A novel use of TAT-EGFP to validate techniques to alter osteosarcoma cell surface glycosaminoglycan expression.
Several methods to alter cell surface glycosaminoglycan (GAG) expression have previously been described, including treatments with chlorate to reduce the addition of charged sulfate groups, xyloside compounds to displace GAGs from their core proteins, and GAG lyases, such as heparinase and chondroitinase, to release GAG fragments from the cell layer. While these methods are useful in identifying cellular mechanisms which are dependent on GAGs, they must be stringently validated to assess results in the appropriate context. To determine the most useful technique for the evaluation of GAG function in osteogenesis, MG-63 osteosarcoma cells were systematically treated with these agents and evaluated for changes in cell surface GAGs using a TAT-EGFP fusion protein. TAT, a protein transduction domain from the HIV-1 virus, requires cell surface GAGs to traverse cell membranes. The EGFP component provides a method to assess protein entry into cells in both qualitative and quantitative tests. Here, TAT-EGFP transduction analysis confirmed radiochemical and physiological data that chlorate effectively disrupts GAG expression. TAT-EGFP entry into cells was also inhibited by the exogenous application of commercial heparin and GAGs extracted from MG-63 cells as well as by the pre-treatment of cells with chondroitinase ABC. However, neither heparinase III treatment nor the addition of exogenous chondroitin-6-sulfate affected TAT-EGFP entry into cells. In addition, xyloside-beta-D-naphthol and xyloside-beta-D-cis/trans-decahydro-2-naphthol treatment could not induce significant phenotypic change in these cells, and the unaffected TAT-EGFP transduction confirmed that this was due to an inability to efficiently prime GAG synthesis. The use of TAT-EGFP is thus a useful technique to specifically evaluate cell surface GAG expression in a simple, quantifiable manner, and avoids the complications involved with conventional radiochemical assays or analytical chromatography.
Kumarasuriyar
Arjuna
A
Institute of Molecular and Cell Biology, Singapore, Singapore.
Dombrowski
Christian
C
Rider
David A
DA
Nurcombe
Victor
V
Cool
Simon M
SM
D001692:Biological Transport / Q000187:drug effects; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002808:Chondroitin Lyases / Q000494:pharmacology; D002809:Chondroitin Sulfates / Q000494:pharmacology; D005434:Flow Cytometry; D006025:Glycosaminoglycans / Q000378:metabolism*; D006027:Glycosides / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D019763:Heparin Lyase / Q000494:pharmacology; D006801:Humans; D018613:Microscopy, Confocal; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
D006025:Glycosaminoglycans; D006027:Glycosides; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C010807:xylosides; D049452:Green Fluorescent Proteins; D006493:Heparin; D002809:Chondroitin Sulfates; D002808:Chondroitin Lyases; D019763:Heparin Lyase