprotein-GAGdocking,canprovideadditionalinsightsintoGAG-proteininteractions.

HOWSPECIFICAREGLYCOSAMINOGLYCAN-PROTEIN

INTERACTIONS?

ThediscoveryofmultipleGAG-bindingproteinsledanumberofinvestigatorstoexaminewhetherthereisaconsensusaminoacidsequenceforGAGbinding.Inretrospect,thisstrategywasoverlysimplisticbecauseitassumedthatallGAG-bindingproteinswouldrecognizethesameoligosaccharidesequencewithinheparin,oratleast,sequencesthatwouldsharemanycommonfeatures.WenowknowthatsomeGAG-bindingproteinsinteractwithdifferentoligosaccharidesequences.Thebindingsitesintheproteinalwayscontainbasicaminoacids(lysineandarginine)whosepositivechargespresumablyinteractwiththenegativelychargedsulfatesandcarboxylatesoftheGAGchains.However,thearrangementofthesebasicaminoacidscanbequitevariable,consistentwiththevariablepositioningofsulfogroupsintheGAGpartner.

Mostproteinsareformedfromα-helices,β-strands,andloops.Therefore,toengagealinearGAGchain,thepositivelychargedaminoacidresiduesmustalignalongthesamesideoftheproteinsegment.α-Heliceshaveperiodicitiesof3.4residuesperturn,whichwouldrequirethebasicresiduestooccureverythirdorfourthpositionalongthehelixinordertoalignwithanoligosaccharide.Inβ-strands,thesidechainsalternatesideseveryotherresidue.Thus,tobindaGAGchain,thepositivelychargedresiduesinaβ-strandwouldbelocatedquitedifferentlythaninanα-helix.Onthebasisofthestructureofseveralheparin-bindingproteinsthatwereavailablein1991,AlanCardinandHerschelWeintraubproposedthattypicalheparin-bindingsiteshadthesequenceXBBXBXorXBBBXXBX,whereBislysineorarginineandXisanyotheraminoacid.Fromthestructuralargumentsprovidedabove,itshouldbeobviousthatonlysomeofthebasicresiduesinthesesequencescouldparticipateinGAGbinding,theactualnumberbeingdeterminedbywhetherthepeptidesequenceexistsasanα-helixoraβ-sheet.Wenowknowthatthepresenceofthesesequencesinaproteinmerelysuggestsapossibleinteractionwithheparin(oranotherGAGchain),butitdoesnotprovethattheinteractionoccursunderphysiologicalconditions.Infact,thepredictedbindingsitesforheparininfibroblastgrowthfactor2(FGF2)turnedouttobeincorrectoncethecrystalstructurewasdetermined.Itislikelythatbindinginvolvesmultipleproteinsegmentsthatjuxtaposepositivelychargedresiduesintoathree-dimensionalturn-richrecognitionsite.Inmanycasesthebindinginvolvesloopswhichmakethepositioningmorevariable.AnexampleofthisphenomenonisobservedinthechemokineCCL5,whichcontainsaBBXBmotifinaloop.Thespecificarrangementofresiduesshouldvaryaccordingtothetypeandfinestructureofthoseoligosaccharidesinvolvedinbinding.Inplantandanimallectins,andinantibodiesthatrecognizeglycans,theglycanrecognitiondomainsaretypicallyshallowpocketsthatengagetheterminalsugarsoftheoligosaccharidechain(Chapters29,30and37).InGAG-bindingproteins,theproteinusuallybindstosugarresiduesthatliewithinthechainorneartheterminus.Therefore,thebindingsitesinGAG-bindingproteinsconsistofcleftsorsetsofjuxtaposedsurfaceresiduesratherthanpockets.TheseGAG-bindingsitesontheproteinsurfacegiverisetomorerapidGAG-proteinbindingkineticsthanaretypicallyobservedforprotein-proteininteractions.GiventhatGAGchainsgenerallyexistinahelicalconformation,onlythoseresiduesonthefacetowardtheproteininteractwithaminoacidresidues;theonesontheothersideofthehelixarepotentiallyfreetointeractwithasecondligand(e.g.,asobservedinFGFdimers).Alternatively,residuesinabindingcleftcouldinteractwithbothsidesofthehelix(e.g.indengueenvelopeprotein).Finally,oneshouldkeepinmindthatbindingoccurstoonlyasmallsegmentoftheGAGchain.Thus,asingleGAGchaincanpotentiallybindmultipleproteinligandsfacilitatingcooperativebindingthatcanleadtoproteinoligomerization(e.g.somechemokines).

ANTITHROMBIN-HEPARIN:APARADIGMFORSTUDYING

GLYCOSAMINOGLYCAN-BINDINGPROTEINS

Perhapsthebest-studiedexampleofprotein-GAGinteractionisthebindingofantithrombintoheparinandHS(seecoverimageandFigure38.2).Thisinteractionisofgreatpharmacologicalimportancebecauseheparinisusedclinicallyasananticoagulant.Bindingofantithrombintoheparinhasadualeffect:First,itcausesaconformationalchangeintheproteinandactivationoftheproteaseinhibitingaction,resultingina1000-foldenhancementintherateatwhichitinactivatesthrombinandFactorXa.Second,theheparinchainactsasatemplate,enhancingthephysicalappositionofthrombinandantithrombin.Thus,boththeprotease(thrombin)andtheinhibitorhaveGAG-bindingsites.Heparinactsasacatalystinthesereactionsbyenhancingtherateofthereactionthroughappositionofsubstratesandconformationalchange.Aftertheinactivationofthrombinbyantithrombinoccurs,thecomplexlosesaffinityforheparinanddissociates.Theheparinisthenavailabletoparticipateinanotheractivation/inactivationcycle.Antithrombinisamemberoftheserpinfamilyofproteaseinhibitors,manyofwhichbindtoheparin.Earlystudiesusingaffinityfractionationschemesshowedthatonlyaboutone-thirdofthechainsinaheparinpreparationactuallybindwithhighaffinitytoantithrombin.Comparingthesequenceoftheboundchainswiththosethatdidnotbindfailedtorevealanysubstantialdifferencesincomposition,consistentwiththelaterdiscoverythatthebindingsiteconsistsofonlyfivesugarresidues(Figure38.2)(theaverageheparinchainisabout50sugarresidues).ThisobservationcanbeextendedtovirtuallyallGAG-bindingproteins,inferringthatthebindingsitesrepresentaverysmallsegmentofthechains.CrystalsofantithrombinwerepreparedandanalyzedbyX-raydiffractionto2.6-Åresolution.ThedockingsitefortheheparinpentasaccharideisformedbytheappositionofhelicesAandD,whichbothcontaincriticalarginineandlysineresiduesattheinterface.ThesequenceintheDhelix(124AKLNCRLYRKANKSSKLVSANR145)placesmanyofthepositivelychargedresiduesononefaceofthehelix,inproximitytothearginineresiduesintheAhelix(41PEATNRRVW49)(Figure38.2).ThepentasaccharideissufficienttoactivateantithrombinbindingtowardFactorXa,butitwillnotfacilitatetheinactivationofthrombin.Forthistooccur,alargeroligosaccharideofatleast18residuesisneeded.Asmentionedabove,thrombinalsocontainsaheparin-bindingsite,andthelargerheparinoligosaccharideisthoughttoactasatemplatefortheformationofaternarycomplexwiththrombinandantithrombin.Incontrasttoantithrombin,thrombinexhibitslittleoligosaccharidespecificity.Asmightbeexpected,addinghighconcentrationsofheparinactuallyinhibitsthereaction,becausetheformationofbinarycomplexesofheparinandthrombinorheparinandantithrombinpredominate.Thisimportantprincipleof“activationatlowconcentrationsand

inhibitionathighconcentrations”alsooccursinothersystemswhereternarycomplexesform(Chapters29and30).HeparinisapharmaceuticalformulationproducedbypartialfractionationofnaturalGAGsderivedprimarilyfromporcineintestines(Chapter17).MastcellsareknowntoproduceahighlysulfatedversionofHSthatresemblesheparin;highlysulfated,iduronicacid–richheparinoligosaccharidesarealsopresentinHSisolatedfromothertissuesaswell,especiallytheskin.Althoughheparinhasproventobeofgreattherapeuticuse,itsroleinvivoremainsunclear.Heparinandchondroitinsulfateareoftenfoundinstoragegranulesalongwithbiogenicamines,proteases,andotherproteins,possiblyenablingefficientstorage.Mastcellsdegranulateinresponsetospecificantigenstimulation,resultinginreleaseofstoredheparin,histamine,andproteases.Whenthisoccurs,localanticoagulationmightoccur,butlocalizedcoagulationdefectshavenotbeendescribedinanimalsbearingmutationsthataltermastcellsorheparin.AsmallpercentageofendothelialcellHScontainsantithrombin-bindingsequencesaswell.However,thesebindingsitesappeartobelocatedontheabluminalsideofbloodvessels,andmicelackingthecentral3-O-sulfatedGlcNSunit,ahallmarkoftheantithrombin-bindingsequence(Figure38.2),donotexhibitanysystemiccoagulopathyafterbirth.Nevertheless,antithrombindeficiencycausesmassivedisseminatedcoagulopathy.Perhapsthesefindingsindicatethatlower-affinitybindingsequencesaresufficienttoactivateantithrombin.Thissystemillustratesanimportantcaveat:onecannotnecessarilyascribefunctionstoendogenousproteoglycansbasedontheeffectsofGAGsaddedinvitrotoexperimentalsystems.

FGF-HEPARININTERACTIONSENHANCESTIMULATIONOFFGF

RECEPTORSIGNALTRANSDUCTION

Alargenumberofgrowthfactorscanbepurifiedbasedontheiraffinityforheparin.Theheparin-bindingfamilyoffibroblastgrowthfactorshasgrowntomorethan22membersandincludestheprototypeFGF2,otherwiseknownasbasicfibroblastgrowthfactor.FGF2hasaveryhighaffinityforheparin(Kd~10−9M)andrequires1.5–2MNaCltoelutefromheparin-Sepharose.FGF2haspotentmitogenicactivityincellsthatexpressoneoftheFGFsignalingreceptors(fourFGFRgenesareknownandmultiplesplicevariantsexist).Cell-surfaceHSbindstobothFGF2andFGFR,facilitatingtheformationofaternarycomplex.BothbindingandthemitogenicresponsearegreatlystimulatedbyheparinorHS,whichspromotedimerizationoftheligand-receptorcomplex.ThecostimulatoryroleofHS(andheparin)inthissystemisreminiscentoftheheparin/antithrombin/thrombinstory.Indeed,theminimalbindingsequenceforFGF2alsoconsistsofapentasaccharide.However,thispentasaccharideisnotsufficienttotriggerabiologicalresponse(mitogenesis).Forthistooccur,alonger

oligosaccharide(10mer)containingtheminimalsequenceandadditional6-O-sulfogroupsareneededtobindFGFR.ThesequencethatbindstobothFGF2andFGFRisprevalentinheparinbutrareinHS.TherequirementforthisrarebindingsequencereducestheprobabilityoffindingthisparticulararrangementinnaturallyoccurringHSchains.Thus,somepreparationsofHSareinactiveinmitogenesis,andthosecontainingonlyonehalfofthebipartitebindingsequenceareactuallyinhibitory.ThestructureofFGF2cocrystallizedwithaheparinhexasaccharidehassincebeenobtained(Figure38.3).Theheparinfragment([GlcNS6Sα1-4IdoA2Sα1-4]3)washelicalandboundtoaturn-richheparin-bindingsiteonthesurfaceofFGF2.OnlyoneN-sulfogroupandthe2-O-sulfogroupfromtheadjacentiduronicacidareboundtothegrowthfactorintheturn-richbindingdomain,andthenextGlcNSresidueisboundtoasecondsite,consistentwiththeminimalbindingsequencedeterminedwitholigosaccharidefragments.NosignificantconformationalchangeinFGF2occursuponheparinbinding,consistentwiththeideathatheparinprimarilyservestodimerizeFGF2andjuxtaposecomponentsoftheFGFsignal-transductionpathway.ThecrystalstructureofacidicFGF(FGF1)hasalsobeensolvedandshowssimilarsequencesonitssurface.However,theoligosaccharidesequencethatbindswithhighaffinitytoFGF1contains6-O-sulfogroups.Thecocrystalstructureofthecomplexof(FGF2-FGFR)2,firstsolvedintheabsenceofheparin/HSligand,showedacanyonofpositivelychargedaminoacidresidues,suggestiveofanunoccupiedheparin-bindingsite.Subsequently,theheparin-oligosaccharide-containingcomplexwassolvedafterintroductionofheparinoligosaccharides,suggestinga2:2:2complexofFGF2:FGFR:HS(Figure38.3).Anotherimportantfeatureofthiscomplexistheorientationofthenon-reducingendsoftheHSchainsthatterminateinanN-sulfoglucosamineresidue,whicharisesbyendolyticcleavageofchainsbytheenzymeheparanase(Chapter17).ThestructureoftheFGF-FGFR-HScomplexisnotwithoutcontroversy;structuralanalysisofcomplexesformedinsolutionandpurifiedbygelfiltrationhassuggestedaverydifferentstructureconsistingofa2:2:1complex(Figure38.3).

OTHERATTRIBUTESOFGLYCOSAMINOGLYCAN-PROTEIN

INTERACTIONS

Insomecases,theinteractionofGAGchainswithproteinsmaydependonmetalcofactors.Forexample,L-andP-selectinshavebeenshowntobindtoasubfractionofHSchainsandheparininadivalent-cation-dependentmanner.Thisobservationraisesthepossibilitythatotherexamplesofcation-dependentinteractionswithGAGchainsmayexist.GAGbindingtoL-selectinhelpsinleukocyterolling.Furthermore,theinteractioncanbepharmacologicallymanipulatedbyexogenousheparin,includingchemicallymodifiedderivativesthatlackanticoagulantactivity.

CSproteoglycansinthecentralnervoussystem(CNS)influencecellmigrationandaxonpathfindingandregulateneuriteoutgrowth.Theinteractionofrare,highlysulfateddisaccharidesequencesinCSchainswithmorphogensandgrowthfactorsimpactCNSdevelopmentandplayrolesinCNSpathology.HSproteoglycansareoftenexpressedinaspatiallyandtemporallylimitedfashion.ThetemporaryplacementofanHSproteoglycanataspecifictissuesitemightormightnotcoincidewiththepresenceofitsappropriateproteinligand.Furthermore,ifthebindingpartnerhasnoaccesstotheHSproteoglycan,itcannotinteract—addinganadditionallevelofspecificity.RecentstudiesdemonstratethatthefinestructureofHSchainsalsochangesduringdevelopment,thusenablingordisablingspecificassociationsbetweenligandsandreceptors.Gradientsofmorphogens,factorsthatdeterminecellfatesbasedonconcentration,alsodeterminethepatternsofcellandtissueorganizationduringdevelopment(Chapter27).Themechanismofmorphogengradientformationiscontroversial,butinterestingly,virtuallyallmorphogenscaninteractwithheparinandHS.Theseinteractionscanaffecttransportofligands,receptorinteractions,endocytosis,anddegradation,whichtogethermayhavearoleindeterminingtherobustnessofthegradient.TheGAGchainsofproteoglycansalsoofferalineardomainoverwhichproteinscandiffuse.Bylimitingthespaceavailabletotheseproteinsfromthethree-dimensionalspaceofextracellularfluidsandtheextracellularmatrixtoone-dimensionalspacealongthechains,thechanceofencountersamongheparin-bindingproteins,suchasFGFanditsreceptor(FGFR),maybeenhanced.Thus,thecriticalroleofHSproteoglycansmaybeincontrollingthekineticsofprotein–proteininteractionsratherthanthethermodynamicsofsuchencounters.

ACKNOWLEDGEMENTS

TheauthorsappreciatehelpfulcommentsandsuggestionsfromKristianSaied,EathenRyan,PatienceWrightandKristinStanford.

FURTHERREADING

LiW,JohnsonDJ,EsmonCT,HuntingtonJA.2004.Structureoftheantithrombin-thrombin-heparinternarycomplexrevealstheantithromboticmechanismofheparin.NatStructMolBiol11:857-862.

MohammadiM,OlsenSK,GoetzR.2005.AproteincanyonintheFGF-FGFreceptordimerselectsfromanalacartemenuofheparansulfatemotifs.CurrOpinStructBiol15:506-516.

DuchesneL,OcteauV,BearonRN,BeckettA,PriorIA,LounisB,FernigDG.2012.Transportoffibroblastgrowthfactor2inthepericellularmatrixiscontrolledbythespatialdistributionofitsbindingsitesinheparansulfate.PLoSBiol10:e1001361.

KamhiE,JooEJ,DordickJS,LinhardtRJ.2013.Glycosaminoglycansininfectiousdisease.BiolRevCambPhilosSoc88:928-943.

ThackerBE,XuD,LawrenceR,EskoJD.2013.Heparansulfate3-O-sulfation:Araremodificationinsearchofafunction.MatrixBiol35:60-72.

XuD,EskoJD.2014.Demystifyingheparansulfate-proteininteractions.AnnuRevBiochem83:129-157.

MizumotoS,YamadaS,SugaharaK.2015.Molecularinteractionsbetweenchondroitin-dermatansulfateandgrowthfactors/receptors/matrixproteins.CurrOpinStructBiol34:35-42.

PominVH,MulloyB.2015.Currentstructuralbiologyoftheheparininteractome.CurrOpinStructBiol34:17-25.

SmithPD,Coulson-ThomasVJ,FoscarinS,KwokJC,FawcettJW.2015."GAG-ingwiththeneuron":Theroleofglycosaminoglycanpatterninginthecentralnervoussystem.ExpNeurol274:100-114.

DeshauerC,MorganAM,RyanEO,HandelTM,PrestegardJH,WangX.2015.InteractionsofthechemokineCCL5/RANTESwithmedium-sizedchondroitinsulfateligands.Structure(London,England:1993)23:1066-1077.

FigureLegends

FIGURE38.1.Conformationofheparinoligosaccharides.(A)Glucosamine(GlcN)andglucuronicacid(GlcA)existinthe4C1conformation,whereasiduronicacid(IdoA)existsinequallyenergeticconformationsdesignated1C4and2S0.(B)Space-fillingmodelofaheparinoligosaccharide(14mer)deducedbynuclearmagneticresonance.(C)Thesamestructureinstickrepresentation.TherenderingsinBandCweremadewithRASMOLusingdatafromtheMolecularModelingDatabase(MMDBId:3448)attheNationalCenterforBiotechnologyInformation(NCBI).

FIGURE38.2.Crystalstructureoftheantithrombin-pentasaccharidecomplex(fromProteinDataBank).(A,D)α-Helicesthatmakecontactwithheparin;(RCL)thereactivecenterloopthatinactivatesthrombinandFactorX;(F)anotherα-helixintheprotein.(Lowerpanel)Interactionsbetweenkeyaminoacidresiduesandindividualelementsinthepentasaccharide.(Solidlines)Electrostaticinteractionsbetweenpositivelychargedresiduesandsulfategroups;(brokenlines)hydrogenbonds;(alternatelybrokenandsolidline)bridgingwatermolecule.

FIGURE38.3.CrystalandNMRsolutionstructuresofGAG-proteincomplexes.(A)Crystalstructureofthe2:2:2FGF2:FGFR1:heparincomplex(sideview)anda2:2:1complex;(B)StructureofthedimericV-C1domainsofRAGE(receptorforadvancedglycationendproducts)(PDB4IM8).Thedodecasaccharideismanuallymodeledintothestructureonthebasisoftheobservedpartialelectrondensity;(C)StructureofthedimericE2domainofamyloidprecursor–likeprotein1(APLP-1)andboundoligosaccharide(PDB3QMK);(D)Structureofdimericinterleukin-8(PDB2IL8)andamodeledoligosaccharide(degreeofpolymerization:20);(E)ArepresentativeframefromthemostenergeticallyfavoredmodelsoftheCCL5-chondroitinsulfatecomplexdeducedbyNMR.Theribbonrepresentationofthecomplexwithsidechainsofselectiveaminoacidsisshowningray.Thechondroitin-4-sulfate(dp6)ligandisshowninthestickrepresentationwiththenon-reducingendandthereducingendsugarlabeledGlcA1andGalNAc3,respectively(fromDeshaueretal.(2015)Structure23,1066–1077)

TABLE38.1Examplesofglycosaminoglycan-bindingproteinsandtheirbiologicalactivity

Class ExamplesPhysiological/pathophysiologicaleffectsofbinding

Enzymes glycosaminoglycanbiosyntheticenzymes,thrombinandcoagulationfactors(proteases),complementproteins(esterases),extracellularsuperoxidedismutase,lipases

multiple

Enzymeinhibitors

antithrombinIII,heparincofactorII,secretoryleukocyteproteinaseinhibitor,C1-esteraseinhibitor

coagulation,inflammation,complementregulation

Celladhesionproteins

P-selectin,L-selectin,someintegrins

celladhesion,inflammation,metastasis

Extracellularmatrixproteins

laminin,fibronectin,collagens,thrombospondin,vitronectin,tenascin

celladhesion,matrixorganization

Chemokines plateletfactorIV,γ-andβ-interferons,interleukins

chemotaxis,signaling,inflammation

Growthfactors fibroblastgrowthfactors,hepatocytegrowthfactor,vascularendothelialgrowthfactor,insulin-likegrowthfactor–bindingproteins,TGF-β-bindingproteins

mitogenesis,cellmigration

Morphogens hedgehogs,TGF-βfamilymembers,wnts

cellspecification,tissuedifferentiation,development

Guidancefactors

Slits,ROBOreceptors,neuropilins

axonguidance,endothelialtubeformation

Tyrosine-kinasegrowthfactorreceptorsandcoreceptors

fibroblastgrowthfactorreceptors,vascularendotheliumgrowthfactorreceptor,receptorforadvancedglycationendproducts(RAGE),receptorproteintyrosinephosphatases(RPTPs)

Mitogenesis,axonguidance,inflammation

Lipid-bindingproteins

apolipoproteinsEandB,lipoproteinlipase,hepatic

lipidmetabolism,cellmembranefunctions

lipase,annexinsPlaqueproteins

prionproteins,amyloidproteins

plaqueformation

Nuclearproteins

histones,transcriptionfactors

unknown

Pathogensurfaceproteins

malariacircumsporozoiteprotein

pathogeninfections

Viralenvelopeproteins

herpessimplexvirus,denguevirus,humanimmunodeficiencyvirus,hepatitisCvirus,vacciniaviruscomplementcontrolprotein(VCP)

viralinfections

TABLE38.2Methodstomeasureglycosaminoglycan-proteininteractionMethod Type Throughput PrincipleAffinitychromatography

M H/I immobilizedligandorglycosaminoglycanchainsoncolumnmatrix

Affinitycoelectrophoresis

M/S I gelretardationthroughprotein-impregnatedgel

Analyticalultracentrifugation

S L equilibriumsedimentationatdifferentcarbohydrate:proteinratios

Circulardichroism S I/L changeinrotationofplane-polarizedlightuponbinding

CompetitionELISA M H solution-andsolid-phaseligandscompeteforbinding

Computational S L calculatescomplexstructureandbindingenergy

Fluorescencespectroscopy

S H conformationalchange,ligandbindinginduceschangeinfluorescence

Ionmobilitymassspectrometry

G I/L Measurescomplexshapeandstoichiometry

Isothermaltitrationcalorimetry

S I measuresenthalpyofbindingdirectlyandKdvalues

Laserlightscattering S I intrinsicscatteringintensitiesofcarbohydrate-proteincomplexusedtocalculatestoichiometry

Nuclearmagneticresonance

S L chemicalshift,couplingconstant,andnuclearOverhausereffecttodeterminecontactpoints,distances,andconformation

Surfaceplasmonresonance

M H/I mass-inducedrefractiveindexchangeinrealtimefordirectmeasurementofassociationanddissociationrateconstants

Xray M L solidstatecocrystalstructure(M)Mixedphase;(S)solutionphase;(G)gasphase;(H)high;(I)intermediate;(L)low.

Lectin (handout)