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Transcript
Chapter 28 Discovery and Classification of GlycanBinding Proteins Essentials of Glycobiology 3rd edition DiscoveryandClassificationofGlycan-BindingProteins Glycansserveavarietyofbiologicalfunctionsbyvirtueoftheirmass,shape,charge, andotherphysicalproperties.Manyoftheirmorespecificbiologicalrolesare mediatedviarecognitionbycomplementaryglycan-bindingproteins(GBPs).Nature hastakenadvantageofthediversityofglycansexpressedinorganismsbyevolving proteinmodulestorecognizediscreteglycansthatmediatespecificphysiologicalor pathologicalprocesses.Thischapterprovidesageneralclassificationandoverview ofnaturallyoccurringGBPs,thehistoryoftheirdiscovery,someoftheirbiological functionsandwaysinwhichnewGBPsareidentified.Chaptersthatfollowdescribe theanalysisofglycan–proteininteractions(Chapter29),thephysicalprinciples involved(Chapter30)andthestructuresandbiologicalfunctionsofseveralGBPs subclasses(Chapters31-38). TWODISTINCTCLASSESOFGBPs GBPsarefoundinalllivingorganisms,andfallintotwooverarchinggroups–lectins andsulfatedglycosaminoglycan(GAG)-bindingproteins(onlineAppendix28A). Lectinsarefurtherclassifiedintoevolutionarily-relatedfamiliesidentifiedby “carbohydrate-recognitiondomains”(CRDs)basedonprimaryaminoacidand/or three-dimensionalstructuralsimilarities(Figure28.1).CRDscanexistasstandaloneproteinsorasdomainswithinlargermulti-domainproteins.Theytypically recognizeterminalgroupsonglycans,whichfitintoshallowbutwell-defined bindingpockets(Chapters29,30).Incontrast,proteinsthatbindtosulfatedGAGs (heparan,chondroitin,dermatanandkeratansulfates,Chapter17)dosoviaclusters ofpositivelychargedaminoacidsthatbindspecificarrangementsofcarboxylicacid andsulfategroupsalongGAGchains(Chapter38).Mostoftheseproteinsare evolutionarilyunrelated.GBPsthatbindtothenon-sulfatedGAGhyaluronicacid (hyaladherins)shareanevolutionarilyconservedfoldthatbindstoshortsegments oftheinvarianthyaluronanrepeatingdisaccharide(Chapter16),soarebest classifiedaslectinsratherthangroupedwithsulfatedGAG-bindingproteins.The restofthischapterconsiderslectins,differentfamiliesofwhicharedetailedin Chapters31-37. DISCOVERYANDHISTORYOFLECTINS Lectinswerediscoveredinplantsin1888whenextractsofcastorbeanseedswere foundtoagglutinateanimalredbloodcells.Subsequentlyseedsofmanyplantswere foundtocontainsuch"agglutinins",laterrenamedlectins(Latinfor“select”)when theywerefoundtodistinguishhumanABObloodgroups(Chapter14)importantfor bloodtransfusions.Lectinsareparticularlycommonintheseedsofleguminous plantsandthese"L-type"lectins,includingconcanavalinAandphytohemagglutinin, havebeenextensivelystudied.Althoughtheirspecificglycan-bindingactivities makeplantlectinsextremelyusefulscientifictools,theirbiologicalfunctionsin plantsremainmostlyunknown. Thefirstanimallectindiscoveredwastheasialoglycoproteinreceptor(ASGPR) identifiedbyAnatolMorellandGilbertAshwellinthelate1960’sduringtheir investigationsoftheturnoverofaserumglycoprotein,ceruloplasmin.Likemost glycoproteinscirculatinginblood,ceruloplasminhascomplexN-glycanswithsialic acidtermini.Toprepareradiolabeledceruloplasmin,theterminalsialicacidswere removed,leavinganexposedgalactose.Surprisingly,asialoceruloplasminhada circulationhalf-life(inrabbits)ofminuteswhereasintactceruloplasminremained inthebloodforhours.GlycoproteinswithexposedGalresidueswererapidly clearedintolivercellsviaanendocyticcellsurfacereceptorthatspecificallybound toterminalβ-linkedGalorGalNAc.ASGPRwaspurifiedbyaffinitychromatography usingacolumnofimmobilizedasialoglycoprotein. Otherglycan-specificreceptorsinvolvedinglycoproteinclearanceandtargeting weresubsequentlydiscovered,includingmannose6-phosphatereceptorsfor targetinglysosomalenzymestothelysosomesandmannosereceptorsthatclear glycoproteinswithterminalmannoseorGlcNAcresiduesfromtheblood.Small solublelectinsspecificforβ-linkedgalactose(nowcalled“galectins”,Chapter36) wereisolatedbyaffinitychromatographyinextractsfrommanybiologicalsources rangingfromtheslimemoldDictyosteliumdiscoideumtomammaliantissues.Bythe 1980’s,theconceptofvertebratelectinsthatrecognizespecificglycanswaswell established.Althoughthefirstanimallectinsidentifiedwerespecificforendogenous glycans,manylectinsspecificforexogenousglycansofmicroorganismswerelater found.Lectinsrecognizingexogenousglycansincludesolubleproteinsthatcirculate inthebloodofmanyspeciesaswellasmembrane-boundreceptorsoncellsofthe immunesystem. Lectinsarealsowidespreadinmicroorganisms,althoughtheytendtobecalledby othernamessuchashemagglutininsandadhesins.Theinfluenzavirus hemagglutinin,whichbindstosialicacidonhostcells(Chapter15)wasthefirstGBP isolatedfromamicroorganism.Theviralhemagglutinins,likemanyplantlectins, canagglutinateredbloodcells.Manybacteriallectinshavebeendescribed.Theyfall intotwogeneralclasses:adhesinsonbacterialsurfacesthatrecognizeglycanson hostcellmembraneglycolipidsorglycoproteinstofacilitatebacterialadhesionand colonization,andsecretedbacterialtoxins(Chapter37). DISCOVERYOFSULFATEDGAG-BINDINGPROTEINS AlargegroupofGBPsthatdefyclassificationbasedonsequenceorstructure recognizesulfatedGAGs(Chapter38).Thebest-studiedexampleistheinteractionof heparinwithantithrombin.Heparinwasdiscoveredin1916byJayMcLean,a medicalstudent,butitwasnotuntil1939thatheparinwasshowntobean anticoagulantinthepresenceof“heparincofactor”,whichwasthenidentifiedas antithrombininthe1950s.ManyothersulfatedGAG-bindingproteinswerelater discoveredbyaffinitychromatographyoncolumnsofimmobilizedheparin.Growth factorsandcytokinesbearingclustersofpositivelychargedaminoacidsalongtheir proteinsurfaceinteractwithsulfatedGAGsinalooserfashion—i.e.,theydonot alwaysshowthehighspecificityseenwithantithrombin.However,insomecases, specificGAGsequencesmediatetheformationofhigher-ordercomplexes,actingas atemplateforoligomerizationorpositioningofproteinssuchasFGFanditscell surfacereceptor. MAJORBIOLOGICALFUNCTIONSOFGBPs GBPsfunctionincommunicationbetweencellsinmulticellularorganismsandin interactionsbetweenmicrobesandhostsandcanalsobeinvolvedinbinding growthfactorsorcytokines.Theseinteractionscantakevariousforms,resultingin movementofmolecules,cells,andinformation. Trafficking,targetingandclearanceofproteins Directingmovementofglycoproteinswithinandbetweencellsisacommon functionforlectinsinmanyorganisms.Ineukaryoticcells,includingyeastaswellas highereukaryotes,severalgroupsoflectinsareimportantinglycoprotein biosynthesisandintracellularmovement(Chapter39).Intheendoplasmic reticulum(ER),twolectins,calnexinandcalreticulinbindmonoglucosylatedhigh mannoseglycanspresentonnewlysynthesizedglycoproteins,formingpartofa qualitycontrolsystemforproteinfolding.Bindingtocalnexinorcalreticulinkeeps proteinsintheERuntiltheyarecorrectlyfolded.OthergroupsoflectinsintheER, includingM-typelectinsandproteinscontainingmannose6-phosphatereceptor homologydomainstakepartintheprocessofER-associatedglycoprotein degradation(ERAD),bindingpartiallyprocessedhighmannoseglycanson terminallymisfoldedglycoproteins,causingthemtoberetrotranslocatedintothe cytoplasmfordeglycosylation,followedbydegradationintheproteasome.Oneof thebestcharacterizedfunctionsofGBPsisindeliveryofnewlysynthesized lysosomalenzymesfromthetrans-GolgitolysosomesbyP-typelectins(Chapter33) thatrecognizemannose6-phosphateresiduesthathavebeenaddedtoN-glycanson lysosomalenzymesintheGolgiapparatus,targettingthemtoendosomesforfusion withlysosomes. Oncereleasedfromcells,glycoproteinscanalsobetakenupfordegradationin lysosomes.Asnotedabove,theASGPRonmammalianhepatocytescontrols turnoverofmanyserumglycoproteinsbyrecognitionofterminalGalorGalNAc residues.Similarly,themannosereceptoronmacrophagesandsinusoidalcellsof theliverbindsandclearsglycoproteinswitholigomannoseN-glycansthatare releasedfromcellsduringinflammationandtissuedamage. NotallGBP-mediatedtargetingleadstodegradation.Glycan-bindingsubunitsof secretedbacterialandplanttoxinstargetthemtoglycolipidsoncellsurfacesand facilitateentryofthetoxinsintocells(Chapter37).Manyenzymescontainglycanbindingdomainsthatbringanotherdomainwithenzymeactivityintoclose proximitywithitssubstrates.Onenotablegroupincludesbacterialcellulasesin whichcellulose-bindingmodulespositiontheenzymaticdomainforoptimal degradationofcellulosefibers.Usingasimilarprinciple,GalNAc-bindingdomainsin polypeptide-N-acetylgalactosaminyltransferasesthatinitiateO-linkedglycosylation inanimalspositiontheseenzymestoaddfurtherGalNAcresiduestoregionsof polypeptidesthatalreadybearO-glycans(Chapter10). Celladhesion Distinctiveglycansonthesurfacesofdifferentcells,botheukaryoticand prokaryotic,makethemtargetsforGBPs.Bindingofglycansonthesurfaceofone cellbyGBPsonanothercellcaninducerecognitionandadhesion,whereas crosslinkingglycansondifferentcellsbymultivalentsolubleGBPsprovidesan alternativemechanism.Suchinteractionsareexploitedinspecializedsituations exemplifiedbytransientcontactsbetweenmovingcells.Theselectins,three receptorsthatfunctionininteractionsbetweenwhitebloodcells,plateletsand endothelia,providethebestcharacterizedexampleoflectin-glycaninteractionsin cell-celladhesion(Chapter34).Forexample,L-selectinonlymphocytesbinds glycansonthespecializedendothelialcellsoflymphnodestoinducelymphocytehoming,whereincirculatinglymphocytesleavethebloodstreamandenterthe lymphnode.OthermammalianGBPsthatmediatebindingofcellstoeachotheror thatcrosslinkligandsonthesamecellsurfaceincludeSiglecs(Chapter35)and galectins(Chapter36).Lectinsinmulticellularorganismsalsoforminteractions betweencellsandtheextracellularmatrixandsupporttheorganizationofmatrix components.Forexample,proteinscontaining“linkmodules”thatbindspecifically tohyaluronanincartilage(andothertissues)areessentialforstructuringthe extracellularmatrix(Chapter38)andotherextracellularproteinsbindtosulfated GAGstoorganizecell-cellandcell-matrixinteractions(Chapter38). Manybacteriaalsouselectinstoadheretoglycansonhostcellsinsituationsin whichtheywouldotherwisegetwashedaway.Thelectinsareusuallypresentatthe endsoflongstructurescalledpiliorfimbriaethatprojectfromthesurfaceofthe bacteria(Chapter37).Adhesioncanbepartoftheinfectionprocess.Forexample,a mannose-specificadhesinonpathogenicstrainsofEscherichiacolithatcause urinaryinfectionsbindstoepithelialcellsoftheurinarytract.Otherglycan-protein interactionsbetweenhostcellsandbacteriaprovideanormalmechanismofcoexistence.Severalbacterialspeciesthatarepartofthenormalgutfloraincluding non-pathogenE.coliuseadhesinstobindtoglycolipidspresentoncellsliningthe largeintestine. Immunityandinfection Manylectinsareinvolvedinimmuneresponses,in“lower”vertebratesand invertebratesaswellasinmammals.Differencesinglycansonhostandmicrobial cellsurfacesarecommonlythebasisforinnateimmuneresponses.Phagocytosisisa commonoutcomeofthebindingofglycan-specificreceptorsonmacrophagesto glycanscommontobacteria,fungiandviruses.Otherlectinscirculatingintheblood, suchasserummannose-bindingproteinandficolins,bindtopathogencellsurfaces andactivatethecomplementcascade,leadingtocomplement-mediatedkilling. Bindingofglycanstolectinsonimmunecellscanalsotriggerintracellularsignaling thatactivatesorsuppressescellularresponses.Receptorsthatrecognizeselfglycanssuchassialicacid,aswellasseveralthatarespecificforglycans characteristicofmicro-organismscaninitiatesuchsignaling.Forexample,binding ofα2-6linkedsialicsacidtoCD22,amemberoftheSiglecfamilyofvertebrate lectinsfoundonBlymphocytes,initiatessignalingthatinhibitsactivationtoprevent self-reactivity(Chapter35).Incontrast,bindingoftrehalosedimycolate,aglycolipid foundinthecellwallofMycobacteriumtuberculosistothemacrophageC-typelectin mincle,inducesasignalingpathwaythatcausesthemacrophagetosecrete proinflammatorycytokines. Finally,virusesoftenuseGBPstoattachtohostcellsduringinfection(Chapter37). Proteinsonvirussurfaces,includingthoseoninfluenzavirus,reovirus,Sendaivirus, andpolyomavirus,bindtosialicacids.Inadditiontobringingthevirusintocontact withtheircelltargets,thesehemagglutininstypicallyinducemembranefusion, facilitatingvirusentryanddeliveryofnucleicacidsintothecytosol.Glycan-binding receptorsonvirusesareoftenhighlyspecificforaparticularlinkage;human influenzavirusbindstosialicacidslinkedα2-6toGal,whereasbirdinfluenzavirus bindstoα2-3linkedsialicacid.Otherviruses,suchasherpessimplexvirus,have GAG-bindingproteinsthatbindtoheparansulfateproteoglycansoncellsurfaces. ORGANIZATIONOFLECTINS Animportantconceptinidentifying,definingandclassifyinglectinsisthatsugarbindingactivityisembodiedindiscreteproteinmodulesordomains,referredtoas carbohydrate-recognitiondomains(CRDs).CRDsaretypicallyindependentlyfolding segmentsofproteins;oftenonecanseparatethesugar-bindingactivityfromother activitiesoftheproteinbyexpressingitsCRDinisolation.Insomecases,theCRDs constitutetheentireGBP(Figure28.2). WhenalectiniscomprisedsimplyofitsCRD,itsfunctionsoftenaredependenton multivalency,whichendowslectinswiththeabilitytocross-linksugar-containing structures.Thisarrangementexplainstheabilityofmanyplantlectinstoagglutinate cellsandtoclusterglycoproteinsoncellsurfaces,whichcaninducemitogenesis. OtherGBPsthatfunctionthiswayincludethegalectins,whichcanbridgeglycanson onecellsurfaceorbetweencells.Sometimesotheractivitiesareencodedwithinthe structureofthesamedomainthatbindssugars;somecytokinescomprisedofa singlefoldeddomainmayhavedistinctsitesforbindingglycansandothertarget receptors.Morecommonly,otheractivitiesoflectinsresideinseparatemodulesin multi-domainproteins(Figure28.2).Sucharrangementsarewidespreadandthe domainsassociatedwithCRDsperformmanydifferentfunctions,includingbinding othertypesofligands,performingenzymaticreactions,anchoringproteinsto membranesanddirectingoligomerization.GBPsoftencontainmultiplemodules, combiningseveralfunctionsinoneprotein. Membraneanchorsinlectinscantakemultipleforms,buttheyoftenspanthe membrane,linkingextracellularCRDswithcytoplasmicdomains.Thisarrangement facilitatestheflowofinformationbetweenglycan-bindingsitesontheextracellular surfaceandthecytoplasm.Simplesequencemotifsinthecytoplasmicdomainsof transmembranelectinsoftencontroltraffickingofreceptorsandtheirboundglycan ligands.Commonfunctionsofsuchintracellularmovementsareinternalizationof cellsurfacereceptors,directingboundligandstoendosomesandlysosomes,and movementthroughintracellularcompartmentssuchastheendoplasmicreticulum andGolgiapparatustothecellsurface.Flowofinformationintheoppositedirection canleadtostimulationofsignalingcomplexesonthecytoplasmicsideofthe membraneinresponsetobindingofglycansatthecellsurface. Clusteringofglycan-bindingsites(multivalency)isoftencriticaltobothrecognition andbiologicalfunctionsoflectins.Clusteringofsitesisachievedindifferentways, byformationofsimpleoligomersofCRDs,asaresultofthepresenceofmultiple CRDsinasinglereceptorpolypeptideandthroughassociationofCRD-containing polypeptidesthroughindependentoligomerizationdomains.Someoligomersare stable,whileothers,suchasthoseformedbysomegalectins,areinequilibriumwith monomers.Thesearrangementsfacilitatemultivalentbindingtoincreaseavidity anddirectthegeometricalarrangementofbindingsites.MultipleCRDsmayfacein thesamedirectionforsurfacerecognitionorinoppositedirectionstofacilitate crosslinking.MultivalentCRDsmayhavefixedspacingorflexiblespacingto accommodatedifferenttargetglycans.Insomecases,oligomerizationdomainsalso formstructuralfeatures,servingsasstalksthatprojectCRDsfromthecellsurface. Oligomerizationdomainscanalsoembodyotherfunctions,suchastheproteasebindingsitesinthecollagen-likedomainsofmannose-bindingprotein. CLASSIFICATIONOFLECTINSBASEDONSTRUCTURAL SIMILARITIES ItisconvenienttoclassifylectinsbasedonthestructuresoftheCRDsthatthey contain(Figure28.3).CRDsarefoundinalargenumberofdifferentstructural categories,indicatingthatmanydifferentproteinfoldscanaccommodateglycan binding(Chapter30).Basedonthisobservation,sugar-recognitionmusthave evolvedindependentlymanytimesandthediversityofCRDstructuresmusthave arisentoaddressadiversityoffunctions. GBPsappearacrossallkingdomsoflife,butthetypesoflectinsineachkingdom varyconsiderably.Severalfamiliesappearinbothprokaryotesandeukaryotes,but theirdistributionssuggestdifferentevolutionaryhistories.Themalectindomain, althoughconservedinstructureandwidelydistributedinprokaryotes,plantsand animals,isfoundinproteinswithdistinctdomainorganizationanddifferent functionsinthethreegroups.Animalmalectinisamembrane-anchoredCRDofthe endoplasmicreticulumthatbindsN-linkedglycansduringglycoprotein biosynthesis.Inplants,themalectinCRDisexpressedatthecellsurfaceandis linkedtoacytoplasmickinasedomain.BacterialmalectinsconsistofCRDs associatedwithglycohydrolasedomains.Similarly,R-typeCRDs(Chapter31)in plantsformthecellsurface-bindingcomponentoftoxinssuchasricinandarelinked toglycohydrolasegenesinbacteria,butinanimalstheyappearintwodistinct contexts:inpolypeptide-N-acetylgalactosaminyltransferasesthatinitiateO-GalNAc glycans(Chapter10)andinthemannosereceptorfamily.AlthoughtheseCRDshave beenadaptedtoservedifferentfunctionsindifferentkingdoms,asugar-binding functionappearstohaveevolvedearlyandbeenpreservedinsubsequentlineages. IncontrasttoCRDswithbroadevolutionarydistribution,twoothergroupsof lectinshavesporadicdistributions.B-lectindomainsarebroadlydistributedin bacteriainassociationwithhydrolasedomains,arefoundasisolatedortandem CRDsinmonocotplantsbutnotinotherplants,inbonyfishesbutnotinother animals,andinandsomefungi.TheF-typelectinsappearinbacteriaandina limitednumberofanimalspecies.Inthesecases,thepresenceofrelateddomainsin evolutionarilydistantspeciesmayreflectlateralgenetransferratherthanthe presenceofaprecursorlectininthedistantcommonancestorthattheyshare.A differentpatternofevolutionisobservedforPA14domains,theonlyothertypeof CRDfoundinbothbacteriaandeukaryotes.AlthoughthePA14foldisrelatively widespread,suggestingthatitoriginatedearlyandwasretainedacrossspecies,only asubsethavebeenshowntohavesugar-bindingactivity:CRDsassociatedwith bacterialglycohydrolasesandinadhesinsandflocculationfactorsonthesurfaceof yeast. Theintracellularsortinglectinsmentionedearlier,suchascalnexin,calreticulin,and M-typelectins,arethemostbroadlydistributedlectinsthatevolvedfromacommon eukaryoticancestor.Theirdistributionandtheconservationoftheirfunctions probablyreflectanancientandconservedroleinintracellulartraffickingof glycoproteinsineukaryotes.TwoothergroupsofCRDsappeartobefoundin metazoansbutnotsimplereukaryotes.TheL-typeCRDshavedivergedinfunction betweenanimals,wheretheyfunctioninintracellularglycoproteinsortingand trafficking,andplants,wheretheyserveaprotectivefunction(Chapter32). Chitinase-likesugar-bindingdomainsacrossarangeofspeciesretaintheabilityto bindpolymersofGlcNAc,buttheirbiologicalfunctionsarenotwellunderstood,soit isuncleariftheyhavesharedrolesinplantsandanimals. Inadditiontothewidelydistributedfamilies,certainCRDfamiliesareevolutionarily restricted.Inadditiontoanimal-specificandvertebrate-specificlectingroups,there arealsogroupssuchastheI-typelectinsfoundonlyinmammals(Chapter35).The patternofevolutionofanimal-specificlectinsvaries.Galectinsseemtobesimilarin organizationinvertebratesandinvertebratesanditmaybepossibletoidentify orthologsinquitediversespecies(Chapter36).Incontrast,C-typeCRDshave undergoneindependentradiationinvertebratesandinvertebrates,andidentifying orthologsevenbetweenmouseandhumanproteinsinsomecasesisdifficult (Chapter34).Ofthetwelvedifferentproteinfoldsfoundinplantlectins,nineappear tobeuniquetoplants.Itisalsonoteworthythatvirusesseemtohavedeveloped theirownapproachestobindingglycansratherthanborrowingfromhosts(Chapter 37). InadditiontofamiliesofproteinsthatshareevolutionarilyrelatedCRDs,thereare individualproteinsthatbindsugarsthroughdomainsthatarenotrelatedtoCRDsin otherproteins.Examplesincludeproteinswithdedicatedsugarbindingdomains, suchassomelamininGdomains,whichrecognizeglycansonα-dystroglycan (Chapter45),pentraxins,whichbindmodifiedandphosphorylatedsugars,and macrophageαMβ2integrin,whichbindsfungalglucansandexposedGlcNAcresidues onglycoproteins.Otherproteinsbindtosugarsthroughdomainsthatalsohave otherligands:annexinVbindsbisectingGlcNAcresiduesaswellasphospholipids andseveralcytokineshavebeenreportedtobindsugarsaswellastheirtarget receptors.Sulfated-GAGbindingproteinshavealsolargelyevolvedbyconvergent evolution. IDENTIFYINGGBPsBYBIOLOGICALANDBIOCHEMICALFUNCTION ANDSTRUCTURALSIMILARITY Therearemultiplewaysinwhichglycanrecognitioncanbeimplicatedinspecific biologicalprocesses.Onecommonapproachistodemonstratetheabilityofsimple monosaccharidesorsmallglycanstocompetewithaprocess.Informationcanoften alsobegainedbymodifyingsugarsoncellsandglycoproteinswithenzymesthat addorremovesugars,bygeneticmanipulation,andbychemicalinhibitorsofglycan metabolism.Thesestrategieshaveprovidedinformationabouttheglycansinvolved, forexamplethoseneededforvirusortoxinbindingorthoserequiredfor endocytosisofglycoproteins.Basedonthisinformation,itisthenpossibletolook forGBPsthattargettheseparticularsugarsandwhichcanthenbelinkedtothe biologicalprocess. Theabilitytobindspecificsugars,assessedinvariousbiochemicalassays,hasoften beenthebasisfordirectidentificationofnovelGBPswithoutreferencetoa particularbiologicalfunction.Inadditiontoformingabasisforbindingand competitionassays,thebindingactivityiscommonlyusedasameansofisolating theseproteinsbyemployingaffinitychromatographyonappropriateimmobilized glycanligands.Awidevarietyofmethodsforcouplingmonosaccharidesand complexglycanstocreateaffinityresinshavebeendeveloped.Asmentionedabove, manysulfated-GAGbindingproteinshavebeendiscoveredbyaffinity chromatographyonimmobilizedGAGchains.Alimitationoftheseapproachesis thatbindingactivitydoesnotdirectlyindicateabiologicalfunctionandtherolesof manywell-characterizedGBPshavenotbeenfullydetermined. Theobservationthatmanylectinsfallintostructuralfamiliesprovidesan alternativewaytoidentifynovelGBPsthroughanalysisofproteinsequences. SequencemotifscharacteristicofCRDsareroutinelyusedtoscreensequencesfrom wholegenomesequencing.ThesemotifscanalsobeusedtoscreenspecificcDNA andgenesequencesofinterestbecauseoftheirassociationwithbiological functions.Detectionofanappropriatemotifsuggeststhepresenceofafunctional CRD,andstructuralknowledgeofknownsugar-bindingsitescansuggestwhethera novelproteinislikelytoretainglycan-bindingactivity.Insomecases,itcaneven suggestpotentialligands.Suchpredictionsoftenmotivatetestingforsugarbinding activity,eitherbyspecificallyexaminingbindingtopredictedligandsorby screeningmoregenerallyusingglycanarrays. Althoughstructure-basedpredictionsdonotdirectlyyieldinformationabout biologicalfunction,theorganizationofCRDsandtheirassociationwithother domainsoftenprovideinformationaboutpotentialfunctions.Thistypeoftop-down analysisislimitedtodiscoveryofGBPsthatcontaindomainsresemblingknown CRDs.Asglycanarrayscreeningbecomesmorewidelyaccessible,morebroad-based screeningcanbeenvisioned. NATURALLIGANDSFORGBPs Monosaccharidesorsmalloligosaccharidesinisolationtendtobelow-affinity ligandsforGBPs,oftenwithdissociationconstantsinthemillimolarrange.These intrinsicaffinitiesareenhancedinseveralways(Figure28.4).Atthelevelof individualglycans,affinitycanbeenhancedbylinkingthesugartoothertypesof structures.Typicalconjugationofglycanstoproteinsandlipidscanleadto enhancedCRDbinding.Forexample,someGBPssuchasthemacrophagereceptor minclebindtoglycolipidswithmuchhigheraffinitythantheybindtofree oligosaccharides.Inthiscase,enhancedaffinitycanresultfromthepresenceofan extendedoraccessorybindingsiteinaCRDadjacenttothesugar-bindingsite, whichisabletoaccommodatethehydrophobictailofthelipid.OtherGBPsbind selectivelytoaparticularglycanconjugatedtoaspecificpolypeptidemotif.Optimal bindingofP-selectintotheligandPSGL-1requiresanO-linkedglycanbearinga sialylLewisxstructureonapeptidewithadjacentacidicresiduesandsulfated tyrosines(Chapter34).Inyetothercases,glycanrecognitioniscombinedwithother bindingdomainsonaprotein.ThemannosereceptorcontainsC-typeCRDsthat bindhighmannoseoligosaccharidesandafibronectintypeIIrepeatthatbindsto triplehelicalpolypeptides.Together,thesetwomodalitiesfacilitatebindingto fragmentsofcollagenreleasedatsitesofinflammation. Amajordeterminantofbindingtonaturalligandsistheinteractionofmultivalent glycanswithclusteredCRDs,resultinginhighaviditybinding.Clusteringofligands canresultfromthepresenceofmultiplebindingepitopesinasingleoligosaccharide orpolysaccharide,thepresenceofmultipleglycansattachedtoasingleprotein scaffoldorthepresenceofadjacentglycoproteinsorglycolipidsinacellmembrane. Similarly,clusteringofCRDscanreflectthepresenceofmultipleCRDsinasingle polypeptide,formationofoligomersofpolypeptidethateachcontainsasingleCRD andfromclusteringofCRD-containingproteinsinthecellmembrane.Eachofthese levelsoforganizationofCRDshasthepotentialtoplacegeometricalconstraintson theoptimalarrangementofligands,dependingonthedegreetowhichCRDsare heldinafixedarrangementorareflexiblylinked.Clusteringofglycansattachedtoa singlepolypeptide,particularlyinheavilyO-glycosylatedproteinssuchasmucins, canalsoaffecttheirabilitytotakeondifferentconformations.SinceGBPstypically interactwithasingleconformation,selectingoneofmultipleaccessible conformations,thereisanentropicpenaltyassociatedwithbindingwhichmaybe reducedwhentheglycanhasfewerpotentialconformations.Invitrobiochemical assays,includingglycanarrays,reflectonlysomeofthesetypesofclusteringof CRDsandligands,sotheymustbeinterpretedwithsomecaution.Insomecases, bindingofaCRDtoisolatedglycansmaybeessentiallyundetectableeventhough bindingoftheintactCRD-containingproteintoitsendogenousglycoconjugatemay behighlyselectiveandquitestrong.Caremustalsobeexercisedinuseoftheterm ligand,todistinguishtheglycanpartofaligandfromtheentirenatural glycoconjugateorevencellsurface. TERMINOLOGYFORSPECIFICGBPLIGANDS Basedontheaboveconsiderations,GBPsmaybindselectivelytoaparticularglycan onlywhenitisconjugatedtoaparticularglycoprotein.TheGBPligandisneitherthe glycanitselfnortheproteinitself.ExamplesincludeP-selectinbindingtosialyl LewisxonPSGL-1(seeabove)andE-selectinbindingtothesameglycan(sialyl Lewisx)carriedonavariantformoftheproteinCD44.Thereisatpresentno consistentwaytodesignateaglycanonparticularproteinasaligandforaspecific lectin.SayingthatsialylLewisxorthatPSGL-1(protein)isthe“ligand”forP-Selectin isnotaccurate.TheE-selectin-bindingformofCD44wasgivenadifferentname (HCELL,hematopoieticstemcellligandforE-selectin)thatfailstoidentifythe polypeptidecarrier.Thismatterhasyettoberesolved,especiallywhenasingle proteinmightbetherequiredpolypeptidescaffoldthatcarriesglycansfordifferent GBPs.Atthispoint,theconceptthatglycansareoftenligandsforGBPsonlyinthe contextoftheirproteinorlipidcarriershasbeenwellestablished. FIGURELEGENDS FIGURE28.1.Representativestructuresfromfourcommonanimallectinfamilies. Theemphasisisontheextracellulardomainstructureandtopology.Thefollowing arethedefinedcarbohydrate-bindingdomains(CRDs)shown:(CL)C-typelectin; (GL)Galectin;(MP)P-typelectin;(IL)I-typelectin.Otherdomainsare(EG)EGF-like domain;(IG2)immunoglobulinC2-setdomain;(TM)transmembranedomain;and (C3)complementregulatoryrepeat.ThenumberofdomainsaccompanyingtheCRD variesamongfamilymembers. FIGURE28.2.Arrangementsofcarbohydrate-recognitiondomains(CRDs)inGBPs. ProteinscontainingjustCRDsorCRDsassociatedwithothertypesoffunctional domains,withmembraneanchorsorwitholigomerizationdomainsaredepicted schematically.AsingleGBPcancontainalloftheseadditionaldomains. agglutinin;EDEM,Endoplasmicreticulum-associateddegradation-enhancingαmannosidase-likeproteins;GH,glycohydrolase;MRH,mannosereceptorhomology. FIGURE28.4.SourcesofenhancedbindingofnaturalligandstoGBPs.Within individualCRDs,secondaryinteractionsbeyondtheprimarybindingsitecanbe withsugar,proteinorlipidportionsofglycoconjugateligands.Multivalent interactionscanreflectinteractionofsinglebranchedoligosaccharidesormultiple oligosaccharidesattachedtoaglycoproteinwithmultipleCRDsbroughttogether withinreceptoroligomersorinGBPclustersonthecellsurface. FURTHERREADING StillmarkH.Inauguraldissertation.UniversityofDorpat,Dorpat(nowTartu); Estonia:1888.UberRicin,EingiftigesFermentausdenSamenvonRicinuscommunis L.undeinigenanderenEuphoribiaceen. GoldsteinIJ,HughesRC,MonsignyM,OsawaT,SharonN.Whatshouldbecalleda lectin?Nature.1980;285:66. AshwellG,HarfordJ.Carbohydrate-specificreceptorsoftheliver.AnnuRevBiochem. 1982;51:531–554.PubMedPMID:6287920. DrickamerK.Twodistinctclassesofcarbohydrate-recognitiondomainsinanimal lectins.JBiolChem.1988;263:9557–9560.PubMedPMID:3290208 Powell,LD,Varki,A.I-typelectins.JBiolChem.1995;270:14243–14246. LeeR.T.andLeeY.C.2000.Affinityenhancementbymultivalentlectin-carbohydrate interaction.Glycoconj.J.17:543-551. Casu,B,Lindahl,U.Structureandbiologicalinteractionsofheparinandheparan sulfate.AdvCarbohydrChemBiochem.2001;57:159–206. Esko,JD,Selleck,SB.Orderoutofchaos:assemblyofligandbindingsitesinheparan sulfate.AnnuRevBiochem.2002;71:435–471. DrickamerK.andTaylorM.E.2003.Identificationoflectinsfromgenomicsequence data.MethodsEnzymol.2003,362:592-599. RigdenD.J.,MelloL.V.,and,GalperinM.Y.2004.ThePA14domain,aconservedallbetadomaininbacterialtoxins,enzymes,adhesinsandsignalingmolecules. TrendsBiochem.Sci.29:335–339. SharonN.andLisH.2004.Historyoflectins:fromhemagglutininstobiological recognitionmolecules.Glycobiology14:53R-62R. Lee,J.K.,Baum,L.G.,Moremen,K.,andPierce,M.2004.TheX-lectins:anewfamily withhomologytotheXenopuslaevisoocytelectinXL-35.Glycoconj.J.21:443450 BlundellC.D.,AlmondA.,MahoneyD.J.,DeAngelisP.L.,CampbellI.D.,andDayAJ. 2005.TowardsastructureforaTSG-6.hyaluronancomplexbymodelingand NMRspectroscopy:insightsintoothermembersofthelinkmodulesuperfamily. J.Biol.Chem.280:18189-201 Varki,A,Angata,T.Siglecs--themajorsubfamilyofI-typelectins.Glycobiology. 2006;16(1):1R–27R. SchallusT.,JaeckhC.,FehérK.,PalmaA.S.,LiuY.,SimpsonJ.C.,MackeenM.,StierG., GibsonT.J.,FeiziT.,PielerT.,andMuhle-GollC.2008.Malectin:anovel carbohydrate-bindingproteinoftheendoplasmicreticulumandacandidate playintheearlystepsofproteinN-glycosylation.Mol.Biol.Cell.19:3404-3414. VanDamme,E.J.M.,Lannoo,N.,andPeumans,W.J.2008.PlantLectins.Adv.Bot.Res. 48:107–209 TaylorM.E.andDrickamerK.2009.Structuralinsightsintowhatglycanarraystell usabouthowglycan-bindingproteinsinteractwiththeirligands.Glycobiology 19:1155-1162. DamT.K.,GerkenT.A.,andBrewerC.F.2009.Thermodynamicsofmultivalent carbohydrate-lectincrosslinkinginteractions:importanceofentropyinthebind andjumpmechanism.Biochemistry48:3822-3827. LindnerH.,MüllerL.M.,Boisson-DernierA.,andGrossniklausU.2012.CrRLK1L receptor-likekinases:notjustanotherbrickinthewall.Curr.Opin.PlantBiol. 15:659-669. GhequireM.G.K.,LorisR.,andDeMotR.2012.MMBLproteins:fromlectinto bacteriocin.Biochem.Soc.Trans.40:1553-1559. GilbertH.J.,KnoxJ.P.,andBorastonA.B.2013.Advancesinunderstandingthe molecularbasisofplantcellwallpolysacchariderecognitionbycarbohydratebindingmodules.Curr.Opin.Struct.Biol.2013:669-677. AdrangiS.andFaramarziM.A.2013.Frombacteriatohuman:ajourneyintothe worldofchitinases.Biotechnol.Adv.31:1786–1795 TaylorM.E.andDrickamerK.2014.Convergentanddivergentmechanismsofsugar recognitionacrosskingdoms.Curr.Opin.Struct.Biol.28:14–22. NagaeM.andYamaguchiY.2014.Three-dimensionalstructuralaspectsofprotein– polysaccharideinteractions.Int.J.Mol.Sci.15:3768-3783. DrickamerK.andTaylorM.E.2015.Recentinsightsintostructuresandfunctionsof C-typelectinsintheimmunesystem.Curr.Opin.Struct.Biol.34:26-34. BishnoiR.,KhatriI.,SubramanianS.,andRamya,T.N.C.2015.PrevalenceoftheFtypelectindomain.Glycobiology25:888–901. Table1.Appendix26A Comparisonoftwomajorclassesofglycan-bindingproteins Glycosaminoglycan-binding Lectinsa proteinsb Sharedevolutionaryorigins Sharedstructuralfeatures DefiningAAresidues involvedinbinding Typeofglycansrecognized yes(withineachgroup) yes(withineachgroup) oftentypicalforeachgroup no no patchofbasicaminoacidresidues N-glycans,O-glycans, differenttypesofsulfatedglycosaminoglycans glycosphingolipids(afew alsorecognizesulfated glycosaminoglycans) Locationofcognateresidues typicallyinsequencesat typicallyinsequencesinternaltoanextended withinglycans outerendsofglycanchains sulfatedglycosaminoglycanchain Specificityforglycans stereospecificityhighfor oftenrecognizearangeofrelatedsulfated recognized specificglycanstructures glycosaminoglycanstructures Single-sitebindingaffinity oftenlow;highavidity oftenmoderatetohigh generatedbymultivalency Valencyofbindingsites multivalencycommon oftenmonovalent (eitherwithinnative structureorbyclustering) Subgroups C-typelectins,galectins,Pheparansulfate–bindingproteins,chondroitin typelectins,I-typelectins,L- sulfate–bindingproteins,dermatansulfate– typelectins,R-typelectins bindingproteins etc. Typesofglycansrecognized canbesimilar(e.g., classificationitselfisbasedontypeof withineachgroup galectins)orvariable(e.g., glycosaminoglycanchainrecognized C-typelectins) ModifiedfromVarkiA.andAngataT.2006.Glycobiology16:1R–27R. aThereareotheranimalproteinsthatrecognizeglycansinalectin-likemanneranddonotappeartofallintoone ofthewell-recognizedclasses(e.g.,variouscytokines). bHyaluronan(HA)-bindingproteins(hyaloadherins)fallinbetweenthesetwoclasses.Ontheonehand,some (butnotall)ofthehyaloadherinshavesharedevolutionaryorigins.Ontheotherhand,recognitioninvolves internalregionsofHA,whichisanonsulfatedglycosaminoglycan. Chapter 38 Proteins that Bind Sulfated Glycosaminoglycans Essentials of Glycobiology, 3rd edition ProteinsthatBindSulfatedGlycosaminoglycans Authors:JeffreyD.Esko,JamesPrestegardandRobertJ.Linhardt Glycosaminoglycansbindtomanydifferentclassesofproteinsmostlythrough electrostaticinteractionsbetweennegativelychargedsulfategroupsanduronic acidsandpositivelychargedaminoacidsintheprotein.Thischapterfocuseson examplesofglycosaminoglycan-bindingproteins,methodsformeasuring glycosaminoglycan-proteininteraction,andinformationaboutthree-dimensional structuresofthecomplexes. GLYCOSAMINOGLYCAN-BINDINGPROTEINSARECOMMON Severalhundredglycosaminoglycan(GAG)-bindingproteinshavebeendiscovered, whichmakeuptheGAG-interactomeandfallintothebroadclassespresentedin Table38.1.Toalargeextent,studiesoftheGAG-interactomehavefocusedon proteininteractionswithheparin,amorehighlysulfated,iduronicacid(IdoA)-rich formofheparansulfate(HS;Chapter17).Thisbiasreflectsinpartthecommercial availabilityofheparinandheparin-Sepharose,whicharefrequentlyusedfor fractionationstudies,andtheassumptionthatbindingtoheparinmimicsbindingto HSpresentoncellsurfacesandintheextracellularmatrix.Incomparison,relatively fewproteinsareknowntointeractwithchondroitinsulfate(CS)orkeratansulfate (KS)withcomparableavidityandaffinity.Insomecases,CSandtherelatedGAG, dermatansulfate(DS),maybephysiologicallyrelevantbindingpartnersbecause theseGAGspredominateinmanytissues.Determiningthephysiologicalrelevance oftheseinteractionsisamajorareaofresearch. Incontrasttolectins,whichtendtofallintoevolutionarilyconservedfamilies (Chapters28-37),GAG-bindingproteinsdonothavecommonfoldsandinstead appeartohaveevolvedbyconvergentevolution.AsshowninTable38.1,the interactionbetweenGAGsandproteinscanhaveprofoundphysiologicaleffectson processessuchashemostasis,lipidtransportandabsorption,cellgrowthand migration,anddevelopment.BindingtoGAGscanresultinimmobilizationof proteinsattheirsitesofproductionorintheextracellularmatrixforfuture mobilization;regulationofenzymeactivity;bindingofligandstotheirreceptors; proteinoligomerization;andprotectionofproteinsagainstdegradation.Insome cases,theinteractionmayreflectcomplementarityofcharge(e.g.,histone-heparin interactions)ratherthananyspecificbiologicallyrelevantinteraction.Inother cases,theinteractionhasbeenshowntodependonrarebutveryspecificsequences ofmodifiedsugarsintheGAGchain(e.g.,antithrombinbinding). METHODSFORMEASURINGGLYCOSAMINOGLYCAN-PROTEIN BINDING NumerousmethodsareavailableforanalyzingGAG-proteininteractions,andsome provideadirectmeasurementofKdvalues.Acommonmethodinvolvesaffinity fractionationofproteinsonSepharosecolumnscontainingcovalentlylinkedGAG chains,usuallyheparin.Theboundproteinsareelutedwithdifferentconcentrations ofsodiumchloride,andtheconcentrationrequiredforelutionisgenerally proportionaltotheKd.High-affinityinteractionsrequireatleast1MNaClto displaceboundligand,whichtranslatesintoKdvaluesof10−7–10−9M(determined underphysiologicalsaltconcentrationsbyequilibriumbinding).Proteinswithlow affinity(10−4–10−6M)eitherdonotbindunder“normal”conditions(0.15MNaCl)or requireonly0.3–0.5MNaCltoelute.Thismethodisbasedontheassumptionthat GAG-proteininteractionisentirelyionic,whichisnotentirelycorrect.Nevertheless, itcanprovideanassessmentofrelativeaffinity,whencomparingdifferentGAGbindingproteins. Anumberofmoresophisticatedmethodsarenowinusethatprovidedetailed thermodynamicdata(ΔH[changeinenthalpy],ΔS[changeinentropy],ΔCp[change inmolarheatcapacity],etc.),kineticdata(associationanddissociationrates),and high-resolutiondataonatomiccontactsinGAG-proteininteractions(Table38.2). Regardlessofthetechniqueoneuses,itmustbekeptinmindthatinvitrobinding measurementsarenotlikelytobethesameasthosewhentheproteinbindsto proteoglycansonthecellsurfaceorintheextracellularmatrix,wherethedensity andvarietyofGAG-bindingproteins,proteoglycansandotherinteractingfactors variesgreatly.Todeterminethephysiologicalrelevanceoftheinteraction,one shouldconsidermeasuringbindingunderconditionsthatcanleadtoabiological response.Forexample,onecanmeasurebindingtocellswithalteredGAG composition(Chapter49)oraftertreatmentwithspecificlyasestoremoveGAG chainsfromthecellsurface(Chapter17)andthendeterminewhetherthesame responseoccursasobservedinthepresenceofGAGchains.Theinteractioncanthen bestudiedmoreintensivelyusingtheinvitroassaysdescribedabove. CONFORMATIONALANDSEQUENCECONSIDERATIONS Asmentionedabove,mostGAG-bindingproteinsinteractwithHSorheparin.The likelybasisforthispreferenceisgreatersequenceheterogeneityandmorevariable sulfation,comparedtootherGAGs.Theunusualconformationalflexibilityof iduronicacid,whichisfoundinheparin,HS,andDS,alsohasaroleintheirabilityto bindproteins.GAGsarelinearhelicalstructures,consistingofalternatingresidues ofN-acetylglucosamine(GlcNAc)orN-acetylgalactosamine(GalNAc)with glucuronicacid(GlcA)oriduronicacid(IdoA)(withtheexceptionofkeratan sulfates,whichconsistofalternatingGlcNAcandgalactoseresidues;Chapter17). Inspectionofheparinoligosaccharidescontaininghighlymodifieddomains ([GlcNS6S-IdoA2S]n)showsthattheN-sulfoand6-O-sulfogroupsofeach disacchariderepeatlieonoppositesidesofthehelixfromthe2-O-sulfoand carboxylgroups(Figure38.1).Analysisoftheconformationofindividualsugars showsthatGlcNAcandGlcAresiduesassumeapreferredconformationinsolution, designated4C1(indicatingthatcarbon4isabovetheplanedefinedbycarbons2,3, and5andtheringoxygen,andthatcarbon1isbelowtheplane;Chapter2).In contrast,IdoA2Sassumesthe1C4orthe2SOconformation(Figure38.1),which reorientsthepositionofthesulfosubstituents,therebycreatingadifferent orientationofchargedgroups.InmanycaseswhenaproteinbindstoanHSchain,it inducesachangeinconformationoftheIdoA2Sresidueresultinginabetterfitand enhancedbinding.IdoA2SresidueshavealwaysbeenfoundindomainsrichinNsulfoandO-sulfogroups(forbiosyntheticreasons;Chapter17),whichisalsowhere proteinsusuallybind.Thus,thegreaterdegreeofconformationalflexibilityinthese modifiedregionsmayexplainwhysomanymoreproteinsbindwithhighaffinityto heparin,HS,andDSthantootherGAGs.ThepresenceofanN-acetylgroupinan GlcNAcresiduechangesthepreferredconformationoftheneighboringIdoA2S residue,showingthatevenminormodificationscaninfluenceconformationand chainflexibility.BindingtoGAGsthathavealowdegreeofsulfationmayrequire largerdomainsintheproteintointeractwithlongerstretchesofanoligosaccharide. Moleculardynamicsimulationsonlargeheparinoligosaccharidesarepossiblewith theavailabilityofsupercomputers(seeSimulation35.1ontheaccompanying website).Suchsimulationscanbeusedtopredicttheconformationalflexibilityof differentdomainswithinthechainandwhencombinedwithrecentadvancesin protein-GAGdocking,canprovideadditionalinsightsintoGAG-proteininteractions. HOWSPECIFICAREGLYCOSAMINOGLYCAN-PROTEIN INTERACTIONS? ThediscoveryofmultipleGAG-bindingproteinsledanumberofinvestigatorsto examinewhetherthereisaconsensusaminoacidsequenceforGAGbinding.In retrospect,thisstrategywasoverlysimplisticbecauseitassumedthatallGAGbindingproteinswouldrecognizethesameoligosaccharidesequencewithin heparin,oratleast,sequencesthatwouldsharemanycommonfeatures.Wenow knowthatsomeGAG-bindingproteinsinteractwithdifferentoligosaccharide sequences.Thebindingsitesintheproteinalwayscontainbasicaminoacids(lysine andarginine)whosepositivechargespresumablyinteractwiththenegatively chargedsulfatesandcarboxylatesoftheGAGchains.However,thearrangementof thesebasicaminoacidscanbequitevariable,consistentwiththevariable positioningofsulfogroupsintheGAGpartner. Mostproteinsareformedfromα-helices,β-strands,andloops.Therefore,toengage alinearGAGchain,thepositivelychargedaminoacidresiduesmustalignalongthe samesideoftheproteinsegment.α-Heliceshaveperiodicitiesof3.4residuesper turn,whichwouldrequirethebasicresiduestooccureverythirdorfourthposition alongthehelixinordertoalignwithanoligosaccharide.Inβ-strands,theside chainsalternatesideseveryotherresidue.Thus,tobindaGAGchain,thepositively chargedresiduesinaβ-strandwouldbelocatedquitedifferentlythaninanα-helix. Onthebasisofthestructureofseveralheparin-bindingproteinsthatwereavailable in1991,AlanCardinandHerschelWeintraubproposedthattypicalheparin-binding siteshadthesequenceXBBXBXorXBBBXXBX,whereBislysineorarginineandXis anyotheraminoacid.Fromthestructuralargumentsprovidedabove,itshouldbe obviousthatonlysomeofthebasicresiduesinthesesequencescouldparticipatein GAGbinding,theactualnumberbeingdeterminedbywhetherthepeptidesequence existsasanα-helixoraβ-sheet.Wenowknowthatthepresenceofthesesequences inaproteinmerelysuggestsapossibleinteractionwithheparin(oranotherGAG chain),butitdoesnotprovethattheinteractionoccursunderphysiological conditions.Infact,thepredictedbindingsitesforheparininfibroblastgrowthfactor 2(FGF2)turnedouttobeincorrectoncethecrystalstructurewasdetermined.Itis likelythatbindinginvolvesmultipleproteinsegmentsthatjuxtaposepositively chargedresiduesintoathree-dimensionalturn-richrecognitionsite.Inmanycases thebindinginvolvesloopswhichmakethepositioningmorevariable.Anexampleof thisphenomenonisobservedinthechemokineCCL5,whichcontainsaBBXBmotif inaloop.Thespecificarrangementofresiduesshouldvaryaccordingtothetype andfinestructureofthoseoligosaccharidesinvolvedinbinding. Inplantandanimallectins,andinantibodiesthatrecognizeglycans,theglycan recognitiondomainsaretypicallyshallowpocketsthatengagetheterminalsugars oftheoligosaccharidechain(Chapters29,30and37).InGAG-bindingproteins,the proteinusuallybindstosugarresiduesthatliewithinthechainornearthe terminus.Therefore,thebindingsitesinGAG-bindingproteinsconsistofcleftsor setsofjuxtaposedsurfaceresiduesratherthanpockets.TheseGAG-bindingsiteson theproteinsurfacegiverisetomorerapidGAG-proteinbindingkineticsthanare typicallyobservedforprotein-proteininteractions.GiventhatGAGchainsgenerally existinahelicalconformation,onlythoseresiduesonthefacetowardtheprotein interactwithaminoacidresidues;theonesontheothersideofthehelixare potentiallyfreetointeractwithasecondligand(e.g.,asobservedinFGFdimers). Alternatively,residuesinabindingcleftcouldinteractwithbothsidesofthehelix (e.g.indengueenvelopeprotein).Finally,oneshouldkeepinmindthatbinding occurstoonlyasmallsegmentoftheGAGchain.Thus,asingleGAGchaincan potentiallybindmultipleproteinligandsfacilitatingcooperativebindingthatcan leadtoproteinoligomerization(e.g.somechemokines). ANTITHROMBIN-HEPARIN:APARADIGMFORSTUDYING GLYCOSAMINOGLYCAN-BINDINGPROTEINS Perhapsthebest-studiedexampleofprotein-GAGinteractionisthebindingof antithrombintoheparinandHS(seecoverimageandFigure38.2).Thisinteraction isofgreatpharmacologicalimportancebecauseheparinisusedclinicallyasan anticoagulant.Bindingofantithrombintoheparinhasadualeffect:First,itcausesa conformationalchangeintheproteinandactivationoftheproteaseinhibiting action,resultingina1000-foldenhancementintherateatwhichitinactivates thrombinandFactorXa.Second,theheparinchainactsasatemplate,enhancingthe physicalappositionofthrombinandantithrombin.Thus,boththeprotease (thrombin)andtheinhibitorhaveGAG-bindingsites.Heparinactsasacatalystin thesereactionsbyenhancingtherateofthereactionthroughappositionof substratesandconformationalchange.Aftertheinactivationofthrombinby antithrombinoccurs,thecomplexlosesaffinityforheparinanddissociates.The heparinisthenavailabletoparticipateinanotheractivation/inactivationcycle. Antithrombinisamemberoftheserpinfamilyofproteaseinhibitors,manyofwhich bindtoheparin. Earlystudiesusingaffinityfractionationschemesshowedthatonlyaboutone-third ofthechainsinaheparinpreparationactuallybindwithhighaffinityto antithrombin.Comparingthesequenceoftheboundchainswiththosethatdidnot bindfailedtorevealanysubstantialdifferencesincomposition,consistentwiththe laterdiscoverythatthebindingsiteconsistsofonlyfivesugarresidues(Figure 38.2)(theaverageheparinchainisabout50sugarresidues).Thisobservationcan beextendedtovirtuallyallGAG-bindingproteins,inferringthatthebindingsites representaverysmallsegmentofthechains. CrystalsofantithrombinwerepreparedandanalyzedbyX-raydiffractionto2.6-Å resolution.Thedockingsitefortheheparinpentasaccharideisformedbythe appositionofhelicesAandD,whichbothcontaincriticalarginineandlysine residuesattheinterface.ThesequenceintheDhelix (124AKLNCRLYRKANKSSKLVSANR145)placesmanyofthepositivelycharged residuesononefaceofthehelix,inproximitytothearginineresiduesintheAhelix (41PEATNRRVW49)(Figure38.2).Thepentasaccharideissufficienttoactivate antithrombinbindingtowardFactorXa,butitwillnotfacilitatetheinactivationof thrombin.Forthistooccur,alargeroligosaccharideofatleast18residuesis needed.Asmentionedabove,thrombinalsocontainsaheparin-bindingsite,andthe largerheparinoligosaccharideisthoughttoactasatemplatefortheformationofa ternarycomplexwiththrombinandantithrombin.Incontrasttoantithrombin, thrombinexhibitslittleoligosaccharidespecificity.Asmightbeexpected,adding highconcentrationsofheparinactuallyinhibitsthereaction,becausetheformation ofbinarycomplexesofheparinandthrombinorheparinandantithrombin predominate.Thisimportantprincipleof“activationatlowconcentrationsand inhibitionathighconcentrations”alsooccursinothersystemswhereternary complexesform(Chapters29and30). Heparinisapharmaceuticalformulationproducedbypartialfractionationof naturalGAGsderivedprimarilyfromporcineintestines(Chapter17).Mastcellsare knowntoproduceahighlysulfatedversionofHSthatresemblesheparin;highly sulfated,iduronicacid–richheparinoligosaccharidesarealsopresentinHSisolated fromothertissuesaswell,especiallytheskin.Althoughheparinhasproventobeof greattherapeuticuse,itsroleinvivoremainsunclear.Heparinandchondroitin sulfateareoftenfoundinstoragegranulesalongwithbiogenicamines,proteases, andotherproteins,possiblyenablingefficientstorage.Mastcellsdegranulatein responsetospecificantigenstimulation,resultinginreleaseofstoredheparin, histamine,andproteases.Whenthisoccurs,localanticoagulationmightoccur,but localizedcoagulationdefectshavenotbeendescribedinanimalsbearingmutations thataltermastcellsorheparin.AsmallpercentageofendothelialcellHScontains antithrombin-bindingsequencesaswell.However,thesebindingsitesappeartobe locatedontheabluminalsideofbloodvessels,andmicelackingthecentral3-OsulfatedGlcNSunit,ahallmarkoftheantithrombin-bindingsequence(Figure38.2), donotexhibitanysystemiccoagulopathyafterbirth.Nevertheless,antithrombin deficiencycausesmassivedisseminatedcoagulopathy.Perhapsthesefindings indicatethatlower-affinitybindingsequencesaresufficienttoactivate antithrombin.Thissystemillustratesanimportantcaveat:onecannotnecessarily ascribefunctionstoendogenousproteoglycansbasedontheeffectsofGAGsadded invitrotoexperimentalsystems. FGF-HEPARININTERACTIONSENHANCESTIMULATIONOFFGF RECEPTORSIGNALTRANSDUCTION Alargenumberofgrowthfactorscanbepurifiedbasedontheiraffinityforheparin. Theheparin-bindingfamilyoffibroblastgrowthfactorshasgrowntomorethan22 membersandincludestheprototypeFGF2,otherwiseknownasbasicfibroblast growthfactor.FGF2hasaveryhighaffinityforheparin(Kd~10−9M)andrequires 1.5–2MNaCltoelutefromheparin-Sepharose.FGF2haspotentmitogenicactivityin cellsthatexpressoneoftheFGFsignalingreceptors(fourFGFRgenesareknown andmultiplesplicevariantsexist).Cell-surfaceHSbindstobothFGF2andFGFR, facilitatingtheformationofaternarycomplex.Bothbindingandthemitogenic responsearegreatlystimulatedbyheparinorHS,whichspromotedimerizationof theligand-receptorcomplex. ThecostimulatoryroleofHS(andheparin)inthissystemisreminiscentofthe heparin/antithrombin/thrombinstory.Indeed,theminimalbindingsequencefor FGF2alsoconsistsofapentasaccharide.However,thispentasaccharideisnot sufficienttotriggerabiologicalresponse(mitogenesis).Forthistooccur,alonger oligosaccharide(10mer)containingtheminimalsequenceandadditional6-O-sulfo groupsareneededtobindFGFR.ThesequencethatbindstobothFGF2andFGFRis prevalentinheparinbutrareinHS.Therequirementforthisrarebindingsequence reducestheprobabilityoffindingthisparticulararrangementinnaturallyoccurring HSchains.Thus,somepreparationsofHSareinactiveinmitogenesis,andthose containingonlyonehalfofthebipartitebindingsequenceareactuallyinhibitory. ThestructureofFGF2cocrystallizedwithaheparinhexasaccharidehassincebeen obtained(Figure38.3).Theheparinfragment([GlcNS6Sα1-4IdoA2Sα1-4]3)was helicalandboundtoaturn-richheparin-bindingsiteonthesurfaceofFGF2.Only oneN-sulfogroupandthe2-O-sulfogroupfromtheadjacentiduronicacidare boundtothegrowthfactorintheturn-richbindingdomain,andthenextGlcNS residueisboundtoasecondsite,consistentwiththeminimalbindingsequence determinedwitholigosaccharidefragments.Nosignificantconformationalchangein FGF2occursuponheparinbinding,consistentwiththeideathatheparinprimarily servestodimerizeFGF2andjuxtaposecomponentsoftheFGFsignal-transduction pathway.ThecrystalstructureofacidicFGF(FGF1)hasalsobeensolvedandshows similarsequencesonitssurface.However,theoligosaccharidesequencethatbinds withhighaffinitytoFGF1contains6-O-sulfogroups. Thecocrystalstructureofthecomplexof(FGF2-FGFR)2,firstsolvedintheabsence ofheparin/HSligand,showedacanyonofpositivelychargedaminoacidresidues, suggestiveofanunoccupiedheparin-bindingsite.Subsequently,theheparinoligosaccharide-containingcomplexwassolvedafterintroductionofheparin oligosaccharides,suggestinga2:2:2complexofFGF2:FGFR:HS(Figure38.3). Anotherimportantfeatureofthiscomplexistheorientationofthenon-reducing endsoftheHSchainsthatterminateinanN-sulfoglucosamineresidue,whicharises byendolyticcleavageofchainsbytheenzymeheparanase(Chapter17).The structureoftheFGF-FGFR-HScomplexisnotwithoutcontroversy;structural analysisofcomplexesformedinsolutionandpurifiedbygelfiltrationhassuggested averydifferentstructureconsistingofa2:2:1complex(Figure38.3). OTHERATTRIBUTESOFGLYCOSAMINOGLYCAN-PROTEIN INTERACTIONS Insomecases,theinteractionofGAGchainswithproteinsmaydependonmetal cofactors.Forexample,L-andP-selectinshavebeenshowntobindtoasubfraction ofHSchainsandheparininadivalent-cation-dependentmanner.Thisobservation raisesthepossibilitythatotherexamplesofcation-dependentinteractionswithGAG chainsmayexist.GAGbindingtoL-selectinhelpsinleukocyterolling.Furthermore, theinteractioncanbepharmacologicallymanipulatedbyexogenousheparin, includingchemicallymodifiedderivativesthatlackanticoagulantactivity. CSproteoglycansinthecentralnervoussystem(CNS)influencecellmigrationand axonpathfindingandregulateneuriteoutgrowth.Theinteractionofrare,highly sulfateddisaccharidesequencesinCSchainswithmorphogensandgrowthfactors impactCNSdevelopmentandplayrolesinCNSpathology. HSproteoglycansareoftenexpressedinaspatiallyandtemporallylimitedfashion. ThetemporaryplacementofanHSproteoglycanataspecifictissuesitemightor mightnotcoincidewiththepresenceofitsappropriateproteinligand.Furthermore, ifthebindingpartnerhasnoaccesstotheHSproteoglycan,itcannotinteract— addinganadditionallevelofspecificity.Recentstudiesdemonstratethatthefine structureofHSchainsalsochangesduringdevelopment,thusenablingordisabling specificassociationsbetweenligandsandreceptors. Gradientsofmorphogens,factorsthatdeterminecellfatesbasedonconcentration, alsodeterminethepatternsofcellandtissueorganizationduringdevelopment (Chapter27).Themechanismofmorphogengradientformationiscontroversial,but interestingly,virtuallyallmorphogenscaninteractwithheparinandHS.These interactionscanaffecttransportofligands,receptorinteractions,endocytosis,and degradation,whichtogethermayhavearoleindeterminingtherobustnessofthe gradient.TheGAGchainsofproteoglycansalsoofferalineardomainoverwhich proteinscandiffuse.Bylimitingthespaceavailabletotheseproteinsfromthethreedimensionalspaceofextracellularfluidsandtheextracellularmatrixtoonedimensionalspacealongthechains,thechanceofencountersamongheparinbindingproteins,suchasFGFanditsreceptor(FGFR),maybeenhanced.Thus,the criticalroleofHSproteoglycansmaybeincontrollingthekineticsofprotein– proteininteractionsratherthanthethermodynamicsofsuchencounters. ACKNOWLEDGEMENTS TheauthorsappreciatehelpfulcommentsandsuggestionsfromKristianSaied, EathenRyan,PatienceWrightandKristinStanford. FURTHERREADING LiW,JohnsonDJ,EsmonCT,HuntingtonJA.2004.Structureoftheantithrombinthrombin-heparinternarycomplexrevealstheantithromboticmechanismof heparin.NatStructMolBiol11:857-862. MohammadiM,OlsenSK,GoetzR.2005.AproteincanyonintheFGF-FGFreceptor dimerselectsfromanalacartemenuofheparansulfatemotifs.CurrOpin StructBiol15:506-516. DuchesneL,OcteauV,BearonRN,BeckettA,PriorIA,LounisB,FernigDG.2012. Transportoffibroblastgrowthfactor2inthepericellularmatrixiscontrolled bythespatialdistributionofitsbindingsitesinheparansulfate.PLoSBiol10: e1001361. KamhiE,JooEJ,DordickJS,LinhardtRJ.2013.Glycosaminoglycansininfectious disease.BiolRevCambPhilosSoc88:928-943. ThackerBE,XuD,LawrenceR,EskoJD.2013.Heparansulfate3-O-sulfation:Arare modificationinsearchofafunction.MatrixBiol35:60-72. XuD,EskoJD.2014.Demystifyingheparansulfate-proteininteractions.AnnuRev Biochem83:129-157. MizumotoS,YamadaS,SugaharaK.2015.Molecularinteractionsbetween chondroitin-dermatansulfateandgrowthfactors/receptors/matrixproteins. CurrOpinStructBiol34:35-42. PominVH,MulloyB.2015.Currentstructuralbiologyoftheheparininteractome. CurrOpinStructBiol34:17-25. SmithPD,Coulson-ThomasVJ,FoscarinS,KwokJC,FawcettJW.2015."GAG-ingwith theneuron":Theroleofglycosaminoglycanpatterninginthecentralnervous system.ExpNeurol274:100-114. DeshauerC,MorganAM,RyanEO,HandelTM,PrestegardJH,WangX.2015. InteractionsofthechemokineCCL5/RANTESwithmedium-sizedchondroitin sulfateligands.Structure(London,England:1993)23:1066-1077. FigureLegends FIGURE38.1.Conformationofheparinoligosaccharides.(A)Glucosamine(GlcN)and glucuronicacid(GlcA)existinthe4C1conformation,whereasiduronicacid(IdoA) existsinequallyenergeticconformationsdesignated1C4and2S0.(B)Space-filling modelofaheparinoligosaccharide(14mer)deducedbynuclearmagnetic resonance.(C)Thesamestructureinstickrepresentation.TherenderingsinBandC weremadewithRASMOLusingdatafromtheMolecularModelingDatabase(MMDB Id:3448)attheNationalCenterforBiotechnologyInformation(NCBI). FIGURE38.2.Crystalstructureoftheantithrombin-pentasaccharidecomplex(from ProteinDataBank).(A,D)α-Helicesthatmakecontactwithheparin;(RCL)the reactivecenterloopthatinactivatesthrombinandFactorX;(F)anotherα-helixin theprotein.(Lowerpanel)Interactionsbetweenkeyaminoacidresiduesand individualelementsinthepentasaccharide.(Solidlines)Electrostaticinteractions betweenpositivelychargedresiduesandsulfategroups;(brokenlines)hydrogen bonds;(alternatelybrokenandsolidline)bridgingwatermolecule. FIGURE38.3.CrystalandNMRsolutionstructuresofGAG-proteincomplexes.(A) Crystalstructureofthe2:2:2FGF2:FGFR1:heparincomplex(sideview)anda2:2:1 complex;(B)StructureofthedimericV-C1domainsofRAGE(receptorforadvanced glycationendproducts)(PDB4IM8).Thedodecasaccharideismanuallymodeled intothestructureonthebasisoftheobservedpartialelectrondensity;(C)Structure ofthedimericE2domainofamyloidprecursor–likeprotein1(APLP-1)andbound oligosaccharide(PDB3QMK);(D)Structureofdimericinterleukin-8(PDB2IL8)and amodeledoligosaccharide(degreeofpolymerization:20);(E)Arepresentative framefromthemostenergeticallyfavoredmodelsoftheCCL5-chondroitinsulfate complexdeducedbyNMR.Theribbonrepresentationofthecomplexwithside chainsofselectiveaminoacidsisshowningray.Thechondroitin-4-sulfate(dp6) ligandisshowninthestickrepresentationwiththenon-reducingendandthe reducingendsugarlabeledGlcA1andGalNAc3,respectively(fromDeshaueretal. (2015)Structure23,1066–1077) TABLE38.1Examplesofglycosaminoglycan-bindingproteinsandtheir biologicalactivity Physiological/pathophysiological Class Examples effectsofbinding Enzymes glycosaminoglycan multiple biosyntheticenzymes, thrombinandcoagulation factors(proteases), complementproteins (esterases),extracellular superoxidedismutase, lipases Enzyme antithrombinIII,heparin coagulation,inflammation, inhibitors cofactorII,secretory complementregulation leukocyteproteinase inhibitor,C1-esterase inhibitor Celladhesion P-selectin,L-selectin,some celladhesion,inflammation, proteins integrins metastasis Extracellular laminin,fibronectin, celladhesion,matrixorganization matrix collagens,thrombospondin, proteins vitronectin,tenascin Chemokines plateletfactorIV,γ-andβ- chemotaxis,signaling,inflammation interferons,interleukins Growthfactors fibroblastgrowthfactors, mitogenesis,cellmigration hepatocytegrowthfactor, vascularendothelial growthfactor,insulin-like growthfactor–binding proteins,TGF-β-binding proteins Morphogens hedgehogs,TGF-βfamily cellspecification,tissue members,wnts differentiation,development Guidance Slits,ROBOreceptors, axonguidance,endothelialtube factors neuropilins formation Tyrosinefibroblastgrowthfactor Mitogenesis,axonguidance, kinasegrowth receptors,vascular inflammation factor endotheliumgrowthfactor receptorsand receptor,receptorfor coreceptors advancedglycation endproducts(RAGE), receptorproteintyrosine phosphatases(RPTPs) Lipid-binding apolipoproteinsEandB, lipidmetabolism,cellmembrane proteins lipoproteinlipase,hepatic functions Plaque proteins Nuclear proteins Pathogen surface proteins Viralenvelope proteins lipase,annexins prionproteins,amyloid proteins histones,transcription factors malariacircumsporozoite protein herpessimplexvirus, denguevirus,human immunodeficiencyvirus, hepatitisCvirus,vaccinia viruscomplementcontrol protein(VCP) plaqueformation unknown pathogeninfections viralinfections TABLE38.2Methodstomeasureglycosaminoglycan-proteininteraction Method Type Throughput Principle Affinity M H/I immobilizedligandor chromatography glycosaminoglycanchainsoncolumn matrix Affinity M/S I gelretardationthroughproteincoelectrophoresis impregnatedgel Analytical S L equilibriumsedimentationat ultracentrifugation differentcarbohydrate:proteinratios Circulardichroism S I/L changeinrotationofplane-polarized lightuponbinding CompetitionELISA M H solution-andsolid-phaseligands competeforbinding Computational S L calculatescomplexstructureand bindingenergy Fluorescence S H conformationalchange,ligandbinding spectroscopy induceschangeinfluorescence Ionmobilitymass G I/L Measurescomplexshapeand spectrometry stoichiometry Isothermaltitration S I measuresenthalpyofbindingdirectly calorimetry andKdvalues Laserlightscattering S I intrinsicscatteringintensitiesof carbohydrate-proteincomplexused tocalculatestoichiometry Nuclearmagnetic S L chemicalshift,couplingconstant,and resonance nuclearOverhausereffectto determinecontactpoints,distances, andconformation Surfaceplasmon resonance M H/I Xray M L mass-inducedrefractiveindexchange inrealtimefordirectmeasurementof associationanddissociationrate constants solidstatecocrystalstructure (M)Mixedphase;(S)solutionphase;(G)gasphase;(H)high;(I)intermediate;(L)low. Lectin (handout)