Carbohydrate-carbohydrate interaction provides adhesion force and specificity for cellular recognition and adhesion

Carbohydrates at the cell surface have been proposed as mediators in cell-cell recognition events involved in embryogenesis, metastasis, and other proliferation processes by calcium-dependent carbohydrate to carbohydrate interactions. They are the most prominently exposed structures on the surface of living cells, and with flexible chains and many binding sites are ideal to serve as the major players in initiating these cellular events. However, biological relevance of these type interactions is often questioned because of the very low affinity binding of single carbohydrate molecules and that they manifest themselves only through the contact of a large number of molecules tightly arranged in the membrane. Weak interactions are considerably more difficult to study and only a few biologically significant examples of direct carbohydrate-carbohydrate interactions have been reported, e.g. pioneering work showing glycosphingolipid self-interactions through multivalent interaction of Lewis X epitopes. However, there are no reports on the existence of specific proteoglycan self-interactions through carbohydrate-carbohydrate interactions in cellular recognition system, as it has been done with glycosphingolipids. Here, we used sponges, organisms on which the first proteoglycan-mediated cell-cell recognition in the animal kingdom was demonstrated, as a model system to study carbohydrate-mediated cellular recognition. We show that the interaction between single oligosaccharides from surface proteoglycans is relatively strong and comparable to protein-carbohydrate interactions, highly specific, and dependent on Ca2+-ions. 200 kDa glycans from the core protein of Microciona prolifera cell surface proteoglycans have been previously shown to mediate homotypic Microciona proteoglycan-proteoglycan interactions. Here, 200 kDa glycans from four different sponge species: Microciona prolifera, Halichondria panicea, Suberites fuscus and Cliona celata were purified and investigated for species-specific interactions. Selective recognition of glycans by live cells was studied to confirm the existence of glycan-glycan recognition system in biologically relevant situations. Mature sponge cells have the ability to reaggregate species-specifically and form homogenous aggregates on a shaker at the right shear forces in the presence of physiological 10 mM Ca2+. Live cells were allowed to aggregate with glycan-coated beads similar in size to small sponge cells in the presence of calcium. They specifically recognized beads coated with their own glycans and did not mix but separated from beads coated with glycans isolated from different species. The glycan-glycan recognition assay was developed to mimic species-specific cellcell recognition in sponges. 200 kDa glycans immobilized onto beads similar in size to small sponge cells assembled species-specifically in the presence of physiological calcium, at the same shear forces as in cell-cell aggregation. Glycans coated on beads aggregated with glycans from the same species coated on beads, and separated from glycans from other species. The glycan density necessary for specific live cellcell recognition in sponges is 828 molecules/μm2. In our studies, the glycan density necessary for specific glycan-coated bead was very similar: ~810 molecules/μm2. Mature live cells demonstrated specific recognition of 200 kDa glycans during selective-binding to glycans coated on surfaces in the presence of calcium. They strongly adhered to glycans from their own surface proteoglycans coated onto a solid polystyrene phase, while the binding to glycans from different proteoglycans was 3 - 5 times lower. Moreover, homotypic adhesion to glycan-coated plates enhanced sponge cell differentiation and formation of mineral skeleton (spicules). Larval cells, after settlement and spreading of larvae, can fuse species-specifically in nature. In our studies, live larval cells recognized and adhered specifically to glycans purified from adhesion proteoglycans from their "mother sponge". They showed almost no interaction with glycans from other species. As in cell-glycan adhesion assays, highly species-specific adhesion of 200 kDa glycans to glycan-coated surfaces could be observed in the presence of physiological calcium. Tested glycans bound strongly to glycans from the same species and showed up to a six fold reduction in binding to glycans from other species. Atomic force microscopy (AFM) was performed to measure for the first time adhesion forces between single glycan molecules obtained from different surface proteoglycans. Measurements revealed equally strong adhesion forces in the range of several hundred piconewtons (pN) between glycan molecules as between proteins and glycans measured in another recognition system. Moreover, statistically significant differences (p value < 0.01) were seen between homotypic (glycans from the same species) and heterotypic (glycans from different species) interactions. Moreover, the polyvalent character of binding characterized mainly interactions between glycans from the same species. This indicates that not only the higher adhesion force per binding site as such but also the higher amount of multiple interactions between glycans from the same species versus mixture of glycans from different species guaranteed the specificity of the glycan-mediated recognition. These findings confirm for the first time the existence of specific glycan-glycan recognition system between cell surface proteoglycans. We propose that these cell's outermost surface structures serve as important players in initiating the very first contacts between cells through highly species-specific and flexible carbohydratecarbohydrate interactions

Carbohydrate-carbohydrate interaction provides adhesion force and specificity for cellular recognition and adhesion

Carbohydrates at the cell surface have been proposed as mediators in cell-cell recognition events involved in embryogenesis, metastasis, and other proliferation processes by calcium-dependent carbohydrate to carbohydrate interactions. They are the most prominently exposed structures on the surface of living cells, and with flexible chains and many binding sites are ideal to serve as the major players in initiating these cellular events. However, biological relevance of these type interactions is often questioned because of the very low affinity binding of single carbohydrate molecules and that they manifest themselves only through the contact of a large number of molecules tightly arranged in the membrane. Weak interactions are considerably more difficult to study and only a few biologically significant examples of direct carbohydrate-carbohydrate interactions have been reported, e.g. pioneering work showing glycosphingolipid self-interactions through multivalent interaction of Lewis X epitopes. However, there are no reports on the existence of specific proteoglycan self-interactions through carbohydrate-carbohydrate interactions in cellular recognition system, as it has been done with glycosphingolipids. Here, we used sponges, organisms on which the first proteoglycan-mediated cell-cell recognition in the animal kingdom was demonstrated, as a model system to study carbohydrate-mediated cellular recognition. We show that the interaction between single oligosaccharides from surface proteoglycans is relatively strong and comparable to protein-carbohydrate interactions, highly specific, and dependent on Ca2+-ions. 200 kDa glycans from the core protein of Microciona prolifera cell surface proteoglycans have been previously shown to mediate homotypic Microciona proteoglycan-proteoglycan interactions. Here, 200 kDa glycans from four different sponge species: Microciona prolifera, Halichondria panicea, Suberites fuscus and Cliona celata were purified and investigated for species-specific interactions. Selective recognition of glycans by live cells was studied to confirm the existence of glycan-glycan recognition system in biologically relevant situations. Mature sponge cells have the ability to reaggregate species-specifically and form homogenous aggregates on a shaker at the right shear forces in the presence of physiological 10 mM Ca2+. Live cells were allowed to aggregate with glycan-coated beads similar in size to small sponge cells in the presence of calcium. They specifically recognized beads coated with their own glycans and did not mix but separated from beads coated with glycans isolated from different species. The glycan-glycan recognition assay was developed to mimic species-specific cellcell recognition in sponges. 200 kDa glycans immobilized onto beads similar in size to small sponge cells assembled species-specifically in the presence of physiological calcium, at the same shear forces as in cell-cell aggregation. Glycans coated on beads aggregated with glycans from the same species coated on beads, and separated from glycans from other species. The glycan density necessary for specific live cellcell recognition in sponges is 828 molecules/μm2. In our studies, the glycan density necessary for specific glycan-coated bead was very similar: ~810 molecules/μm2. Mature live cells demonstrated specific recognition of 200 kDa glycans during selective-binding to glycans coated on surfaces in the presence of calcium. They strongly adhered to glycans from their own surface proteoglycans coated onto a solid polystyrene phase, while the binding to glycans from different proteoglycans was 3 - 5 times lower. Moreover, homotypic adhesion to glycan-coated plates enhanced sponge cell differentiation and formation of mineral skeleton (spicules). Larval cells, after settlement and spreading of larvae, can fuse species-specifically in nature. In our studies, live larval cells recognized and adhered specifically to glycans purified from adhesion proteoglycans from their "mother sponge". They showed almost no interaction with glycans from other species. As in cell-glycan adhesion assays, highly species-specific adhesion of 200 kDa glycans to glycan-coated surfaces could be observed in the presence of physiological calcium. Tested glycans bound strongly to glycans from the same species and showed up to a six fold reduction in binding to glycans from other species. Atomic force microscopy (AFM) was performed to measure for the first time adhesion forces between single glycan molecules obtained from different surface proteoglycans. Measurements revealed equally strong adhesion forces in the range of several hundred piconewtons (pN) between glycan molecules as between proteins and glycans measured in another recognition system. Moreover, statistically significant differences (p value < 0.01) were seen between homotypic (glycans from the same species) and heterotypic (glycans from different species) interactions. Moreover, the polyvalent character of binding characterized mainly interactions between glycans from the same species. This indicates that not only the higher adhesion force per binding site as such but also the higher amount of multiple interactions between glycans from the same species versus mixture of glycans from different species guaranteed the specificity of the glycan-mediated recognition. These findings confirm for the first time the existence of specific glycan-glycan recognition system between cell surface proteoglycans. We propose that these cell's outermost surface structures serve as important players in initiating the very first contacts between cells through highly species-specific and flexible carbohydratecarbohydrate interactions

Carbohydrate–carbohydrate interaction provides adhesion force and specificity for cellular recognition

© 2004 Bucior et al. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License. The definitive version was published in Journal of Cell Biology 165 (2004): 529-537, doi:10.1083/jcb.200309005.The adhesion force and specificity in the first experimental evidence for cell–cell recognition in the animal kingdom were assigned to marine sponge cell surface proteoglycans. However, the question whether the specificity resided in a protein or carbohydrate moiety could not yet be resolved. Here, the strength and species specificity of cell–cell recognition could be assigned to a direct carbohydrate–carbohydrate interaction. Atomic force microscopy measurements revealed equally strong adhesion forces between glycan molecules (190–310 piconewtons) as between proteins in antibody–antigen interactions (244 piconewtons). Quantitative measurements of adhesion forces between glycans from identical species versus glycans from different species confirmed the species specificity of the interaction. Glycan-coated beads aggregated according to their species of origin, i.e., the same way as live sponge cells did. Live cells also demonstrated species selective binding to glycans coated on surfaces. These findings confirm for the first time the existence of relatively strong and species-specific recognition between surface glycans, a process that may have significant implications in cellular recognition.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 Foundatio

Proteoglycan mechanics studied by single-molecule force spectroscopy of allotypic cell adhesion glycans

Author Posting. © American Society for Biochemistry and Molecular Biology, 2006. This article is posted here by permission of American Society for Biochemistry and Molecular Biology for personal use, not for redistribution. The definitive version was published in Journal of Biological Chemistry 281 (2006): 5992-5999, doi:10.1074/jbc.M507878200.Early Metazoans had to evolve the first cell adhesion system addressed to maintaining stable interactions between cells constituting different individuals. As the oldest extant multicellular animals, sponges are good candidates to have remnants of the molecules responsible for that crucial innovation. Sponge cells associate in a species-specific process through multivalent calcium-dependent interactions of carbohydrate structures on an extracellular membrane-bound proteoglycan termed aggregation factor. Single-molecule force spectroscopy studies of the mechanics of aggregation factor self-binding indicate the existence of intermolecular carbohydrate adhesion domains. A 200-kDa aggregation factor glycan (g200) involved in cell adhesion exhibits interindividual differences in size and epitope content which suggest the existence of allelic variants. We have purified two of these g200 distinct forms from two individuals of the same sponge species. Comparison of allotypic versus isotypic g200 binding forces reveals significant differences. Surface plasmon resonance measurements show that g200 self-adhesion is much stronger than its binding to other unrelated glycans such as chondroitin sulfate. This adhesive specificity through multiple carbohydrate binding domains is a type of cooperative interaction that can contribute to explain some functions of modular proteoglycans in general. From our results it can be deduced that the binding strength/surface area between two aggregation factor molecules is comparable with that of focal contacts in vertebrate cells, indicating that strong carbohydrate-based cell adhesions evolved at the very start of Metazoan history.This work was supported in part by Grants BIO2002-00128 and BIO2005-01591 (both to X. F.-B.) from the Ministerio de Educacio´n y Ciencia, Spain, which included Fondo Europeo de Desarrollo Regional funds

Pseudomonas aeruginosa Pili and Flagella Mediate Distinct Binding and Signaling Events at the Apical and Basolateral Surface of Airway Epithelium

Pseudomonas aeruginosa, an important opportunistic pathogen of man, exploits numerous factors for initial attachment to the host, an event required to establish bacterial infection. In this paper, we rigorously explore the role of two major bacterial adhesins, type IV pili (Tfp) and flagella, in bacterial adherence to distinct host receptors at the apical (AP) and basolateral (BL) surfaces of polarized lung epithelial cells and induction of subsequent host signaling and pathogenic events. Using an isogenic mutant of P. aeruginosa that lacks flagella or utilizing beads coated with purified Tfp, we establish that Tfp are necessary and sufficient for maximal binding to host N-glycans at the AP surface of polarized epithelium. In contrast, experiments utilizing a P. aeruginosa isogenic mutant that lacks Tfp or using beads coated with purified flagella demonstrate that flagella are necessary and sufficient for maximal binding to heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPGs) at the BL surface of polarized epithelium. Using two different cell-free systems, we demonstrate that Tfp-coated beads show highest binding affinity to complex N-glycan chains coated onto plastic plates and preferentially aggregate with beads coated with N-glycans, but not with single sugars or HS. In contrast, flagella-coated beads bind to or aggregate preferentially with HS or HSPGs, but demonstrate little binding to N-glycans. We further show that Tfp-mediated binding to host N-glycans results in activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway and bacterial entry at the AP surface. At the BL surface, flagella-mediated binding to HS activates the epidermal growth factor receptor (EGFR), adaptor protein Shc, and PI3K/Akt, and induces bacterial entry. Remarkably, flagella-coated beads alone can activate EGFR and Shc. Together, this work provides new insights into the intricate interactions between P. aeruginosa and lung epithelium that may be potentially useful in the development of novel treatments for P. aeruginosa infections

Pseudomonas aeruginosa-Mediated Damage Requires Distinct Receptors at the Apical and Basolateral Surfaces of the Polarized Epithelium ▿ †

Pseudomonas aeruginosa, an important opportunistic pathogen of humans, exploits epithelial damage to establish infection. We have rigorously explored the role of N-glycoproteins and heparan sulfate proteoglycans (HSPGs) in P. aeruginosa-mediated attachment and subsequent downstream events at the apical (AP) and basolateral (BL) surfaces of polarized epithelium. We demonstrate that the N-glycan chains at the AP surface are necessary and sufficient for binding, invasion, and cytotoxicity to kidney (MDCK) and airway (Calu-3) cells grown at various states of polarization on Transwell filters. Upregulation of N-glycosylation enhanced binding, whereas pharmacologic inhibition of N-glycosylation or infection of MDCK cells defective in N-glycosylation resulted in decreased binding. In contrast, at the BL surface, the HS moiety of HSPGs mediated P. aeruginosa binding, cytotoxicity, and invasion. In incompletely polarized epithelium, HSPG abundance was increased at the AP surface, explaining its increased susceptibility to P. aeruginosa colonization and damage. Using MDCK cells grown as three-dimensional cysts as a model for epithelial organs, we show that P. aeruginosa specifically colocalized with HS-rich areas at the BL membrane but with complex N-glycans at the AP surface. Finally, P. aeruginosa bound to HS chains and N-glycans coated on plastic surfaces, showing the highest binding affinity toward isolated HS chains. Together, these findings demonstrate that P. aeruginosa recognizes distinct receptors on the AP and BL surfaces of polarized epithelium. Changes in the composition of N-glycan chains and/or in the distribution of HSPGs may explain the enhanced susceptibility of damaged epithelium to P. aeruginosa

Flagella are sufficient to phosphorylate EGFR and Shc.

<p>Calu-3 cells were grown as well polarized monolayers on Transwells for 9 days and treated with heparinase III (hepIII), tunicamycin (tun), or EGFR inhibitor (AG1478). As a control, cell were left untreated (un); flagella-, Tfp-, or, as a negative control, BSA-coated beads were added to the AP or BL chamber for 1 h. Lysates were immunoprecipitated with Akt or EGFR antibody or directly immunoblotted with (<b>A, B</b>) phospho-Akt, (<b>C–F</b>) phospho-EGFR, or (<b>G–H</b>) phospho-Shc (three different isoforms p46, p52, and p66). Representative gels (A, C, E, G) and quantification by densitometry of three gels (B, D, F, H) are shown. The ratio of phospho-Akt to total-Akt, phospho-EGFR to total-EGFR, or phospho-Shc to total-Shc for untreated cells was set to 1. Shown is the mean +/− SD for 3 independent experiments. ^**P<0.05 compared to cells BL infected with PAO1.</p

<i>P. aeruginosa</i> internalization at the AP surface of polarized epithelium is mediated by Tfp-dependent binding to N-glycans and subsequent PI3K activation and, at the BL surface, by flagella-dependent binding to HS and subsequent EGFR/PI3K activation.

<p>Calu-3 cells were grown as well polarized monolayers on Transwells for 9 days and treated with heparin, heparinase III (hepIII), mannose (Man), tunicamycin (tun), EGFR inhibitor (AG1478), PI3K inhibitor (LY29004), or in combination. (<b>A</b>) PAO1, (<b>B</b>) PAO1Δ<i>pilA</i> or (<b>C</b>) PAO1Δ<i>fliC</i> were added to the AP or BL chamber for 2 h and standard invasion assays were performed. Shown is the mean +/− SD for 4 independent experiments. ^*P<0.05 compared to cells infected with PAO1 at the AP surface (black bar). ^**P<0.05 compared to cells infected with PAO1 at the BL surface (black bar).</p

Akt is phosphorylated upon flagella-mediated <i>P. aeruginosa</i> entry at the BL surface and upon Tfp-mediated entry at the AP surface of polarized epithelium.

<p>Calu-3 cells were grown as well polarized monolayers on Transwells for 9 days and treated with heparinase III (hepIII), tunicamycin (tun), EGFR inhibitor (AG1478), or PI3K inhibitor (LY29004). As a control, cells were left untreated (un) or, as a positive control, cells were treated with HB-EGF. (<b>A</b>, <b>B</b>) PAO1, (<b>C</b>, <b>D</b>) PAO1Δ<i>pilA</i>, or (<b>E</b>, <b>F</b>) PAO1Δ<i>fliC</i> were added to the AP or BL chamber for 1 h. Lysates were immunoprecipitated with Akt antibody followed by immunoblotting with phospho- or total-Akt antibodies. Representative gels (A, C, E) and quantification by densitometry of three gels (B, D, F) are shown. The ratio of phospho-Akt to total-Akt for untreated cells was set to 1. Shown is the mean +/− SD for 3 independent experiments. ⁺P<0.05 compared to cells AP infected with PAO1. ⁺⁺P<0.05 compared to cells BL infected with PAO1. ^*P<0.05 compared to cells AP infected with PAO1 (B), PAO1Δ<i>pilA</i> (D), or PAO1Δ<i>fliC</i> (F). ^**P<0.05 compared to cells BL infected with PAO1 (B), PAO1Δ<i>pilA</i> (D), or PAO1Δ<i>fliC</i> (F).</p