Stimulation with Porphyromonas gingivalis enhances malignancy and initiates anoikis resistance in immortalized oral keratinocytes.

The aim of this study was to get new insights into molecular processes involved in tumor propagation of immortalized oral keratinocytes induced by the keystone pathogen Porphyromonas gingivalis. Cell culture experiments with immortalized OKF6 cells were performed to analyze cellular effects caused by bacterial stimulation focusing on altered gene expression, signaling pathways, proliferation rate, cell viability, migration and invasion behavior, and on the development of antiapoptotic pathways. Gene and protein expression were analyzed using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, western blot, and protein arrays. Trypan blue staining was used to analyze proliferation and viability, transwell assays for cellular migration, Matrigel assays for invasion, and anoikis-assays for evaluating anoikis resistance. Stimulation of OKF6 cells with Porphyromonas gingivalis led to an alteration in the molecular repertoire of proteins which are involved in cell proliferation, epithelial-mesenchymal transition, stem cell formation, migration, invasion, and anoikis resistance. Higher proliferation rates were detected in conjunction with an activation of PI3K/Akt signaling and the mTOR-pathway. Additionally, inhibition of glycogen-synthase-kinase3-ß led to stabilization of ß-catenin and Snail, which resulted in a switch from predominant E-cadherin to N-cadherin expression and increased expression of the stem cell markers Oct3/4, Sox2, and Nanog. Enhanced biosynthesis and enzyme activity of matrix metalloproteinase-9 was accompanied by elevated invasion behavior. Finally, anoikis resistance was detected in stimulated keratinocytes by decreased apoptosis of nonadherent cells and elevated expression of epidermal growth factor receptor and c-Met. Hence, Porphyromonas gingivalis is able to induce a more aggressive tumor-like phenotype in immortalized oral keratinocytes, thus contributing to enhanced tumor features.

Response of MG63 osteoblasts on bacterial challenge is dependent on the state of differentiation.

The present in vitro study examines molecular processes that are relevant during bone homeostasis after Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis infection with a focus on the differentiation level of osteoblasts. Regenerative processes are often hindered by the recurrence of bacterial infections, which can ultimately provoke a severe destruction of bone tissue. To obtain more detailed insights into such a complex scenario, we have used undifferentiated MG63 osteoblast-like cells as an experimental paradigm to examine the impact of two oral pathogens, A. actinomycetemcomitans and P. gingivalis, on proliferation, cytotoxicity and osteogenic differentiation. Cell culture experiments were performed to analyze cellular behavior. The level of genes interfering with bone tissue integrity (matrix metalloproteinases and their tissue inhibitors) and osteogenic markers (alkaline phosphatase, Runx2, human ß-defensin-2) was compared in undifferentiated versus differentiated MG63 cells using real-time polymerase chain reaction. Functional activity of matrix metalloproteinases was quantified by zymography. Western blot analysis was used to verify the phosphorylation state of mitogen-activated protein kinases p38 and extracellular-signal-regulated kinases 1/2. When co-cultured with undifferentiated MG63 cells, oral pathogens provoked distinct cellular effects. Only A. actinomycetemcomitans reduced cell proliferation, increased cell death, and induced osteogenic differentiation. A comparison of matrix metalloproteinase network stability in the presence of oral pathogens revealed a partial sensitivity towards P. gingivalis but not A. actinomycetemcomitans. So, beside the proof of concept that MG63 cells co-cultured with oral pathogens can serve as an in vitro model for mimicking destructive and regenerative events after bacterial infections, our data indicate that double infections might counterbalance otherwise positive effects.

Oral pathogens change proliferation properties of oral tumor cells by affecting gene expression of human defensins.

The impact of oral pathogens onto the generation and variability of oral tumors has only recently been investigated. To get further insights, oral cancer cells were treated with pathogens and additionally, as a result of this bacterial cellular infection, with human defensins, which are as anti-microbial peptide members of the innate immune system. After cell stimulation, proliferation behavior, expression analysis of oncogenic relevant defensin genes, and effects on EGFR signaling were investigated. The expression of oncogenic relevant anti-microbial peptides was analyzed with real-time PCR and immunohistochemistry. Cell culture experiments were performed to examine cellular impacts caused by stimulation, i.e., altered gene expression, proliferation rate, and EGF receptor-dependent signaling. Incubation of oral tumor cells with an oral pathogen (Porphyromonas gingivalis) and human α-defensins led to an increase in cell proliferation. In contrast, another oral bacterium used, Aggregatibacter actinomycetemcomitans, enhanced cell death. The bacteria and anti-microbial peptides exhibited diverse effects on the transcript levels of oncogenic relevant defensin genes and epidermal growth factor receptor signaling. These two oral pathogens exhibited opposite primary effects on the proliferation behavior of oral tumor cells. Nevertheless, both microbe species led to similar secondary impacts on the proliferation rate by modifying expression levels of oncogenic relevant α-defensin genes. In this respect, oral pathogens exerted multiplying effects on tumor cell proliferation. Additionally, human defensins were shown to differently influence epidermal growth factor receptor signaling, supporting the hypothesis that these anti-microbial peptides serve as ligands of EGFR, thus modifying the proliferation behavior of oral tumor cells.

Tenascin-R is expressed by Schwann cells in the peripheral nervous system.

The extracellular matrix glycoprotein tenascin-R (TN-R) has been implicated in a variety of cell-matrix interactions involved in the molecular control of axon guidance and neural cell migration during development and regeneration of the central nervous system (CNS). Whereas TN-R is amply expressed in the early postnatal and adult mammalian CNS, the protein has so far not been detected in different compartments of the peripheral nervous system (PNS). Here we provide first evidence that TN-R (predominantly TN-R 160 isoform) is transiently expressed in the sciatic nerve of late embryonic (E14-18) and neonatal mice, while at later developmental stages, both protein and mRNA are downregulated. In vitro, TN-R protein was found to be expressed by both undifferentiated and neuronally differentiated PC12 cells and by L1-positive Schwann cells (SC), but not by other neural and non-neural cell types in cell cultures derived from embryonic (E17/18) hindlimbs and neonatal sciatic nerves. In the developing PNS, TN-R expression correlated with axon growth and SC migration during the period of skeletal muscle innervation. Based on different in vitro approaches, we found that the substrate-bound glycoprotein selectively inhibits the fibronectin-dependent: (1) neurite outgrowth from dorsal root ganglion neurons (strongly expressing alpha5beta1 integrin and the disialoganglioside GD3) by a ganglioside-sensitive signaling mechanism; and (2) migration of primary myoblasts and other non-neuronal cells in a ganglioside-independent manner. Our findings suggest the functional role of TN-R in PNS pattern formation during distinct stages of axon pathfinding and skeletal muscle innervation.

Nerve growth factor-mediated expression of galectin-3 in mouse dorsal root ganglion neurons.

Galectin-3, a member of the galectin family of beta-galactoside-specific lectins has been found to be expressed by subsets of dorsal root ganglion (DRG) neurons during development and in adulthood. Here we show that (i) after 3-7 days in vitro, DRG neurons derived from neonatal mice express galectin-3 intra- and extracellularly and (ii) lectin expression requires the presence of nerve growth factor (NGF). After 3 days in vitro, a higher number of DRG neurons expressed galectin-3 in the presence of NGF (65 +/- 7%) than in the presence of brain-derived neurotrophic factor (BDNF, 30 +/- 3%) or neurotrophin-3 (NT-3, 34 +/- 3%). After 7 days in vitro, these numbers dropped to 51 +/- 3% (for NGF), 0% (for BDNF) and 8 +/- 4% (for NT-3), respectively. Our findings provide first evidence for the contribution of a neurotrophin to the neuronal expression of galectins and suggest an NGF/TrkA-mediated expression of galectin-3 by early postnatal DRG neurons.

Galectin-3 is upregulated in microglial cells in response to ischemic brain lesions, but not to facial nerve axotomy.

We have recently demonstrated that the beta-galactoside-specific lectin galectin-3 is expressed by microglial cells in vitro, but not by normal resting microglia in vivo. In the present study, we have analyzed the expression of galectin-3 by microglia under traumatic conditions in vivo using two experimental rat models which substantially differ in the severity of lesion related to a breakdown of the blood-brain barrier (BBB) and the occurrence of inflammatory processes. These two features are absent after peripheral nerve lesion and present after cerebral ischemia. Here we show that, following facial nerve axotomy under conditions allowing (nerve anastomosis) or not subsequent regeneration (nerve resection), galectin-3 is not expressed by microglia in the corresponding facial nucleus 1-112 days after lesion. Galectin-3 is also absent in microglia at sites of a defective BBB in the normal brain, such as the circumventricular organs. Following experimental ischemia (i.e., permanent occlusion of the middle cerebral artery), in contrast, galectin-3 becomes strongly expressed by activated microglia as early as 48 hours after trauma, as determined by immunohistochemistry and Western blot analysis. Our findings suggest that the expression of galectin-3 by microglia in vivo correlates with the state of microglial activation.

Association of tenascin-R with murine brain myelin membranes: involvement of divalent cations.

In the central nervous system (CNS), tenascin-R (TN-R) is mainly expressed by oligodendrocytes and in white matter tracts. Here, we have examined the molecular association of TN-R with CNS myelin by incubation of myelin membranes (MM) purified from adult mouse brain under different ionic conditions. By Western blot analysis, the 160 kDa isoform was the main TN-R component detectable in MM as a dimer which became degraded to monomers of 160 kDa and major fragments of 125 and 80 kDa in the absence of protease inhibitors. In the presence of chelating agents, TN-R was completely extracted from MM. Calcium ions promoted the dissociation of TN-R while zinc or copper blocked it. TN-R release from MM was sensitive to heat suggesting the involvement of calcium-dependent myelin protease(s) in this process. In addition, 1,10-phenanthroline (a metalloprotease blocker) partially inhibited TN-R release in the presence of calcium ions. We conclude that divalent metal ions stabilize the association of TN-R with CNS myelin and upon damage, the protein can be released and degraded by endogenous proteases, suggesting the implication of myelin-derived TN-R in axon growth inhibition and demyelinating diseases.

Involvement of chondroitin sulfates on brain-derived tenascin-R in carbohydrate-dependent interactions with fibronectin and tenascin-C.

Tenascin-R (TN-R), a matrix glycoprotein of the central nervous system (CNS), has been implicated in a variety of cell-matrix interactions involved in the control of axon growth, myelination and cell adhesion to fibronectin during development and regeneration. While most of the functional analyses have concentrated exclusively on the role of the core protein, the contribution of TN-R glycoconjugates present on many potential sites for N- and O-glycosylation is presently unknown. Here we provide evidence that TN-R derived from adult mouse brain expresses chondroitin sulfate (CS) glycosaminoglycans (GAGs), i.e. C-6S and C-4S, that are recognized by the CS/dermatan sulfate-specific monoclonal antibodies 473 HD and CS-56. Using ligand-binding, cell adhesion and neurite outgrowth assays, we show that TN-R-linked CS GAGs (i) are involved in the interaction with the heparin-binding sites of fibronectin and are responsible for TN-R-mediated inhibition of cell adhesion to a 33/66-kD heparin-binding fibronectin fragment or to FN-C/H I and FN-C/H II peptides, known to participate in fibronectin binding to cell surface proteoglycans; and (ii) partially contribute to the interaction between TN-R and TN-C which, however, does not lead to an interference with TN-R- and TN-C-mediated inhibition of neurite outgrowth when the two molecules are offered as a mixed substrate in culture. Our findings suggest the functional implication of TN-R-linked CS GAGs in matrix interactions with fibronectin and TN-C that are likely to contribute to a modulation of cellular behavior and the macromolecular organization of matrix components in the developing or injured adult CNS.

The yin and yang of tenascin-R in CNS development and pathology.

An important biological consequence of the initial interactions between the cell surface and its extracellular environment is the diversity of cellular responses ranging from overt repulsion or avoidance reaction to stable adhesion or final positioning. It is now evident that positive and negative guiding mechanisms are equally relevant to normal pattern formation during development and decisive for the outcome of a regenerative process. In this context, the present review summarizes the knowledge about the extracellular matrix glycoprotein tenascin-R, a member of the tenascin gene family. In contrast to all other known family members, tenascin-R is exclusively expressed in the central nervous system of vertebrates by oligodendrocytes and neuronal subsets at later developmental stages and in adulthood. We focus on the glycoprotein's structure, tissue distribution and functional implications in the molecular control of axon targeting, neural cell adhesion, migration and differentiation during nervous system morphogenesis and pathology.

Chondroitin sulfates expressed on oligodendrocyte-derived tenascin-R are involved in neural cell recognition. Functional implications during CNS development and regeneration.

Tenascin-R (TN-R), an extracellular matrix constituent of the central nervous system (CNS), has been implicated in a variety of cell-matrix interactions underlying axon growth inhibition/guidance, myelination and neural cell migration during development and regeneration. Although most of the functional analyses have concentrated exclusively on the role of the core protein, the contribution of TN-R glycoconjugates present on many potential sites for N- and O-glycosylation is presently unknown. Here we provide first evidence that TN-R derived from whole rat brain or cultured oligodendrocytes expresses chondroitin sulfate (CS) glycosaminoglycans (GAGs), i.e., C-4S and C-6S, that are recognized by CS-56, a CS/dermatan sulfate-specific monoclonal antibody. Based on different in vitro approaches utilizing substrate-bound glycoprotein, we found that TN-R-linked CS GAGs (1) promote oligodendrocyte migration from white matter microexplants and increase the motility of oligodendrocyte lineage cells; (2) similar to soluble CS GAGs, induce the formation of glial scar-like structures by cultured cerebral astrocytes; and (3) contribute to the antiadhesive properties of TN-R for neuronal cell adhesion in an F3/F11-independent manner, but not to neurite outgrowth inhibition, by mechanism(s) sensitive to chondroitinase or CS-56 treatments. Furthermore, after transection of the postcommissural fornix in adult rat, CS-bearing TN-R was found to be stably upregulated at the lesion site. Our findings suggest the functional impact of TN-R-linked CS on neural cell adhesion and migration during brain morphogenesis and the contribution of TN-R to astroglial scar formation (CS-dependent) and axon growth inhibition (CS-independent), i.e., suppression of axon regeneration after CNS injury.

Expression pattern of galectin-3 in neural tumor cell lines.

Galectin-3 is a member of the galectin family of beta-galactoside-specific animal lectins. Here we show that galectin-3 is constitutively expressed in 15 out of 16 glioma cell lines tested, but not by normal or reactive astrocytes, oligodendrocytes, glial O-2A progenitor cells and the oligodendrocyte precursor cell line Oli-neu. Galectin-3 is also expressed by one oligodendroglioma cell line, but not by primitive neuroectodermal tumor and 4 neuroblastoma cell lines tested so far. In all galectin-3 expressing cell lines, the lectin is predominantly, if not exclusively, localized intracellularly and carries an active carbohydrate recognition domain (shown for C6 rat glioma cells). Moreover, in contrast to primary astrocytes, glioma cells do not or only weakly adhere to substratum-bound galectin-3, probably reflecting an unusual glycosylation pattern. Our findings indicate that the expression of galectin-3 selectively correlates with glial cell transformation in the central nervous system and could thus serve as a marker for glial tumor cell lines and glial tumors.

Tenascin-R interferes with integrin-dependent oligodendrocyte precursor cell adhesion by a ganglioside-mediated signalling mechanism.

Oligodendrocyte (OL) lineage progression is characterized by the transient expression of the disialoganglioside GD3 by OL precursor (preOL) cells followed by the sequential expression of myelin-specific lipids and proteins. Whereas GD3+ preOLs are highly motile cells, the migratory capacity of OLs committed to terminal differentiation is strongly reduced, and we have recently shown that the extracellular matrix protein tenascin-R (TN-R) promotes the stable adhesion and differentiation of O4+ OLs by a sulphatide-mediated autocrine mechanism (O4 is a monoclonal antibody recognizing sulphatides/seminolipids expressed by OLs and in myelin). Using culture conditions that allow the isolation of mouse OLs at distinct lineage stages, here we demonstrate that TN-R is antiadhesive for GD3+ preOLs and inhibits their integrin-dependent adhesion to fibronectin (FN) by a disialoganglioside-mediated signalling mechanism affecting the tyrosine phosphorylation of the focal adhesion kinase. This responsive mechanism appears to be common to various cell types expressing disialogangliosides as: (i) disialogangliosides interfered with the inhibition of cell adhesion of different neural and non-neural cells on substrata containing TN-R and FN or RGD-containing FN fragments. TN-R interacted specifically with disialoganglioside-expressing cells or immobilized gangliosides, and ganglioside treatment of TN-R substrata resulted in a delayed preOL cell detachment as a function of time. We conclude that OL response to one and the same signal in the extracellular matrix critically depends on the molecular repertoire expressed by OLs at different lineage stages and could thus define their final positioning.

I-type lectins in the nervous system.

The number of animal lectins, basically defined upon their interaction with specific carbohydrate structures, is growing considerably during the last few years. Among these proteins the recently identified subfamily of I-type lectins consists of mainly transmembranous glycoproteins belonging to the immunoglobulin superfamily. Most of the I-type lectins participate in cell adhesion events, as are the different sialoadhesins recognizing sialylated glycan structures, which represent the best characterized subgroup. I-type lectins are abundant in the nervous system and have been implicated in a number of morphogenetic processes as fundamental as axon growth, myelin formation and growth factor signaling. In the present review, we summarize the structural and functional properties of I-type lectins expressed in neural tissues with a main focus on the sialoadhesin myelin-associated glycoprotein, the neural cell adhesion molecule and the fibroblast growth factor receptors.

Tenascin-C inhibits beta1 integrin-dependent cell adhesion and neurite outgrowth on fibronectin by a disialoganglioside-mediated signaling mechanism.

We have previously shown that the extracellular matrix molecule tenascin-C inhibits fibronectin-mediated cell adhesion and neurite outgrowth by an interaction with a cellular RGD-independent receptor which interferes with the adhesion and neurite outgrowth promoting activities of the fibronectin receptor(s). Here we demonstrate that the inhibitory effect of tenascin-C on beta1integrin-dependent cell adhesion and neurite outgrowth is mediated by the interaction of the protein with membrane-associated disialogangliosides, which interferes with protein kinase C-related signaling pathways. First, in substratum mixtures with fibronectin, an RGD sequence-containing fragment of the molecule or synthetic peptide, tenascin-C inhibited cell adhesion and spreading by a disialoganglioside-dependent, sialidase-sensitive mechanism leading to an inhibition of protein kinase C. Second, the interaction of intact or trypsinized, i.e., cell surface glycoprotein-free, cells with immobilized tenascin-C was strongly inhibited by gangliosides or antibodies to gangliosides and tenascin-C. Third, preincubation of immobilized tenascin-C with soluble disialogangliosides resulted in a delayed cell detachment as a function of time. Similar to tenascin-C, immobilized antibody to GD2 (3F8) or sphingosine, a protein kinase C inhibitor, strongly inhibited RGD-dependent cell spreading. Finally, the degree of tenascin-C-induced inhibition of cell adhesion was proportional to the degree of disialoganglioside levels of expression by different cells suggesting the relevance of such mechanism in modulating integrin-mediated cell-matrix interactions during pattern formation or tumor progression.

Experimental axonal injury triggers interleukin-6 mRNA, protein synthesis and release into cerebrospinal fluid.

Diffuse axonal injury is a frequent pathologic sequel of head trauma, which, despite its devastating consequences for the patients, remains to be fully elucidated. Here we studied the release of interleukin-6 (IL-6) into CSF and serum, as well as the expression of IL-6 messenger ribonucleic acid (mRNA) and protein in a weight drop model of axonal injury in the rat. The IL-6 activity was elevated in CSF within 1 hour and peaked between 2 and 4 hours, reaching maximal values of 82,108 pg/mL, and returned to control values after 24 hours. In serum, the levels of IL-6 remained below increased CSF levels and did not exceed 393 pg/mL. In situ hybridization demonstrated augmented IL-6 mRNA expression in several regions including cortical pyramidal cells, neurons in thalamic nuclei, and macrophages in the basal subarachnoid spaces. A weak constitutive expression of IL-6 protein was shown by immunohistochemical study in control brain. After injury, IL-6 increased at 1 hour and remained elevated through the first 24 hours, returning to normal afterward. Most cells producing IL-6 were cortical, thalamic, and hippocampal neurons as confirmed by staining for the neuronal marker NeuN. These results extend our previous studies showing IL-6 production in the cerebrospinal fluid of patients with severe head trauma and demonstrate that neurons are the main source of IL-6 after experimental axonal injury.

Galectin-3 promotes neural cell adhesion and neurite growth.

Galectin-3 is a member of the galectin family and belongs to a group of soluble beta-galactoside-binding animal lectins. The molecule is expressed by neural and nonneural cells intra- (cytoplasm and nucleus) as well as extra-cellularly (plasma membrane and extracellular space). By using an in vitro cell-substratum adhesion assay, we have addressed the question whether galectin-3 present in the extracellular milieu may support the adhesion and/or neurite outgrowth of neural cells in a manner analogous to cell adhesion molecules. Galectin-3 was immobilized as a substratum and various cell types, N2A (neuroblastoma), PC12 (pheochromocytoma), and TSC (transformed Schwann cells) cell lines, neural cells from early postnatal mouse cerebellum, and dorsal root ganglion neurons from newborn mice were allowed to adhere to the lectin. Here we show that all cell types studied specifically adhered to galectin-3 by the following criteria: 1) the number of adherent cells was dependent on the galectin-3 concentration used for coating; 2) adhesion of cells to galectin-3, but not to collagen type I or laminin was inhibited by polyclonal antibodies to galectin-3; 3) upon addition of asialofetuin (a polyvalent carrier of terminal beta-galactosides) to the cell suspension prior to the adhesion assay, cell adhesion to galectin-3 was inhibited in a dose-dependent manner; and 4) cell adhesion to galectin-3 was abolished by treatment of cells with endo-beta-galactosidase. In addition, the adhesion of dorsal root ganglion neurons to galectin-3 could be inhibited by lactose. Notably, substratum-bound galectin-3 promoted the outgrowth of neurites from dorsal root ganglia explants and this neurite outgrowth promoting activity could be inhibited by polyclonal antibodies to galectin-3.

Tenascin-R is antiadhesive for activated microglia that induce downregulation of the protein after peripheral nerve injury: a new role in neuronal protection.

Microglial activation in response to pathological stimuli is characterized by increased migratory activity and potential cytotoxic action on injured neurons during later stages of neurodegeneration. The initial molecular changes in the CNS favoring neuronofugal migration of microglia remain, however, largely unknown. We report that the extracellular matrix protein tenascin-R (TN-R) present in the intact CNS is antiadhesive for activated microglia, and its downregulation after facial nerve axotomy may account for the loss of motoneuron protection and subsequent neurodegeneration. Studies on the protein expression in the facial and hypoglossal nucleus in rats demonstrate that TN-R is a constituent of the perineuronal net of motoneurons and 7 d after peripheral nerve injury becomes downregulated in the corresponding motor nucleus. This downregulation is reversible under regenerative (nerve suture) conditions and irreversible under degenerative (nerve resection) conditions. In short-term adhesion assays, the unlesioned side of brainstem cryosections from unilaterally operated animals is nonpermissive for activated microglia, and this nonpermissiveness is almost abolished by a monoclonal antibody to TN-R. Microglia-conditioned media and tumor necrosis factor-alpha downregulate TN-R protein and mRNA synthesis by cultured oligodendrocytes, which are one of the sources for TN-R in the brainstem. Our findings suggest a new role for TN-R in neuronal protection against activated microglia and the participation of the latter in perineuronal net destruction, e.g., downregulation of TN-R.

Murine microglial cells express functionally active galectin-3 in vitro.

In the present study we have analyzed the expression of galectin-3, a beta-galactoside-specific soluble animal lectin, by microglial cells in vitro. In enriched microglial cell cultures derived from neonatal mouse brain after 2 to 3 weeks in vitro, almost all microglial cells expressed galectin-3 intracellularly and about 90% expressed the molecule on the cell surface. Western blot analyses of lysates from microglial cells using galectin-3-specific antibodies revealed a single band with an apparent molecular weight of 29 kD. The carbohydrate recognition domain of microglia-derived galectin-3 was functional as the molecule could be affinity purified on lactose-agarose. Upon an incubation with lactose-, but not with sucrose-containing buffers the amount of cell surface expressed galectin-3 was strongly reduced, suggesting that the molecule appears to be associated with the plasma membrane via its carbohydrate recognition domain. The total amount as well as the portion of cell surface expressed galectin-3 increased upon treatment with granulocyte-macrophage colony-stimulating factor. Our findings suggest that galectin-3 expression is subject to regulation by growth factors supposed to be involved in the cascade of microglial activation under pathological conditions.

Homophilic binding properties of galectin-3: involvement of the carbohydrate recognition domain.

Galectin-3, an animal lectin specific for beta-galactosides, is composed of three different domains. The N-terminal half of the molecule (N domain) consists of a short N-terminal segment followed by glycine-, proline-, and tyrosine-rich tandem repeats. The C-terminal domain (C domain) harbors the carbohydrate recognition domain homologous to other members of the galectin family of lectins. Galectin-3 aggregates in solution, and participation of the N domain of the molecule in this process has already been demonstrated. Using a solid-phase radioligand binding assay, which allows the direct analysis of galectin-3 self-association, here we provide evidence that the carbohydrate recognition domain of the lectin is involved in carbohydrate-dependent homophilic interactions: (a) Radiolabeled galectin-3 binds to immobilized galectin-3, and the addition of unlabeled galectin-3 in solution increases the rate of binding of radiolabeled lectin; (b) binding of radiolabeled galectin-3 to immobilized galectin-3 is inhibited by the C domain; (c) binding of radiolabeled galectin-3 to immobilized galectin-3 or the C domain is inhibited by lactose but not by sucrose; and (d) the radiolabeled C domain does not bind to immobilized C domain. Taken together, these data suggest that in addition to the N domain, the homophilic interactions of galectin-3 are mediated by the C domain.