Fluorescent and radiolabeling of polysaccharides: binding and internalization experiments on vascular cells.
Glycosaminoglycans (GAGs) such as heparan sulfates are complex carbohydrate polymers. These structural components of the extracellular matrix are essential for the adhesion, migration, and regulation of cellular growth. To understand the physiological role of GAGs and GAG analogues, a practical approach consists of labeling and detecting them in cell extracts, or analyzing binding domains and their distributions into the cells. We propose a convenient and reliable method for preparing and labeling amino-enriched, polysaccharides with the fluorescent derivative 5-[(4,6-dichlorotriazine-2-yl)amino]-fluorescein (DTAF). Radioiodination is then performed on the DTAF moiety. This method was applied to polysaccharides known to inhibit vascular smooth-muscle cell (SMC) proliferation such as functionalized dextrans derived from poly(alpha 1-6 glucose) and fucan, poly(L-fucose 4-sulfate) extracted from brown seaweed. Using autoradiography and confocal microscopy, we observed the fixation and internalization of labeled antiproliferative products in SMCs from rat aorta. These probes can be useful for the understanding of polysaccharide-cell interactions. In addition, the method presented here can be applied to various synthetic or natural biomedical materials.
Prigent-Richard
S
S
LRM, CNRS, URA 502, IFREMER URM2, University Paris XIII, Villetaneuse, France.
Cansell
M
M
Vassy
J
J
Viron
A
A
Puvion
E
E
Jozefonvicz
J
J
Letourneur
D
D
D000596:Amino Acids; D000818:Animals; D001345:Autoradiography; D002455:Cell Division; D004705:Endocytosis; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D000596:Amino Acids; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D011134:Polysaccharides; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein