title: Cathepsin D cleaves aggrecan at unique sites within the interglobular domain and chondroitin sulfate attachment regions that are also cleaved when cartilage is maintained at acid pH.
abstract: Bovine aggrecan was digested with bovine cathepsin D at pH 5.2 under conditions of partial digestion and the resulting aggrecan core protein fragments were separated by electrophoresis on gradient polyacrylamide gels. The fragments were characterized by their reactivity to specific antibodies and by N-terminal amino acid sequencing. It was also demonstrated that cathepsin D cleaved bovine aggrecan at five sites within the core protein, between residues Phe(342)-Phe(343) in the interglobular domain, Leu(1462)-Val(1463) between the chondroitin sulfate attachment regions 1 and 2 and Leu(1654)-Val(1655), Phe(1754)-Val(1755) and Leu(1854)-Ile(1855) that are located within the chondroitin sulfate attachment region 2 of the core protein. The time course of digestion showed that there was a continued degradation of aggrecan and there was no preferential cleavage of the core protein at any one site. It was shown that cathepsin D digested aggrecan over the pH range 5.2-6.5 resulting in the same products. When bovine cartilage was maintained in explant culture at pH 5.2 there was a rapid loss of both radiolabeled and chemical pools of sulfated glycosaminoglycans into the culture medium and this loss was inhibited by the inclusion in the medium of the aspartic proteinase inhibitor, pepstatin A. The aggrecan core protein fragments appearing in the medium of cultures maintained at pH 5.2 were characterized and it was shown that the fragments had N-terminal sequences starting at Phe(343), Ile(1855), and Val(1755) or Val(1463). This work demonstrates that cathepsin D present within the extracellular matrix of articular cartilage has the potential to contribute to the proteolytic processing of the core protein of aggrecan in this tissue.
authors.affiliation: School of Human Biosciences, La Trobe University, Victoria 3086, Australia. c.handley@latrobe.edu.au
authors.forename: C J
authors.initials: CJ
authors.lastname: Handley
authors.forename: M T
authors.initials: MT
authors.lastname: Mok
authors.forename: M Z
authors.initials: MZ
authors.lastname: Ilic
authors.forename: C
authors.initials: C
authors.lastname: Adcocks
authors.forename: D J
authors.initials: DJ
authors.lastname: Buttle
authors.forename: H C
authors.initials: HC
authors.lastname: Robinson
mesh_terms: D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002358:Cartilage, Articular / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism*; D002417:Cattle; D002809:Chondroitin Sulfates / Q000378:metabolism*; D046508:Culture Techniques; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D006863:Hydrogen-Ion Concentration; D037181:Lectins, C-Type; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism*
chemical_list: D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D010446:Peptide Fragments; D011509:Proteoglycans; D002809:Chondroitin Sulfates; D002402:Cathepsin D