Zuzana Holíková

New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells: Biotinylated galectins as tool to detect specific binding sites

Nuclear presence of galectins suggests a role of these endogenous lectins in the regulation of transcription, pre‐mRNA splicing and transport processes. Therefore, detection and localization of nuclear binding sites for galectins by a new methodological step, has potential to further functional analysis. In the first step of our model study we monitored the nuclear expression of galectins‐1 and −3 in cultured stromal cells of human bone marrow and human/porcine keratinocytes. To enable detection and localization of galectin‐specific binding sites, we used purified galectins biotinylated without loss of activity as cytochemical tool. The degree of labeling of the probes was determined by adapting two‐dimensional gel electrophoresis and calculating pI changes in response to stepwise chemical modification of basic and acidic side chains by the biotinylation reagents. Binding studies revealed positivity for galectin‐1, whereas galectins‐3, −5, and −7 were not reactive with nuclear sites under identical conditions in bone marrow stromal cells and keratinocytes prepared from hair follicle enriched for stem cells. Inhibition by lactose indicated an involvement of the carbohydrate recognition domain in nuclear binding of galectin‐1. Colocalization of the galectin‐1‐dependent signal with the SC35 splicing factor and sensitivity toward RNase treatment argued in favor of galectin binding in nuclear speckles, albeit only for a small fraction of the cells. Epidermal cells positive for galectin‐1‐binding sites expressed ΔNp63 known as a potential marker of stem cells. Based on cytokeratin expression cells with nuclear binding of labeled galectin‐1 were basal and not suprabasal cells. Regarding proliferation, no relationship to the expression of a proliferation marker, Ki‐67, was observed. The nucleolar signal colocalized with fibrillarin and nucleophosmin/B23 as representatives of nucleolar proteins in both types of studied cells. In conclusion, the application of labeled galectins to localize accessible binding sites adds a new aspect to the functional analysis of these lectins in the nucleus.

Coexpression of binding sites for A(B) histo-blood group trisaccharides with galectin-3 and Lag antigen in human Langerhans cells

Galectin‐3 is an immunomodulatory protein with binding capacity for various glycoconjugates including IgE. It has been shown to be produced by epidermal keratinocytes and is present on the surfaces of skin Langerhans cells (LC). Therefore, it may have a role in the pathogenesis of various skin diseases, such as atopic dermatitis. To study the expression of galectin‐3 in LC, we used, in addition to specific antibodies, a panel of synthetic, carrier‐immobilized, specific oligosaccharides of the A‐ and B‐histo‐blood group, which are recognized by this lectin. In the mean time, Birbeck granules were visualized with an anti‐Lag antibody. The double labeling experiments showed a remarkable colocalization of signals for Lag antigen (Birbeck granules) and galectin‐3, as well as the binding sites for A‐ and B‐histo‐blood group trisaccharides. The specificity of the oligosaccharide binding was demonstrated by the lack of binding by Lec, Led (H blood group antigen), and sLex, which are not recognized by galectin‐3. These results suggest that galectin‐3 is present in Birbeck granules, where it retains reactivity for its glycoligands. J. Leukoc. Biol. 66: 644–649; 1999.

Differentiation-Dependent Glycosylation of Cells in Squamous Cell Epithelia Detected by a Mammalian Lectin

The squamous stratified epithelia contain a proliferative (harboring mitotic activity) and a differentiating compartment. Due to the potential of protein-carbohydrate interactions to regulate cellular activities we introduced a mammalian lectin to cyto- and histochemical analysis. We answer the questions of whether and to what extent this new probe can pinpoint differentiation-dependent glycosylation changes in sections and in culture of keratinocytes. Material and Methods: Purification and labeling enabled monitoring of galectin-3 reactivity in frozen sections of human and pig epidermis and basal cell carcinomas as well as in culture of keratinocytes. The staining pattern of the lectin was correlated with the staining profile of other cell markers including desmosomal proteins, β1 integrin, and the proliferation marker Ki-67. The Dolichos biflorus agglutinin (DBA) sharing binding reactivity of galectin-3 to the A type histoblood group epitope was used for comparison. Results: Both lectins exhibit suprabasal binding. However, their profiles were not identical, substantiated by lack of coinhibition. Strong DBA reactivity was also observed in a limited number of basal layer cells, namely in cells without the expression of the proliferation marker Ki-67. Cultured mitotic epidermal cells have no reactivity for DBA. Presence of ligands for this plant lectin was connected with decreased positivity of nuclei for Ki-67 and the occurrence of ring-shaped nucleoli, micronucleoli or absence of nucleoli. Considering colocalization the pattern of galectin-3-binding sites coincided with the presence of desmosomal proteins such as desmoplakin-1 and desmoglein but not β1 integrin, a potential ligand. Interestingly, studied basal cell carcinomas expressed no binding sites for galectin-3, while a limited number of cells were DBA-reactive. Conclusion: The expression of galectin-3-binding sites and also DBA-reactive glycoligands correlates with an increased level of differentiation and/or cessation of proliferation in the examined squamous stratified epithelia. Further application of tissue lectins for characterizing ligand expression and its modulation is an important step to reveal functional relevance.

Defining the glycophenotype of squamous epithelia using plant and mammalian lectins. Differentiation-dependent expression of α2,6- and α2,3-linked N-acetylneuraminic acid in squamous epithelia and carcinomas, and its differential effect on binding of the endogenous lectins galectins-1 and -3

A thorough characterization of the properties of squamous epithelial cells is necessary in order to improve our understanding of the functional aspects of normal development and malignant aberrations. Up to now, studies have focused almost exclusively on monitoring distinct protein markers. With our growing awareness of the coding function of glycan chains of cellular glycoconjugates and their interaction with receptors (lectins) in situ, defining the glycophenotype of these cells has become an important issue. Whereas the commonly applied plant lectins are tools used to map the presence and localization of biochemically defined saccharide epitopes, the introduction of endogenous (mammalian) lectins to this analysis enables us to take the step from monitoring the presence of glycan to understanding the functional implications by revealing ligand properties of the detected epitope for tissue lectin. Thus, in this study we investigated a distinct aspect of glycosylation using plant and mammalian lectins, i.e. the linkage type of sialylation. We first mapped the expression profile of the type of sialylation (α2,3‐ or α2,6‐linked) by plant lectins. Based on the hypothesis that this factor regulates accessibility of ligands for endogenous lectins we introduced two labeled galectins to this study. Galectin‐3 (but not galectin‐1) binding was related to cell differentiation in normal adult and developing epithelia, cultured epidermal cells, and carcinomas derived from these epithelia. The presented data suggest that α2,6‐linked N‐acetyl‐D‐neuraminic acid moieties could serve to mask galectin‐3‐reactive glycoepitopes. As a consequence, monitoring of the linkage type of sialic acid in glycans by plant lectins therefore has implications for the extent of glycan reactivity with endogenous lectins, pointing to a potential function of changes in sialylation type beyond these cell and lectin systems.

Insight into the pathogenesis of sporadic basal cell carcinoma.

Sporadic basal cell carcinoma (BCC) is the commonest human cancer. Although its aggressiveness is low and metastatic potential negligible, the increasing incidence of the tumor in the Western world drives attention to its pathogenesis. In 1996, germ‐line mutations in the patched receptor of the Sonic hedgehog (Shh) signaling pathway were described in the Gorlin–Goltz syndrome in association with multiple nevoid BCCs. Later, the aberrant activation of the Shh was identified in sporadic BCCs as well. Recently, the role of other tumor suppressors and DNA repair gene mutations and their relationship with UV radiation‐induced DNA damage have been elucidated.

Mannosides as crucial part of bioactive supports for cultivation of human epidermal keratinocytes without feeder cells.

Large-scale production of keratinocytes by cell culture is of interest for medical applications. Long-term cultivation of epidermal cells is presently possible with feeder cells, i.e. 3T3 fibroblasts with arrested mitosis, or with specially formulated culture medium. To define refinements for in vitro conditions, the analysis of the natural environment with growth-maintaining/stimulating factors can provide important clues. Cells with proliferative activity are located in the basal layer of the epidermis in close contact with a basement membrane. Employing lectin and reverse lectin histochemistry of skin, muscle fibers and feeder cells, we assumed that the interplay of mannose-binding sites of epidermal cells, detected by a labeled neoglycoprotein, with glycoligands in the feeder cell layer or basement membrane could trigger signaling with relevance for adhesion and growth regulation. Indeed, coating of polystyrene with mannose-containing neoglycoprotein mimicking a mannose-rich cell matrix enabled the cultivation of keratinocytes without feeder cells in a Ca(2+)-dependent manner in serum-containing culture medium. Following this experimental demonstration of specific binding of mannose residues as part of a neoglycoprotein controlled by testing sugar-free carrier protein and other substances, we next synthesized and tested biocompatible polymers. Attachment and proliferation of keratinocytes on the surface of these polymers compared favorably to control experiments using feeder cells. In conclusion, we suggest that these polymers are bioactive offering a perspective for keratinocyte cultivation without feeder cells.

Dendritic cells and their role in skin-induced immune responses.

This article gives a brief review on dendritic cells (DC) with regard to their origin, life cycle and functions. The regulation of immune responses by DC functioning as antigen‐presenting cells is discussed. Special attention is given to epidermal DC, e.g. Langerhans cells. The perspectives of DC‐based therapy are also mentioned.

Analysis of Binding of Mannosides in Relation to Langerin (CD207) in Langerhans Cells of Normal and Transformed Epithelia

Tandem-repeat C-type lectins (pattern-recognition receptors) with specificity for mannosides are intimately involved in antigen recognition, uptake, routing and presentation in macrophages and dendritic cells. In Langerhans cells, Langerin (CD207), a type-II transmembrane protein with a single C-type carbohydrate recognition domain attached to a heptad repeat in the neck region, which is likely to establish oligomers with an α-coiled-coil stalk, has been implicated in endocytosis and the formation of Birbeck granules. The structure of Langerin harbours essential motifs for Ca2+-binding and sugar accommodation. Lectin activity has previously been inferred by diminished antibody binding to cells in the presence of the glycan ligand mannan. In view of the complexity of the C-type lectin/lectin-like network, it is unclear what role Langerin plays for Langerhans cells in binding mannosides. In order to reveal in frozen tissue sections to what extent mannose-binding activity co-localizes with Langerin, we have used a synthetic marker, i.e. a neoglycoprotein carrying mannose maxiclusters, as a histochemical ligand, and computer-assisted fluorescence monitoring in a double-labelling procedure. Mannoside-binding capacity was detected in normal epithelial cells. Double labelling ensured the unambiguous assessment of the binding of the neoglycoprotein in Langerhans cells. Light-microscopically, its localization profile resembled the pattern of immunohistochemical detection of Langerin. This result has implications for suggesting rigorous controls in histochemical analysis of this cell type, because binding of kit reagents, i.e. mannose-rich glycoproteins horseradish peroxidase or avidin, to Langerin (or a spatially closely associated lectin) could yield false-positive signals. To show that recognition of carbohydrate ligands in dendritic cells is not restricted to mannose clusters, we have also documented binding of carrier-immobilized histo-blood group A trisaccharide, a ligand of galectin-3, which was not affected by the presence of a blocking antibody to Langerin. Remarkably, access to the carbohydrate recognition domain of Langerin appeared to be impaired in proliferatively active environments (malignancies, hair follicles), indicating presence of an endogenous ligand with high affinity to saturate the C-type lectin under these conditions.

Biological properties of copolymer of 2-Hydroxyethyl methacrylate with sulfopropyl methacrylate

Interaction of organism with non-toxic implanted polymers depends on the physicochemical properties of the implant surface, which influence the adsorption of bioactive proteins and subsequently adhesion and growth of cells. The synthetic hydrogels are known as poorly adhesive surfaces. In this study we demonstrated the adsorption of albumin, fibrinogen, fibronectin, basic fibroblast growth factor, heparin-binding epidermal growth factor-like growth factor and epidermal growth factor to poly(2-hydroxyethyl methacrylate) (pHEMA) and copolymer of 2-hydroxyethyl methacrylate (HEMA) and potassium salt of 3-sulfopropyl methacrylate (SPMAK). The adhesion and growth of 3T3 cells and human keratinocytes on surface of these polymers was tested without and with pretreatment of polymers with heparin-binding epidermal growth factor-like growth factor. The adhesion of mixture of human granulocytes and monocytes to these surfaces was also tested. The strips of both polymers were subcutaneously and intracerebrally implanted into the rat and the extent of foreign body reaction and brain biocompatibility was evaluated. The results showed the extensive adsorption of basic fibroblast growth factor and heparin-binding epidermal growth factor-like growth factor to copolymer containing SPMAK. However the adhesion (and growth) of cells to this type of copolymers was very low. Preadsorption of human plasma to pHEMA clearly stimulated the leukocyte adhesion in contrary to copolymer containing SPMAK. The extent of foreign-body reaction was significantly higher against the pHEMA compared to tested copolymer p(HEMA-co-SPMAK). In conclusion, the tested copolymer was a poorly adhesive substrate that is only poorly recognized by the non-specific immunity, although the adsorption of basic growth factors to this substrate is highly significant. Both polymers were well tolerated by the brain tissue. The phenotype of surrounding neurons was more close to the control neurons in the brain tissue surrounding the p(HEMA-co-SPMAK) implants.

Dolichos biflorus agglutinin-binding site expression in basal keratinocytes is associated with cell differentiation

A basal layer of squamous epithelia such as epidermis contains stem cells, transit amplifying cells as well as postmitotic differentiating cells. A detailed knowledge of the transition among these cell types in the course of epidermal renewal is important. It would help in better understanding of many pathological processes, including cancer, and in employment of epidermal cells for therapeutic purposes. In this study we analyzed the possible role of Dolichos biflorus agglutinin (DBA)‐reactive α‐N‐acetylgalactosamine glycosylation in behavior of the human epidermal basal cells under in vivo and in vitro conditions. The data received from porcine epidermis were also included. Part of basal cells was positive for DBA‐binding sites and these cells exhibited a lower presence of β1 integrin in their basal surface connected to the basement membrane. The perinuclear Golgi‐like accumulation of β1 integrin was observed in some cultured keratinocytes. The co‐localization of integrin with DBA‐binding sites and 58 kDa protein suggests that α‐N‐acetylgalactosamine glycosylation could be related to β1 integrin retention in the endoplasmatic reticulum Golgi intermediate compartment (ERGIC) at the beginning of the secretory pathway. The lack of anchorage in culture elevated the number of DBA‐binding site positive cells without significant influence on cell growth when cells isolated directly from epidermis were employed in study. Some role of DBA‐reactive glycoligand expressions in a suprabasal movement of differentiated basal cells can be hypothesized.