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Membrane-associated and secreted proteoglycans from a continuous cell line derived from fibrotic schistosomal
L C Silva1, R Borojevic, P A Mourão
1Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Abstract:
Proteoglycans were isolated from a continuous murine cell line (GRX) established from fibrotic granulomas induced in mouse liver by schistosomal infection, representative of liver connective tissue cells. The proteoglycans were labelled with 35SO4, extracted by guanidine-HCl + Triton X-100 in the presence of proteinase inhibitors, and purified by anion-exchange, gel-filtration and affinity-column chromatography. The major fractions of cell-associated and secreted proteoglycans are heparan sulfate proteoglycans. Gel-filtration chromatography on Sephacryl S-400 revealed Kav values of 0.20 and 0.30 for the cell-associated and secreted heparan sulfate proteoglycans, respectively. About 50% of the cell-associated heparan sulfate proteoglycans contained hydrophobic regions, as evidenced by their ability to bind to octyl-Sepharose, while only about 20% of secreted proteoglycans bound to this resin. In addition, no proteoglycan was competitively displaced from the cell surface by heparin. Taken together with other reports on proteoglycan synthesis by a variety of cell types in culture, these observations suggest that cell-surface heparan sulfate proteoglycans possibly contain a hydrophobic domain that functions as a membrane anchor in their attachment to cells. Addition of beta-D-xyloside to the cultures greatly enhanced the release of 35S-dermatan sulfate to the medium. Interestingly, dermatan sulfate is the major glycosaminoglycan found in the schistosoma-induced granuloma, from which the GRX cell line is derived. These studies provide the first biochemical description of the proteoglycans produced by a liver connective tissue cell line derived from schistosomal granulomas.
Insights
Researchers characterized proteoglycans from a murine liver cell line derived from schistosomal granulomas. They found cell-associated heparan sulfate proteoglycans have hydrophobic anchors, and dermatan sulfate release is enhanced by beta-D-xyloside.
Area of Science:
- Biochemistry
- Cell Biology
- Parasitology
Background:
- Liver fibrosis and granuloma formation are key features of schistosomiasis.
- Understanding the extracellular matrix in fibrotic conditions is crucial for therapeutic development.
- Murine cell lines offer a model to study cellular processes in disease contexts.
Purpose of the Study:
- To biochemically characterize proteoglycans from a murine liver cell line (GRX) derived from schistosome-induced granulomas.
- To investigate the nature of cell-associated and secreted proteoglycans.
- To explore the role of proteoglycans in liver connective tissue cells during schistosomal infection.
Main Methods:
- Proteoglycan isolation and labeling with 35SO4.
- Extraction using guanidine-HCl and Triton X-100 with proteinase inhibitors.
- Purification via anion-exchange, gel-filtration (Sephacryl S-400), and affinity chromatography (octyl-Sepharose).
- Analysis of proteoglycan release using beta-D-xyloside.
Main Results:
- Heparan sulfate proteoglycans were the major cell-associated and secreted fractions.
- Cell-associated heparan sulfate proteoglycans showed a higher proportion of hydrophobic regions compared to secreted forms.
- No proteoglycan was displaced from the cell surface by heparin, suggesting a membrane-anchored hydrophobic domain.
- Beta-D-xyloside treatment significantly increased 35S-dermatan sulfate release into the medium.
Conclusions:
- Cell-surface heparan sulfate proteoglycans likely possess a hydrophobic domain anchoring them to the cell membrane.
- Dermatan sulfate, a major component of schistosomal granulomas, can be released from these cells.
- This study provides the first biochemical description of proteoglycans from a liver connective tissue cell line associated with schistosomal granulomas.