Jan Motlík

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.

Nuclear presence of adhesion-/growth-regulatory galectins in normal/malignant cells of squamous epithelial origin

Cellular activities in the regulation of growth or adhesion/migration involve protein (lectin)–carbohydrate recognition at the cell surface. Members of the galectin family of endogenous lectins additionally bind distinct intracellular ligands. These interactions with protein targets explain the relevance of their nuclear and cytoplasmic presence. Expression profiling for galectins and accessible binding sites is a histochemical approach to link localization with cellular growth properties. Non-cross-reactive antibodies for the homodimeric (proto-type) galectins-1, -2 and -7 and the chimera-type galectin-3 (Gal-3) as well as the biotinylated lectins were tested. This analysis was performed with the FaDu squamous carcinoma cell line and long-term cultured human and porcine epidermal cells as models for malignant and normal cells of squamous cell epithelial origin. A set of antibodies was added for phenotypic cell characterization. Strong nuclear and cytoplasmic signals of galectins and the differential reactivity of labeled galectins support the notion of their individual properties. The length of the period of culture was effective in modulating marker expression. Cytochemical expression profiling is a prerequisite for the selection of distinct proteins for targeted modulation of gene expression as a step toward functional analysis.

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.

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.

The Minipig as an Animal Model in Biomedical Stem Cell Research

Pigs and miniature pigs are steadily gaining importance as large animal models in the field of regenerative medicine, including stem cell research. With their size, organ capacity, and physiology resembling in several aspects that of humans, pigs are well suited for preclinical experiments and long-term safety studies. In this chapter, we summarize our experience with the isolation and culture of several somatic stem cell populations from fetal and adult pig tissue and briefly review their potential usefulness in future stem cell-based therapies. We also provide protocols for the isolation of fetal porcine neural stem cells (NSCs), adult bone marrow mesenchymal stem cells (MSCs), and epidermal progenitor cells (EPCs) from adult hair follicles.