The metabolism of 2-fluoro-2-deoxy-D-glucose in human chondrocytes and its incorporation into keratan sulfate proteoglycans.
The incorporation of 2-fluoro-2-deoxy-D-[14C]glucose in proteoglycans was investigated in a cell culture system, where human articular chondrocytes were cultured in high-cell-density thin-layer soft agarose. The proteoglycans were solubilized from the culture medium and the cell layer fraction by extracting with a guanidine hydrochloride buffer and purified by an ion-exchange-chromatography (DEAE-Sepharose CL-6B). With enzymic decomposition experiments concerning the glycosaminoglycan side-chains it could be shown that 65-69% were digestible by keratanase, whereas 21-29% of the 14C-labeled proteoglycans were digested with chondroitinase AC/ABC. The main constituent of the 2-fluoro-2-deoxy-D-[14C]glucose-metabolites present in the glycosaminoglycan side chains of the proteoglycans was 2-fluoro-2-deoxy-D-[14C]galactose. Therefore, 2-fluoro-2-deoxy-D-glucose was preferentially incorporated into keratan sulfate. We investigated the effect of non-radioactive 2-fluoro-2-deoxy-D-glucose on UDP-sugar and proteoglycan biosynthesis after incubation periods of 1-30 h. A high 2-fluoro-2-deoxy-D-glucose concentration in the culture medium did not influence the pool size of UDP-N-acetylhexosamines, but UDP-D-glucose, UDP-D-galactose, UDP-D-glucuronic acid, UDP-2-fluoro-2-deoxy-D-glucose, UDP-2-fluoro-2-deoxy-D-galactose and UDP-2-fluoro-2-deoxy-D-glucuronic acid accumulated in the chondrocytes time dependently. In a pulse/chase experiment the retarded synthesis of fluorinated UDP-sugars was proved. The half-lives (t1/2) for UDP-2-fluoro-2-deoxy-D-glucose and UDP-2-fluoro-2-deoxy-D-galactose were about 7.7 h and 13.3 h, respectively. UDP-2-fluoro-2-deoxy-D-glucuronic acid could be found with delay. The incubation with 2-fluoro-2-deoxy-D-glucose and [14C]glucosamine resulted in a decreased radioactive labelling of chondroitin sulfate and keratan sulfate.
Fedders
G
G
Institute of Clinical Chemistry and Pathobiochemistry, Medical Faculty, University of Technology Aachen, Germany.
Kock
R
R
Van de Leur
E
E
Greiling
H
H
D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D019788:Fluorodeoxyglucose F18; D005944:Glucosamine / Q000378:metabolism; D006026:Glycoside Hydrolases; D006801:Humans; D007632:Keratan Sulfate / Q000378:metabolism*; D000071234:Lumican; D014530:Uridine Diphosphate / Q000378:metabolism; D014539:Uridine Diphosphate Sugars / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D011508:Chondroitin Sulfate Proteoglycans; C000604306:LUM protein, human; D000071234:Lumican; D014539:Uridine Diphosphate Sugars; D019788:Fluorodeoxyglucose F18; D014530:Uridine Diphosphate; D007632:Keratan Sulfate; D003847:Deoxyglucose; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D005944:Glucosamine