Effects of brefeldin A on aggrecan core protein synthesis and maturation in rat chondrosarcoma cells.
In this paper, the effects of the fungal metabolite, brefeldin A, on the synthesis and maturation of aggrecan core protein precursor were studied in rat chondrosarcoma chondrocytes. The aggrecan core protein precursor was partially identified in total protein pools isolated from cell extracts based on its selective cleavage at a single site by the restriction protease factor Xa. During a 2-h labeling period with [3H]serine as precursor, brefeldin A inhibited the synthesis of mature aggrecan from its aggrecan core protein precursor consistent with an inhibition of chondroitin sulfate chain elongation and sulfation as described in the accompanying paper (Calabro, A., and Hascall, V. C. (1994) J. Biol Chem. 269, 22764-22770). This inhibition is presumably the result of the disruption of vesicular transport by brefeldin A, which isolates the aggrecan core protein precursor at the level of the trans-Golgi cisternae from the enzymes for chondroitin sulfate chain elongation and sulfation located in the trans-Golgi network. Brefeldin A also inhibited the exocytosis of all radiolabeled secretory proteins from the cell layer into the medium compartment, which is also consistent with the disruption of vesicular transport attributed to this metabolite. Although total protein synthesis was inhibited by 12% in the presence of brefeldin A, the aggrecan core protein precursor accumulated within the cell layer indicating that the inhibition of chondroitin sulfate synthesis by brefeldin A was not the result of a lack of aggrecan core protein precursor. When the brefeldin A block was removed and cultures chased in the presence of cycloheximide to prevent new protein synthesis, vesicular transport through the cell was re-established and chondroitin sulfate chains were added to a large proportion of the aggrecan core protein precursor that had accumulated during the brefeldin A block. These results suggest that the machinery for chondroitin sulfate synthesis and for protein exocytosis, that were disrupted by brefeldin A treatment, recover after removal of the brefeldin A, even in the presence of cycloheximide, and that the structures involved in these processes reassemble from previously existing proteins. Interestingly, two other proteins with the same relative abundance as the aggrecan core protein precursor were observed. An approximately 210-kDa protein with the characteristics of the fibronectin subunit, and an unidentified approximately 150-kDa protein which was efficiently cleaved by the protease Xa enzyme.
Calabro
A
A
Proteoglycan Chemistry Section, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Hascall
V C
VC
D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D020126:Brefeldin A; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis*; D002813:Chondrosarcoma / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D037181:Lectins, C-Type; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011498:Protein Precursors / Q000096:biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011509:Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D014407:Tumor Cells, Cultured
C508321:Acan protein, rat; D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D003517:Cyclopentanes; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011498:Protein Precursors; D011500:Protein Synthesis Inhibitors; D011509:Proteoglycans; D020126:Brefeldin A