Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 535-545 |
| Number of pages | 11 |
| Journal | Biology of the Cell |
| Volume | 95 |
| Issue number | 8 |
| DOIs | |
| State | Published - Nov 2003 |
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Keywords
- Biotinylation
- Bone marrow stromal cells
- Galectin
- Keratinocytes
- Pre-mRNA splicing
ASJC Scopus subject areas
- Cell Biology
Cite this
New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells : Biotinylated galectins as tool to detect specific binding sites. / Purkrábková, Tereza; Smetana, Karel; Dvořánková, Barbora; Holíková, Zuzana; Böck, Corina; Lensch, Martin; André, Sabine; Pytlík, Robert; Liu, Fu-Tong; Klíma, Jiřá; Smetana, Karel; Motlík, Jan; Gabius, Hans Joachim.
In: Biology of the Cell, Vol. 95, No. 8, 11.2003, p. 535-545.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells
T2 - Biotinylated galectins as tool to detect specific binding sites
AU - Purkrábková, Tereza
AU - Smetana, Karel
AU - Dvořánková, Barbora
AU - Holíková, Zuzana
AU - Böck, Corina
AU - Lensch, Martin
AU - André, Sabine
AU - Pytlík, Robert
AU - Liu, Fu-Tong
AU - Klíma, Jiřá
AU - Smetana, Karel
AU - Motlík, Jan
AU - Gabius, Hans Joachim
PY - 2003/11
Y1 - 2003/11
N2 - 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.
AB - 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.
KW - Biotinylation
KW - Bone marrow stromal cells
KW - Galectin
KW - Keratinocytes
KW - Pre-mRNA splicing
UR - http://www.scopus.com/inward/record.url?scp=0345329271&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0345329271&partnerID=8YFLogxK
U2 - 10.1016/j.biolcel.2003.08.002
DO - 10.1016/j.biolcel.2003.08.002
M3 - Article
C2 - 14630391
AN - SCOPUS:0345329271
VL - 95
SP - 535
EP - 545
JO - Biology of the Cell
JF - Biology of the Cell
SN - 0248-4900
IS - 8
ER -