Evidences for the involvement of the Galectin-1 in the interaction of pig chondrocytes with a lactose-modified chitosan, namely Chitlac, are reported. The Chitlac glycopolymer has been shown to promote pig chondrocyte aggregation and to induce extracellular matrix production. Highly pure Galectin-1 was obtained from pig spleen by affinity chromatography and its identity was determined by ion spray mass spectrometry analysis of tryptic peptide fragments obtained after in-gel digestion. The complete sequence of pig Galectin-1 CDS was obtainedby screening a pig EST database using human Galectin-1 sequence as template. The Galectin-1 cDNA was clonedinto a pGEX-4T-1 expression vector andthe recombinant protein was purified, characterizedandusedto produce a rabbit anti-serum. Recombinant Galectin-1 interacts in a dose-dependent manner with Chitlac as determined with ELISA assay. Expression level of galectin-1 gene, quantifiedby real-time PCR, was significantly higher in chondrocytes cultivatedon Chitlac. In the same way, the presence of Chitlac stimulates secretion of Galectin-1 in culture medium that, by immunohistochemical analysis, revealed to be clustered on the surface of Chitlac-induced aggregates. These data indicate the role of Galectin-1 as a bridging agent between Chitlac andchond rocyte aggregates.
The role of Galectin-1 in the interaction between chondrocytesand a lactose-modified chitosan
MARCON, PATRIZIA;MARSICH, ELEONORA;VETERE, AMEDEO;MOZETIC, PAMELA;CAMPA, CRISTIANA;DONATI, IVAN;VITTUR, FRANCO;GAMINI, AMELIA;PAOLETTI, SERGIO
2005-01-01
Abstract
Evidences for the involvement of the Galectin-1 in the interaction of pig chondrocytes with a lactose-modified chitosan, namely Chitlac, are reported. The Chitlac glycopolymer has been shown to promote pig chondrocyte aggregation and to induce extracellular matrix production. Highly pure Galectin-1 was obtained from pig spleen by affinity chromatography and its identity was determined by ion spray mass spectrometry analysis of tryptic peptide fragments obtained after in-gel digestion. The complete sequence of pig Galectin-1 CDS was obtainedby screening a pig EST database using human Galectin-1 sequence as template. The Galectin-1 cDNA was clonedinto a pGEX-4T-1 expression vector andthe recombinant protein was purified, characterizedandusedto produce a rabbit anti-serum. Recombinant Galectin-1 interacts in a dose-dependent manner with Chitlac as determined with ELISA assay. Expression level of galectin-1 gene, quantifiedby real-time PCR, was significantly higher in chondrocytes cultivatedon Chitlac. In the same way, the presence of Chitlac stimulates secretion of Galectin-1 in culture medium that, by immunohistochemical analysis, revealed to be clustered on the surface of Chitlac-induced aggregates. These data indicate the role of Galectin-1 as a bridging agent between Chitlac andchond rocyte aggregates.Pubblicazioni consigliate
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