Carbohydrate-carbohydrate Interaction Provides Adhesion Force And .

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Carbohydrate-carbohydrate interaction providesadhesion force and specificity forcellular recognition and adhesion.InauguraldissertationzurErlangung der Würde eines Doktors der Philosophievorglegt derPhilosophisch-Naturwissenschaftlichen Fakultätder Universität BaselvonIwona Bucioraus Gdynia, PolenBasel, 2003Canon Schweiz AG

Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultätauf Antrag vonHerrn Prof. Dr. Max M. Burgerund Frau Privatdozentin Dr. Ruth Chiquet-EhrismannBasel, den 8 Juli 2003Prof. Dr. Marcel Tanner(Dekan)

Table of contentsTABLE OF CONTENTSI.LIST OF rbohydrate interaction in cellularrecognition and adhesion6Carbohydrate interactions in vertebrates –glycosphingolipids7Glycosphingolipids in embryonal developmentand in embryonal cell adhesion12Glycosphingolipids in specific recognition betweenlymphoma and melanoma cells13Carbohydrate interactions in invertebrates –proteoglycans15II.1.2.1.Structure and function of sponge proteoglycan18II.1.2.2.Carbohydrate moiety of sponge proteoglycan22II.2.Molecular basis of alent character of carbohydrate interactions27II.2.2.Molecular forces in carbohydrate interactions30III.ABSTRACT33IV.MATERIAL AND METHODS36V.RESULTS46V.1.Glycans obtained by pronase digestion46V.2.Aggregation assays51V.2.1.Cell-cell aggregation is species-specific51V.2.2.Binding of glycans to beads53V.2.3.Cell-glycan aggregation is 1

Table of contentsV.2.4.Glycan-glycan aggregation is species-specific59V.2.5.Calcium uptake65V. 3.Adhesion to glycan-coated plates67V.3.1.Binding to plastic surfaces67V.3.2.Adhesion of live cells to proteoglycan-coated platesis species-specific69Adhesion of live cells to glycan-coated platesis species-specific71Adhesion of live cells to glycan-coated platespromotes cell differentiation73Adhesion of larval cells to glycan-coated platesis species-specific75Adhesion of glycan-coated beads to glycan coated platesis species-specific77Adhesion of glycans to glycan-coated platesis species-specific79Atomic force microscopy measurements of adhesionforces between single glycan molecules81Single carbohydrate-carbohydrate adhesion force is in thepiconewton range81Single carbohydrate-carbohydrate interaction isspecies-specific84Specificity of the carbohydrate-carbohydrate interactionis reflected in the polyvalence86Calcium enhances the strength of the carbohydrate-carbohydrateinteraction90VI.SUMMARY AND 13IX.LIST OF V.4.2.V.4.3.V.4.4.2


List of abbreviationsI. LIST OF ABBREVIATIONSAFMatomic force microscopyBSAbovine serum albuminBSWbicarbonate-buffered seawaterCerceramideCMFBSWcalcium- and magnesium-free bicarbonate-buffered seawaterCMFTSWcalcium- and magnesium-free Tris-buffered seawater; artificial seawaterCSchondroitin sulfateCsClcesium chlorideDSdermatan sulfateEDTAethylenediamidetetraacetic GlcNN-acetylglucosamineGlcAglucuronic acidg-66 kDa glycan obtained from Microciona prolifera proteoglycang-200200 kDa glycan obtained from Microciona prolifera proteoglycanGAGglycosaminoglycanGb4globoside (GalNAcβ1 4Galβ1 4Glcβ1 1Cer)Gg3gangliotriaosylceramide (GalNAcβ1 4Galβ1 4Glcβ1 1Cer)GM3sialosyllactosylceramide (NeuAcα2 3Galβ1 4Glcβ1 1Cer)GSLglycosphingolipidHAhyaluronic acid4

List of abbreviationsHRPhorseradish peroxidaseHSheparan sulfateKSkeratan sulfateLexLewisx determinant (Galβ1 4[Fucα1 3]GlcNAcβ1 3Galβ1 4Glcβ)MAFp3Microciona prolifera protein found in the ring of the proteoglycanMAFp4Microciona prolifera protein found in the arms of the e (Galβ1 4GlcNAcβ1 3Galβ1 4Glcβ1 1Cer)PAGEpolyacrylamide gel-electrophoresisPBSphosphate buffered salineSDSsodiumdodecylsulfateSSEAstage specific embryonic antigenTBETris-borate-EDTA bufferTrisTris(hydroxymethyl)aminomethane5

IntroductionII. INTRODUCTIONII.1. CARBOHYDRATE-CARBOHYDRATE INTERACTIONS INCELLULAR RECOGNITION AND ADHESIONOne of the fundamental features of a living cell is a prompt and adequate behaviorduring formation, maintenance, and pathogenesis of tissues. Thus, duringdevelopment of the nervous system reversible cell extensions and connections areformed1. During embryogenesis adhesive forces between cells are repeatedly beingbuilt up and destroyed2. Lymphoid cells find their homing centers throughintermediate adhesions to the vessel wall3. Cell surface receptors can be misused bymicrobial pathogens4,5,6 or tumor cells7. These short-term adhesion and recognitionevents require reversible but still specific molecular surface interactions, rather thantight and stable adhesions between stationary cells. Carbohydrates, the mostprominently exposed structures on the surface of living cells, with flexible chainsand many binding sites are ideal to serve as the major players in these events. Incontrast to the rapid progress in studies of cell recognition and adhesion throughprotein-protein8 or protein-carbohydrate9 interactions, the number and progress ofstudies on the possible role of carbohydrate-carbohydrate interactions in these eventsis still very small. There are mainly two model systems for studying the occurrenceof carbohydrate-carbohydrate interactions. In vertebrates, glycosphingolipid selfinteractions have been studied in early embryos and embryonic cell lines on onehand, and in tumor cell lines on the other hand. In invertebrates, marine sponge’scell-cell interactions mediated by carbohydrate-rich cell surface proteoglycanmolecules have been studied.6

Introduction: GlycosphingolipidsII.1.1. CARBOHYDRATE INTERACTIONS IN VERTEBRATES– GLYCOSPHINGOLIPIDSEukaryotic cell membranes are characterized by a specific composition GSLs)10. GSLsaremultifunctional molecules and dramatic and continuous changes in their surfaceexpression and composition have been associated with differentiation, developmentand oncogenesis. The functional role of GSLs in these processes is still ociatetheirtoxins13,initiators of signal transduction14, and finally as cell-type-specific antigens in cellularrecognition and regulation of cell growth15 (Fig.1).GSLs can be classified as neutral, acidic (anionic), or basic (cationic). GSLs are alsoclassified into three main series, i.e. ganglio-series, globo-series, type 1 lacto-series,and type 2 lacto-series, according to their core carbohydrate structure which mayinclude one of two hundred different oligosaccharides. The main constituent of theplasma membrane is represented by acidic GSLs: gangliosides containing sialic acid.Other acidic GSLs contain a sulfate group. Gangliosides occur not only as wellknown ganglio-series but also as globo-series or lacto-series gangliosides. Eachganglioside series shows distinctive cell type or tissue type specificity, and they mayplay different functional roles in cell adhesion10.GSLs are present at the surface of the plasma membrane in the form of large clustersindependent from the clusters of transmembrane glycoproteins16 (Fig. 2). These GSLmicrodomains are variously referred to as glycosphingolipid-enriched domains, lipidrafts, or caveolae membranes17,18. Along with GSLs, also present in lipid rafts aresphingomyelin, cholesterol, glycosylphosphatidylinositol (GPI)-anchored proteins,and a variety of signaling molecules. Although the need for cholesterol in promotingassembly of gangliosides has been questioned19, removal of cholesterol from plasma7

Introduction: glycosphingolipidsmembranes profoundly perturbs the physical state of microdomains20 andcompromises their function21.Cell adhesionmediators forlectins (galectins,selectins, allyregulatedantigensReceptors formicrobialexotoxins andmicrobialadhesinsGSLsInitiators ofsignaltransductionthrough GSLsignallingdomainAllogenicantigens(e.g. Lewis)Modulators ofgrowth factor /hormonereceptor functionFig. 1. Glycosphingolipids (GSLs) functions. Six functions are shown schematically:- Cell adhesion mediators (major topic of the introduction): [2, 10, 15, 28, 30, 34]- Tumor-associated antigens: [7, 10, 11, 12, 38]- Allogenic antigens: [10, 30, 105]- Modulators of growth factor receptor function: [10]- Initiators of signal transduction: [2, 14, 15, 19, 33, 35]- Receptors: [4, 10, 13]8

Introduction: glycosphingolipidsAccording to minimum energy conformation model22, a common hydrophobicbackbone of GSLs, i.e. ceramide, is inserted into the lipid bilayer of the plasmamembrane (Fig. 3). The axis of the carbohydrate chain in GSLs is orientedperpendicular to the axis of ceramide. Ceramide consists of a fatty acid chain linkedto the sphingosine base. It holds GSL carbohydrates in defined orientation throughinsertion in plasma membrane, and forms GSL microdomains separately fromglycoprotein microdomains23. In the model, the outer surface of the carbohydratechain, exposed at the cell surface, constitutes a hydrophobic domain surrounded by ahydrophilic area. Various ligands with a complementary structure can bind to thisexposed hydrophobic domain, and two mechanisms of GSL-mediated cell adhesionhave been observed: 1) Mediated by carbohydrate-binding proteins (lectins) thatrecognize specific GSL structures24,25, and by siglecs, receptors with Ig homologywhose N-terminal domain displays lectin activity to recognize various sialylepitopes26; 2) Mediated by complementary carbohydrate moieties of GSLs throughcarbohydrate-to-carbohydrate interaction. In either model, cell adhesion based on thecarbohydrate-carbohydrate interaction is the earliest event in cell recognition,followed by the involvement of adhesive proteins and of integrin receptors (Fig. 4).Hakomori's group was first to show GSLs self-interactions as a possible basis forcellular recognition at the morula stage of mouse embryogenesis, in embryonalcarcinoma cells, in specific aggregation of human embryonal carcinoma cells, and inrecognition between lymphoma and melanoma cells.9

Introduction: glycosphingolipidsFig. 2. Organization and distribution pattern of glycosphingolipids (GSL) and glycoproteins(Gp) at the cell-surface membrane. A, Proposed clustering of GSLs and Gp. B, Freeze-etchelectron micrograph of a human erythrocyte membrane double-labeled with ferritin-wheat germlectin and rabbit anti-globoside staphylococcal protein A colloidal gold. Ferritin-labeled areas (f) arewell separated from the gold-labeled area (large black dots; g) at the external surface (E), indicatingthat these two major glycoconjugates form separate clusters.i, surrounding ice; P, intramebranous particle surface, i.e. P face. The arrowhead indicates thefracture line.Hakomori. S. 1993 (2).10

Introduction: almodelofgloboside(Gb4Cer).Thecarbohydrate chain is orientedperpendicular to the axis of theceramide. The outer surface of thecarbohydrate chain, exposed at thecell surface, consists of ahydrophobic domain surroundedby hydrophilic groups, and aspecificbindingsiteforcomplementary GSLs, lectins, andantibodies.Hakomori, S. 1993 (2).Fig. 4. The model of GSL-dependent cell adhesion based on the membranous organizationof GSLs and glycoproteins (Gp). Compare with Fig. 2. A, The GSL cluster interacts with the Gpcluster; simultaneously, adhesive protein (AP) interacts with the integrin receptor (I). B, Interactionbetween GSL clusters on neighboring cells, subsequently reinforced by adhesive protein-integrinreceptor interaction. S, selectin; I, integrin receptor; AP, adhesive protein.Hakomori, S., et al. 2000 (15).11

Introduction: glycosphingolipidsII.1.1.1. Glycosphingolipids in embryonal development and inembryonal cell adhesionPatterns of GSL expression change greatly during development and differentiation.Early mouse embryos at the early 8- to 32-cell stage specifically express the Lewisx(Lex) determinant (also referred to as stage specific embryonic antigen-1 or SSEA-1)related to the Lewis blood group determinants. Lex shows maximal expression at the16- to 32-cell stage and declines rapidly after compaction27, i.e., tight aggregation ofembryonal cells, beginning approximately after the third division of the fertilizedegg. This pattern indicated a role of Lex in mediating compaction of the mouseembryo at the morula stage. Experimental evidence for a functional role for surfaceLex was provided by the finding that a multivalent derivative of the oligosaccharidelacto-N-fucopentaose III (LNF III), which contains Lex determinant in its structure,caused decompaction of fully compacted mouse embryos28. Compaction is a Ca2 dependent cell adhesion event, the first of many specific cell-cell interactionsoccurring during mammalian embryogenesis. Without this adhesion subsequentdevelopment of the embryo may not occur.A remarkable similarity has been demonstrated between the processes of morulacompaction in early embryogenesis and aggregation of undifferentiated F9 mouseembryonal cells. F9 cells mimic the morula-stage preimplantation embryo and showCa2 -dependent cell aggregation. They also express high levels of cell surface Lex atthe undifferentiated stage, which declines upon differentiation29. It has been foundthat Lex at the cell surface is recognized per se by another Lex, and that this Lex-Lexinteraction mediates aggregation of F9 cells in the presence of a bivalent cation30.Both results, with mouse embryos and mouse embryonal cells, clearly indicated thatLex, a specific carbohydrate structure, is capable of self-interacting, and suggest thatthis carbohydrate-carbohydrate interaction may account for cellular recognition.Many human embryonal carcinoma cells, particularly at the undifferentiated stage,show high expression of globo-series GSLs structures including SSEA-3 and -4,12

Introduction: glycosphingolipidswhich are down-regulated upon differentiation in parallel with a decrease in celladhesion31,32. Undifferentiated human embryonal carcinoma 2102 cells express highamounts of the Lex precursor lactoneotetraosylceramide (nLc4) and SSEA-3 (withthe major epitope GalGb4), and moderate level of globoside (Gb4)33. Expression ofthese GSLs declines in association with a decline of homotypic adhesion during oGb4andgangliotriaosylceramide (Gg3) coated on plates, while they do not adhere to otherGSL epitopes33. Adhesion of 2102 cells to Gb4, which stimulates cell aggregation, isbased on carbohydrate-carbohydrate interaction between nLc4 or GalGb4 (expressedon cells) and Gb4 (coated on plates). Furthermore, binding to Gb4 induces signaltransduction in terms of activation of transcription factors AP1 and CREB. Bindingto Gg3 does not result in any cell activation indicating that there is also somequalitative difference in binding to different GSL layers. These findings prove thatGb4 and globo-series GSLs are involved in cell adhesion, analogous to theinvolvement of Lex in compaction of mouse embryo and aggregation of F9 cells.II.1.1.2. Glycosphingolipids in specific recognition betweenlymphoma and melanoma cellsFurther support for the role of carbohydrate-carbohydrate interactions in cellularrecognition was provided by the demonstration of the specific aggregation of mouselymphoma L5178 cells with mouse melanoma B16 cells based on the interactionbetween two gangliosides: Gg3 and GM334. Gg3 is highly expressed at the surface ofmouse lymphoma cells and GM3 (sialosyllactosylceramide) is expressed at the cellsurface of mouse melanoma cells. The interaction between cells was inhibited bymonoclonal anti-Gg3 and anti-GM3 antibodies. Since these antibodies are highlyspecific for the gangliosides and do not cross-react with carbohydrates onglycoproteins, it has been assumed that the specific cellular recognition between thelymphoma and melanoma cells is indeed based on molecular carbohydratecarbohydrate interactions between Gg3 and GM3. Adhesion of B16 melanoma cells to13

Introduction: glycosphingolipidsGg3-coated surfaces enhanced tyrosine phosphorylation of FAK35. Direct bindingbetween GM3 on the cell surface to Gg3 on the coated plates has been suggested asthe activating mechanism since antibodies against GM3 could activate FAK.The adhesion and spreading of B16 melanoma cells on coated culture dishes wasmostly obvious at early stages of cell plating, which indicates that GSL-mediatedinteraction are very early phenomena in cellular interactions to be overtaken in laterstages by protein- mediated binding36. Furthermore, melanoma cells could adhereand spread faster on glycolipids coated on plates than to extracellular matrixproteins: laminin or fibronectin coated on plates37. Different clones of the B16 linewith different expression levels of the predominant GSL were tested for their bindingbehavior to non-activated endothelial cells. Binding of melanoma cells to endothelialcells was faster but weaker than binding to laminin or fibronectin, and wasdependent on GM3 expression level. Under shear forces, binding strength of themutant B16 cells was still correlated to the expression level of GM3, but it wasstronger than the binding via extracellular proteins. Upon these findings, it has beensuggested that matastatic tumor cells make use of the high expression rates of certainglycolipids to attach to the unstimulated endothelium, before next steps in cellactivation and transmigration are mediated by protein-protein interactions38.GSL-dependent cell adhesion can modulate signal transduction. GM3-dependentadhesion of melanoma cells enhanced tyrosine phosphorylation of cSrc and FAK,and enhanced GTP binding to Rho A and Ras15. Enhanced motility of melanomacells caused by GM3-dependent adhesion to endothelial cells was regarded as theinitial step of melanoma cell metastasis38.14

Introduction: proteoglycansII.1.2. CARBOHYDRATE INTERACTIONS ININVERTEBRATES – PROTEOGLYCANSVirtually all animal cells produce proteoglycans, which vary greatly in structure,expression and functions39,40. They are found in all connective tissues, extracellularmatrix, and on the surfaces of many cell types. Proteoglycans participate in andregulate cellular events and (patho)physiological processes via either theircarbohydrate chains (glycosaminoglycans, GAGs) or their core proteins (Fig. 5). TheGAG chains have the ability to fill the space, bind and organize water molecules andrepel negatively charged molecules. Because of high viscosity and lowcompressibility they are ideal for a lubricating fluid in the joints. On the other handtheir rigidity provides structural integrity to the cells and allows the cell migrationdue to providing the passageways between cells. For example the large quantities ofchondroitin sulfate (CS) and keratan sulfate (KS) found on aggrecan play animportant role in the hydration of cartilage. They give the cartilage its gel-likeproperties and resistance to deformation. Proteoglycans have the ability to regulateproteolytic enzymes and protease inhibitors. Functions of proteoglycans in cell andtissue development and physiology are mediated by specific binding of GAGs orcore proteins to other macromolecules. They bind to signalling molecules, which canlead to the stimulation or prevention of the activity of growth factors. Cell surfaceproteoglycans act as co-receptors, e.g. syndecans serve as a receptor with integrin forfibronectin and other matrix proteins. Despite their structural and functionaldiversity, proteoglycans do have a general propensity to be extracellular matrixcomponents and to mediate specific matrix interactions and biological activitiesrelated to different aspects of cell adhesion41,42, but many of their roles in thesecellular processes are still poorly understood.15

Introduction: proteoglycansReceptors formatrix proteins incell adhesionStimulation orprevention of theactivity of growthfactorsFluid lubricants inthe jointsproteoglycansRegulation ofproteolyticenzymes andproteaseinhibitorsRegulation of thetraffic ofmoleculesFig. 5. Proteoglycans functions. Five different functions are shown schematically and they arereviewed in 39-42, and in 48.A sulfated carbohydrate component of proteoglycan molecules, glycosaminoglycan(GAG), is covalently linked to the core protein (Fig. 6). Core proteins vary in sizefrom 11’000 to about 220’000 Da. The number of GAG chains attached to the coreprotein varies from one to about 100. There are four main types of GAGs: 1) heparin/ heparan sulfate (HS)43, 2) chondroitin sulfate (CS)44 / dermatan sulfate (DS)45, 3)keratan sulfate (KS)46, and 4) hyaluronic acid (HA)47. Each GAG is a polymer of adisaccharide, which in heparin, HS, and HA consists of N-acetyloglucosamine anduronic acid, in CS and DS of N-acetylogalactosamine and uronic acid, and in KS ofN-acetyloglucosamine and galactose. The sugars in GAGs are sulfated to varyingdegrees. An exception is HA, which is not sulfated and is the only GAG present16

Introduction: proteoglycansunder its free form and possessing the ability to aggregate with the class ofproteoglycans termed hyalectans48. There is a potential for an enormous number ofproteoglycans due to the variations in the molecular weight of the core protein, in thetypes and number of GAG chains attached to the protein core, and in sulfationdegree39.Fig. 6. Schematic representation of a proteoglycan structure. A, A single proteoglycan consistsof a core protein molecule to which a large number of glycosaminoglycan chains (GAGs, shown inred) are covalently attached. B, In the cartilage matrix, individual proteoglycans (in box from fig. A)are linked to a non-sulfated GAG, called hyaluronic acid (HA), to form a giant complex with amolecular mass of about 3’000’000. C, Electron micrograph of a proteoglycan complex isolated fromcartilage complex.The very first experimental demonstration of cellular recognition and adhesionphenomena in the animal kingdom was assigned to the cell surface proteoglycan49and came from invertebrates, i.e. from marine sponge model system50,51. Spongesevolved from their unicellular ancestors about 1 billion years ago by developingcellular recognition and adhesion mechanisms to discriminate against "non-self”.17

Introduction: proteoglycansThey are the simplest and earliest multicellular organisms. They do not have anydefined organs or tissues, and only a limited number of cells remain motile withinthe animal. Remarkably, dissociated sponge cells from two different species have thecapacity to reaggregate through surface proteoglycans by sorting out according totheir species of origin, in the same way as mixtures of dissociated embryonic cellsfrom two vertebrate tissues sort out according to their tissue of origin.The purification of proteoglycans is often complicated, but sponge proteoglycans arevery easy to extract in large quantities by removal of extracellular calcium, and theyare abundant in the sponge extracellular matrix. It makes sponges one of the bestpotential models to study proteoglycan structure and function. Consequently, thissimple and highly specific cellular recognition phenomenon of cell-cell aggregationin sponges has been used for almost a century as a model system to study specificcellular recognition and adhesion occurring during tissue and organ formation inmulticellular organisms.II.1.2.1. Structure and function of sponge proteoglycanThe extracellular matrix of the sponge is similarly composed to that of higherMetazoans, containing proteoglycan molecules, collagens and other glycoproteins.Sponge surface proteoglycans52, otherwise known as aggregation factors (AFs), arelarge molecules with an approximate molecular weight raging from 2 x 104 kDa53,54to 1.4 x106 kDa55. Based on their composition and on their electron56 and atomicforce microscope (AFM) images57, sponge cell surface proteoglycan molecules showeither a linear or a sunburst-like core structure with 20-25 radiating arms (Fig. 7).AFM visualization of sponge proteoglycans allowed a better estimation of theirmolecular dimensions and showed remarkable similarities between molecules fromdifferent species (Table 1).Prolonged (4-weeks) EDTA-treatment of the18

Introduction: proteoglycansproteoglycan disrupts the arms from the core structure, indicating that the cation isimportant for the structural integrity of the complex57.Specific cellular recognition of marine sponges is mediated by cell surfaceproteoglycan molecules in a Ca2 -rich environment ( 10 mM, as in seawater). Themodel of proteoglycan-mediated cell-cell adhesion currently used assumes that theproteoglycan molecule is immobilized via its arms onto the cell surface and the corestructure interacts with the core structure immobilized on another cell (Fig. 8). Themolecular basis of the selective cell-cell adhesion in most multicellular animals ismediated by two distinct classes of molecules: a Ca2 -idependent activity like thattypical of the glycoproteins from the Ig superfamily58, and a Ca2 -dependent cell-celladhesion, whose best example is the cadherins59. Interestingly, sponge proteoglycansreunite both functions in the same molecule and mediate species-specific cell-cellrecognition via two functionally distinct domains: 1) a calcium-independent cellbinding domain and 2) a calcium-dependent self-association domainwhich isproviding the intercellular adhesion force.Receptors for sponge proteoglycans are called baseplates. Microciona proliferacellular receptors include membrane-associated glycoproteins of 210 kDa and 68kDa60 with low carbohydrate content, and with a high affinity to both the cells andthe proteoglycan molecules61,62. Geodia cydonium receptors are of low molecularweight (Mr approximately 20’000) and consist chemically of glycoproteins withhigh carbohydrate content63.The binding of surface proteoglycans to sponge cells triggers a wide variety ofcellular responses. The addition of purified proteoglycans to primary cell aggregatesof Geodia cydonium resulted in increased DNA, RNA, and protein synthesis, andin higher mitotic activity64,65. Moreover, binding of the surface proteoglycan toGeodia cells triggered protein phosphorylation66 and ras gene expression67. Finally,involvement of main proteins of Microciona prolifera surface proteoglycan insponge histocompatibility has been suggested68.19

Introduction: proteoglycansMicrociona prolifera proteoglycanHalichondria panicea proteoglycanSuberites fuscus proteoglycanFig. 7. Atomic force microscopy (AFM)images of different proteoglycans.Air-dried surface proteoglycans from threedifferent sponge species were immobilizedon glass and visualized with the AFM.Dimensions of the picture are 3x3 µm. Thecolor-encoded vertical z-scale of the imagescorresponds to 3 nm (dark brown: 0 nmelevation from the surface; white: 3 nmelevationfromthesurface;yellow:intermediate elevation).Table 1. Molecular dimensions of proteoglycans from three different sponge species asestimated from AFM images.proteoglycanM. proliferabackboneshapeRingbackbone length(nm)285H. paniceaRod280140ca. 20S. fuscusRod22080ca. 20Jarchow, J., et al. 2000 (57).20arm length(nm)143number ofarmsca. 20

Introduction: proteoglycansFig. 8. The model of proteoglycan-mediated cell-cell adhesion.A, Proteoglycan molecule with a ring core structure is immobilized via its arms to the cell surfaceand mediates cell-cell interaction via its ring structure. B, Atomic force microscopy (AFM) image of acircular-structure proteoglycan. C, Proteoglycan molecule with a backbone core structure isimmobilized via its arms to the cell surface and mediates cell-cell interaction via its backbonestructure. D, AFM image of a backbone-structure proteoglycan.The protein and carbohydrate content in purified proteoglycans varies amongdifferent sponges. Proteoglycans can consist of as high as 75% protein and just 25% carbohydrates, as in indicated for Geodia cydonium69 and Suberitesdomuncula70. In contrast, Microciona prolifera proteoglycans consist of about 60%carbohydrates and 40% proteins49.21

Introduction: carbohydrate moiety of proteoglycansAt present, the surface proteoglycan from the read beard sponge, Microcionaprolifera, is best characterized. There are two main proteins in Microciona circularproteoglycan, termed MAFp3 (ranging from 30 to 50 kDa) and MAFp4 ( 400kDa)57. Both molecules are extremely polymorphic71. MAFp3 is found exclusively inthe ring structure, while MAFp4 is found exclusively in the arms57. As in most largeproteoglycans, the MAFp4 core protein has a modular structure made of tandemrepeats68. However, MAFp4 does not have significant sequence homologies with anyknown proteoglycans. The closest matches, i.e. 30% identity with MAFp4 repeats,were found in two apparently unrelated proteins: the intracellular loop of Na -Ca exchangers72 and a similar domain-structured endoglucanase from the symbioticbacterium Azorhizobium caulinodans68. Two different carbohydrates with molecularmasses of 6 and 200 kDa are found in the core structure and the arms of adhesionproteoglycans.II.1.2.2. Carbohydrate moiety of sponge proteoglycanMicrociona prolifera surface proteoglycans carry two N-linked glycan molecules:one with a mass of 6.3 kDa73 (termed g-6), believed to bind to a cell surface receptorindependently of Ca2 ions, and one with a mass of 200 kDa74 (termed g-200) thatself associates in a Ca2 -dependent manner75 (Fig. 9). G-6 is the main glycan presenton MAFp4 protein in the arms of proteoglycan molecule, and each arm containsabout 50 g-6 units57. G-200 is the main glycan present on MAFp3 protein in the ringstructure of the proteoglyc

epitopes26; 2) Mediated by complementary carbohydrate moieties of GSLs through carbohydrate-to-carbohydrate interaction. In either model, cell adhesion based on the carbohydrate-carbohydrate interaction is the earliest event in cell recognition, followed by the involvement of adhesive proteins and of integrin receptors (Fig. 4).

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