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Address correspondence to Max M. Burger, Novartis Science Board, Novartis International AG, WKL-125.13.02, CH-4002 Basel, Switzerland. Tel: 41-61-696-7690. Fax: 41-61-696-7693. email: max.burger{at}group.novartis.com
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Abstract |
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Key Words: cellcell recognition; cell surface proteoglycan; carbohydratecarbohydrate interaction; species specificity; adhesion force
Abbreviations used in this paper: AFM, atomic force microscopy; CSW, Ca2+- and Mg2+-free artificial seawater buffered with 20 mM Tris, pH 7.4, supplemented with 2 mM CaCl2; Lex, Lewisx determinant (Galß1
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Introduction |
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The very first experimental demonstration of cellular recognition and adhesion phenomena in the animal kingdom came from an invertebrate system, i.e., from marine sponges (Wilson, 1907), and was later assigned to cell surface proteoglycans (Humphreys, 1963). Dissociated sponge cells from two different species have the capacity to reaggregate through surface proteoglycans (Fernandez-Busquets and Burger, 2003) in a Ca2+-rich environment (10 mM, i.e., physiologic for seawater) by sorting out according to their species of origin, in the same way as dissociated embryonic cells from two different vertebrate tissues sort out according to their tissue of origin. Consequently, this simple and highly specific cellular recognition phenomenon in sponges has been used for almost a century as a model system to study recognition and adhesion events in multicellular organisms. Sponge cellcell aggregation involves Ca2+-independent binding of proteoglycans to a cell surface and Ca2+-dependent self-association of proteoglycans (Turner and Burger, 1973; Jumblatt et al., 1980). A monoclonal antibody raised against the purified proteoglycan from Microciona prolifera sponge inhibited the proteoglycan self-association and the epitopes were identified as short carbohydrate units of the 200-kD glycan (Misevic and Burger, 1993): a sulfated disaccharide (Spillmann et al., 1995) and a pyruvylated trisaccharide (Spillmann et al., 1993). Recently, Vliegenthart's group could demonstrate self-interactions of the sulfated disaccharide using surface plasmon resonance (Haseley et al., 2001). However, species-specific interactions between 200-kD glycans from different sponge species have not yet been demonstrated in order to prove the existence of species-specific carbohydratecarbohydrate recognition system.
200-kD glycan moieties from adhesion proteoglycans from four different marine sponge species were purified here and the species specificity of a glycanglycan interaction was investigated in aggregation and adhesion assays. Atomic force microscopy (AFM) measurements were performed to measure the binding strength between single interacting glycan molecules and to demonstrate quantitative differences in binding forces between different species of 200-kD glycans. Results confirm the concept of the relatively strong and species-specific carbohydratecarbohydrate interaction as an important player in cellular recognition.
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Results |
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Aggregation of glycan-coated beads mimics species-specific cellular aggregation
An assay for glycanglycan recognition was designed, which mimics the classical assay for specific aggregation of sponge cells. Glycan-coated red and green beads the size of small sponge cells were allowed to aggregate under identical shear forces, i.e., rotor speed as used for cellcell recognition assays. Beads coated with glycans from identical proteoglycans formed 6379% yellow aggregates, which are the result of intermingling of red and green beads (Fig. 2). In stark contrast, beads coated with glycans derived from proteoglycans from different species did separate into red and green aggregates. In this case yellow aggregates, i.e., heterotypic mixtures of glycans originating from different species, never formed >12% of aggregated patches.
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As in cellcell and cellglycan recognition, the absence of Ca2+ ions inhibited the glycanglycan recognition. The antibody directed against the carbohydrate epitope of the Microciona proteoglycan inhibited the homotypic interaction between Microciona glycans coated on red and green beads. There was no visible effect of the antibody on homotypic interactions between other species glycans (unpublished data). Results obtained with glycan-coated beads (Fig. 2) reflect thus the same results obtained with live cells (Fig. 1, AD).
It has been reported previously that 400 molecules of Microciona proteoglycan bound per cell cause live cells to aggregate (Jumblatt et al., 1980). The number of 200-kD glycan copies per proteoglycan molecule was determined from the mass of total carbohydrate recovered in 200-kD glycan fractions either after gel electrophoresis or gel filtration (Misevic and Burger, 1993). Because 37% of the total carbohydrate content of the proteoglycan molecule occurred in the form of 200-kD glycan (70% of the proteoglycan mass is carbohydrate; proteoglycan Mr = 2 x 107), one proteoglycan carries
26 copies of this glycan. Therefore,
10,400 glycan molecules (400 proteoglycan molecules) per cell cause living cells to aggregate. In our experiments, binding measurements indicated that
2,500 molecules of 200-kD glycan per bead specifically aggregated glycan-coated beads. The number was calculated from the specific absorbance of stained glycans after reversing the binding to beads (which gave the number of moles: 0.192 x 1011) and Avogadro's number, and was divided by the number of beads (4.5 x 108). Surface areas of the cell and the bead were calculated from diameters, and they were 12.56 µm2 and 3.14 µm2 accordingly. This led to the final assessment that the glycan density per cell and per bead causing species-specific live cell and glycan-coated bead recognition and aggregation is similar: 828 molecules/µm2 for cellcell aggregation and 810 molecules/µm2 for glycan-coated beadbead aggregation.
Live cells and cell surface glycans adhere species specifically to glycans coated on a plastic surface
The binding of live cells to glycans from their surface proteoglycans coated onto a solid polystyrene phase was assessed (Fig. 3, AD). The binding to glycans from proteoglycans from different species of origin was three to five times lower. Cell adhesion showed clear dependence on the quantity of the glycan coated, and could be abolished in the absence of Ca2+ ions. Pretreatment of Microciona cells with the antibody directed against the carbohydrate epitope of their surface proteoglycan inhibited 86% of these cells from adhesion to their own glycan (Fig. 3 E). In this assay, little cross-reactivity of the antibody (Misevic et al., 1987) could be detected because it blocked only 23% or less adhesion between other cells and their 200-kD glycans.
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Specificity of the carbohydratecarbohydrate interaction is also reflected in the polyvalence
The characteristic feature of the glycanglycan interaction is the repetition of interactive sites along the glycan chain, which further increases the strength of the interaction and thus the specificity. The distance between the peak numbers 1 and 2 (2 and 3, etc.) on force curves was measured to produce a histogram of the peak periodicity (Fig. 6 B). 80% of force curves between glycans from the same species of cells showed more than one interaction peak, with a distance between binding motifs of 20 nm. In contrast, <35% of force curves between glycans from different species of cells showed multiple interaction peaks, demonstrating the preference for one rather unspecific binding event during the interaction.
The glycan molecules used here have chain-like structures of an average folded length of 40 nm as imaged by AFM (Jarchow et al., 2000), whereas the extended structure has a length of up to 180 nm (Dammer et al., 1995). 75% of the total lengths of the force curves for the same species glycans were 2050 nm, and in some cases the curves showed extensions up to 130 nm (Fig. 6 C). This then indicates that the interaction sites are located along the carbohydrate chain and not only at its end. In contrast, 70% of the force curves for glycans from two different species showed total interaction lengths of 1030 nm only.
Pronase digestion of glycans is essentially complete
Proteoglycan molecules were subjected to an extensive pronase digestion in order to obtain protein-free glycans. The total 200-kD glycans were separated from free amino acids and peptides by gel filtration and ion-exchange chromatography (Misevic et al., 1987). The 200-kD glycan has an apparent Mr = 200 x 103 ± 40 x 103, and electrophoretic and chromatographic separation techniques indicated that the glycan is a single molecular species with possible charge and size microheterogeneities (Misevic et al., 1987). There have been essentially no losses of carbohydrates during the purification procedures because the carbohydrate yield of the glycan fractions was 97%. Amino acid analysis of glycans from the four sponge species used showed that there was from 0.5 (Cliona celata) to 0.9 (Microciona prolifera) mole of linker aspartate/mol of glycan (Table I). Only trace amounts of a few other amino acids were detected. This indicates that the digestion was complete and that the purification procedure for the 200-kD glycans led to essentially pure glycan fractions, free of any protein contaminations.
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Discussion |
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The examination of carbohydratecarbohydrate interactions at the atomic level is critical in understanding the nature of these interactions and their biological role. Measured adhesive forces between identical glycans (190310 pN) compare well with the range of forces between the entire proteoglycan molecules, which vary from 50 to 400 pN, depending on the number of binding sites ruptured (Dammer et al., 1995; Popescu et al., 2003). Similar values are also reported for other biologically relevant forces, e.g., for single proteinglycan interactions (Fritz et al., 1998; Hanley et al., 2003), when interaction of P-selectin from leukocytes with its carbohydrate ligand from endothelial cells was measured (165 pN), or for single antibodyantigen recognition (Hinterdorfer et al., 1996; Saleh and Sohn, 2003) with rupture forces of 244 pN. The force spectra shown in Fig. 5 exhibit the general shape anticipated for simple entropic polymers that extend until the carbohydratecarbohydrate interaction ruptures. Considering molecular compliance it should be noted that the rupture forces measured here are below those inducing the chair-boat transition, which could have interfered with the interpretation of the results.
The density of glycans on the AFM probe tip and the substrate was adjusted to meet the expectation that the surface layers are neither multilayered nor clustered, and therefore, direct glycanglycan interactions were measured. The exact number of single rupture events between two interacting glycan molecules is difficult to define with certainty because it cannot be excluded that a so-called "single event" may be a composite of more than one bond rupture. Nevertheless, because the statistical analysis of pull-off distances show that the overall lengths of force curves (from 20 nm up to 130 nm) are less than the expected extended length of the glycan (160180 nm), it can be assumed that single glycan molecule and not multiple glycan molecule interactions were measured here.
The specificity of glycan-mediated recognition is guaranteed both by the higher adhesion forces per binding site as well as by the higher amount of polyvalent interactions between glycans from the same species versus glycans from different species. The repetition of the binding motif along the carbohydrate chain ensures sufficient binding strength to function in vivo. Polyvalence can be controlled by various means: e.g., by surface density of presented structures, ionic strength modulating attractive versus repulsive forces, subtle changes in biosynthesis of the carbohydrate sequences, etc. These allow changing the affinity of the interactive molecules and therefore, creating a highly flexible and specific model of recognition system. The model assumes gradual adhesion during initial contact between two different cells or cell and matrix by allowing cells to test surrounding surfaces and first create weak random contacts before releasing or reinforcing adhesion (Burger, 1979). Therefore, interactions between two different species of glycans could still be recorded in AFM measurements, though of lower stability than these between glycans from the same species. Similarly, some amount of heterotypic aggregates consisting of different species of glycans was present in glycan-coated bead aggregation experiments.
Ca2+ ions or other divalent cations are crucial in carbohydratecarbohydrate interactions. Here, the presence of Ca2+ ions was essential. No interaction between cells, cells and surface glycans, and between surface glycans could be observed in the absence of Ca2+. Also no adhesion forces between single glycan molecules could be detected during AFM measurements. However, it has been reported that the presence of Ca2+ ions did not contribute significantly to the adhesion force in LexLex interaction (Tromas et al., 2001). On the other hand, self-aggregation of Lex molecules in aqueous solution, where the molecules move freely, occurred only in the presence of Ca2+ ions (de la Fuente et al., 2001). On the molecular level, Ca2+ ions probably provide coordinating forces (Haseley et al., 2001), though ionic forces cannot be excluded. These Ca2+ interactions are thought to stabilize conformations and can thereby lead to hydrogen bonds and hydrophobic interactions elsewhere in the glycan molecule (Spillmann and Burger, 1996). Further studies are required to resolve the exact role of Ca2+ and other divalent cations in carbohydratecarbohydrate interactions.
Inhibition of sponge cell recognition and aggregation by species-specific carbohydrate epitope antibodies as shown earlier (Misevic et al., 1987; Misevic and Burger, 1993) do not prove glycanglycan interactions to be relevant because they leave the option of glycanprotein interactions open. The same interpretation holds for two of the approaches presented here: species specificity for the glycan-coated bead interaction with live sponge cells (Fig. 1, EH) and for the live cells binding to glycan-coated plastic surfaces (Fig. 3). The specificity found here for glycanglycan interaction (Fig. 4), for glycan-coated bead sorting (Fig. 2), and the force and specificity shown in the AFM measurements (Fig. 6) make, however, a role for carbohydratecarbohydrate in sponge cell recognition and adhesion likely. The fact that the outermost cell surface is made up primarily of a dense layer of hydrophilic glycans supports the notion that upon first contact between cells such reversible and flexible glycanglycan interactions may play a pivotal role in cell recognition processes.
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Materials and methods |
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Analytical methods
For amino acid analyses, dry glycan samples were diluted in 1-ml of ultra pure water and the aliquots of 25 µl were lyophilized and hydrolyzed during 24 h. After hydrolysis, the black residues were suspended in 100 µl of 50 mM HCl containing 50 pmol/µl Sar and Nva each while ultrasonicated for 15 min. After a 15-min centrifugation, the transparent solutions were transferred into new reagent tubes and analyzed on a Hewlett-Packard AminoQuant II analyzer.
Aggregation assay
4.5 x 108 freshly sonified amine-modified beads (1-µm diam; Molecular Probes) were coupled with isolated glycans (1.5 mg/ml) by incubation in Ca2+- and Mg2+-free artificial seawater buffered with 20 mM Tris, pH 7.4, supplemented with 2 mM CaCl2 (CSW), and 2 mg of 5,5'-dithiobis-(2-nitrobenzoic acid) (Molecular Probes) overnight at RT. Coupling efficiency was determined by measuring the glycan concentration (by staining with 1% Toluidine blue) on the beads after reversing the 5,5'-dithiobis-(2-nitrobenzoic acid) cross-linking with the disulfide-reducing agent DTT. The number of 200-kD glycan molecules bound per bead was calculated from the specific absorbance of stained glycans, which gave the number of moles (0.192 x 1011) multiplied by Avogadro's number (6.022 x 1023), and was divided by the number of beads (4.5 x 108).
9 x 106 glycan-coated beads in 400 µl of CSW were allowed to aggregate with cells or other glycan-coated beads on a rotary shaker at 60 rpm for 4 h after the addition of 10 mM CaCl2. Images of aggregates were acquired with a confocal laser-scanning microscope (Leica) equipped with an argon/krypton laser and a 10x objective (PL Fluotar, N.A. 0.3). Image processing was performed using Adobe Photoshop version 6.0. Quantifications were performed using UTHSCSA Image Tool version 2.00 Alpha.
Binding of cells and glycans to glycan-coated plates
Solutions of 200-kD glycans in CSW were placed in each well of a 96-well plastic plate (Falcon; 0.3 ml/well vol). After 2 h, each well was washed with CSW, 100 µl of glycans in 0.1 mg/ml CSW were added and incubated for 2 h at RT, after addition of 10 mM CaCl2. Afterwards, nonbound glycans were washed off with CSW containing 10 mM CaCl2. Bound glycans were stained with 1% Toluidine blue and absorbance was measured at 630 nm. The absorbance of glycans used as a coat was deducted from the total absorbance measured after the addition of glycans to coated wells to give the absorbance of bound glycans.
100 µl of live cells (5 x 103) in CSW were added to each glycan-coated well, supplemented with 200 µl of CSW containing 10 mM CaCl2 and incubated for 2 h at RT. Afterwards, plates were immersed in CSW with 10 mM CaCl2 in a large container, suspended upside down for 10 min with gentle shaking to allow nonadherent cells to sediment out of plates. Bound cells were lysed for 10 min in 2 M NaCl, 20 mM Tris-HCl, pH 7.5. 200 ng Hoechst stain in 20 mM Tris-HCl, pH 7.5, was added to cell lysates and the fluorescence was measured at ex = 360 nm and
em = 450 nm.
AFM
Force measurements were performed with a commercial Nanoscope III AFM (Digital Instruments) equipped with a 162-µm scanner (J-scanner) and oxide-sharpened Si3N4 cantilevers with a thickness of 400 nm and a length of 100 µm. Cantilever spring constants k measured according to Chon et al. (2000) for a series of cantilevers from the same region of the wafer revealed on average k = 0.085 N/m and SD = 0.002 N/m. We observed a variation of <15% for k values from different cantilever batches. However, all measurements were done with cantilevers from the same batch.
AFM supports were built as described previously (Müller et al., 1999). The mica was cleaved using scotch tape, masked using a plastic ring mask with an inner diameter of 5 mm, and brought into the vacuum chamber of the gold sputter coater (Bal-Tec SCD 050). A vacuum of 102 mbar was generated and interspersed by rinsing the chamber with argon gas. At the pressure of 5 x 102 mbar a 20-nm gold layer was deposited on the mica surface and the tip controlled by a quartz thickness and deposition rate monitor (Bal-Tec QSG 050). Gold coated Si3N4 tips and micas allowed covalent chemisorptions of the naturally sulfated carbohydrates. Both the support and the tip were overlaid with CSW containing 10 mM CaCl2 and the goldgold interaction was measured. Afterwards, the support and the tip were incubated with isolated glycans (1 mg/ml) for 15 min at RT. Nonbound glycans were washed off with CSW, and glycanglycan force measurements were performed in CSW containing 10 mM CaCl2. For each measurement, a new tip was coated with 200-kD glycan and a new Au substrate was prepared. The AFM stylus approached and retracted from the surface 100 times with a speed of 200 nm/s. The tip was moved laterally by 50 nm after recording five force-distance curves.
Surface clustering of 200-kD glycans was determined by fluorescence imaging. Glycans were labeled through their amino groups of the amino acid portion with 5(6)-carboxyfluorescein-N-hydroxysuccinimidester (Boehringer). Labeled glycans were separated from free labeling substance via a P-6 sizing column (Amersham Biosciences) in 100 mM pyridine-acetate buffer, pH 5.0.
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Acknowledgments |
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This work was supported by the Friedrich Miescher Institute, branch of the Novartis Research Foundation, the M.E. Müller Foundation, and the Swiss National Research Foundation.
Submitted: 2 September 2003
Accepted: 17 March 2004
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