The intermediates of aggrecanase-dependent cleavage of aggrecan in rat chondrosarcoma cells treated with interleukin-1.
We have examined the abundance and structure of intermediates in the chondrocyte-mediated degradation of aggrecan by aggrecanase(s). Degradation products were identified by Western-blot analysis with antibodies to cleavage-site neoepitopes and to peptides within the globular domains. Rat chondrosarcoma tumour contained full-length aggrecan and all of the individual peptides expected from single independent cleavages at each of the four aggrecanase sites in the chondroitin sulphate (CS) domain. Kinetic analysis of the products present in rat chondrosarcoma cell cultures treated with interleukin-1b showed that the first aggrecanase-mediated cleavages occurred at the four sites within the CS attachment region to generate two stable intermediates, Val(1)-Glu(1459) and Val(1)-Glu(1274). These species were subsequently cleaved at the Glu(373) site in the interglobular domain to form the terminal products, Val(1)-Glu(373), Ala(374)-Glu(1274) and Ala(374)-Glu(1459). It therefore appears that the aggrecanase-mediated processing of native aggrecan by chondrocytes in situ is initiated within the CS-attachment region and completed by cleavage within the interglobular domain. Since it has been shown that digestion of aggrecan monomer in solution with recombinant ADAMTS-4 [Tortorella, Pratta, Liu, Austin, Ross, Abbaszade, Burn and Arner (2000) Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4). J. Biol. Chem. 275, 18566-18573] exhibits similar kinetics, it appears that preferential proteinase cleavage in the CS-rich region is determined by properties inherent in the aggrecan monomer itself, such as preferred peptide sequences for enzyme binding or enhanced accessibility to the core protein at these sites.
Sandy
J D
JD
Shriners Hospital, Tampa Unit, 12502 North Pine Drive, Tampa, FL 33612, USA. jsandy@shctampa.usf.edu
Thompson
V
V
Doege
K
K
Verscharen
C
C
D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002809:Chondroitin Sulfates / Q000378:metabolism; D002813:Chondrosarcoma / Q000201:enzymology / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D016326:Extracellular Matrix Proteins; D007106:Immune Sera / Q000276:immunology; D007375:Interleukin-1 / Q000494:pharmacology*; D007700:Kinetics; D037181:Lectins, C-Type; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
C508321:Acan protein, rat; D053674:Aggrecans; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D007106:Immune Sera; D007375:Interleukin-1; D037181:Lectins, C-Type; D010446:Peptide Fragments; D011509:Proteoglycans; D002809:Chondroitin Sulfates; D010450:Endopeptidases; C083641:aggrecanase