The G1 domain of aggrecan released from porcine articular cartilage forms stable complexes with hyaluronan/link protein

Objective. To raise peptide antibodies recognizing the C-terminal amino acid sequence in the G1 domain of porcine aggrecan, generated by the action of either aggrecanase or neutral metalloproteinase(s), in rabbits and to use them to investigate the release of aggrecan from porcine articular cartilage. Method. An explant culture system was used to investigate the release of the G1 domain of aggrecan from porcine articular cartilage treated with retinoic acid or interleukin 1b and to study how the activity of these agents is modified by the proteinase inhibitor, batimastat (BB94). Results. Retinoic acid and interleukin 1b induced both enzyme activities and the release of the G1 domain into the culture medium. Proteinase activity was significantly reduced when the tissue was incubated in the presence of BB94. The functional properties of the enzyme-generated G1 domain were studied using largepore, agaroseupolyacrylamide gel electrophoresis, and it was shown to interact with hyaluronan and link protein. Conclusions. The results show that there must be a mechanism for removing a functional G1 domain from aggrecan during tissue turnover using this culture system. In normal articular cartilage the rate of protein synthesis is assumed to be equal to the rate of degradation, because the level of extracellular matrix components remains constant. However, changes in the steady state result in pathological destruction of cartilage and loss of function. The primary cause of degradation is elevated levels of proteolytic enzyme activity resulting from an imbalance between proteinases and their inhibitors. The turnover of the proteoglycan aggrecan in articular cartilage has received a considerable amount of attention recently. Although aggrecan is cleaved by a number of matrix metalloproteinases (MMPs) between Asn 341 and Phe 342 in the interglobular domain of the molecule, the major fragments of aggrecan detected in arthritic synovial fluid result from cleavage at the Glu 373 ‐Ala 374 site w1x.