N-terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-treated articular-cartilage cultures. Putative site(s) of enzymic cleavage.
Bovine articular cartilage was cultured both in the presence and in the absence of human recombinant interleukin-1 alpha (IL-1) (100 units/ml). Addition of this cytokine stimulated matrix degradation approx. 3-fold. This increased degradation permitted characterization of the large chondroitin sulphate proteoglycan (aggrecan) fragments accumulating in the media. When compared with controls, the proteoglycans isolated from the medium of cultures treated with IL-1 exhibited a decrease in the Kav. (control 0.25; IL-1-treated 0.37), determined by Sepharose CL-2B chromatography. This decrease in proteoglycan size was accompanied by a decreased ability of these monomers to associate with hyaluronic acid. Thus only 20% of the proteoglycans isolated from the medium of IL-1-treated cultures, compared with 39% for control cultures, had the capacity to form high-M(r) aggregates with hyaluronic acid. SDS/PAGE analysis of the proteoglycans from the media of IL-1-treated cultures demonstrated several large proteoglycan protein-core bands (M(r) 144,000-380,000). The protein-core bands with M(r) 144,000-266,000 exhibited a significantly decreased reactivity with monoclonal antibody 1-C-6 (specific for domains G1 and G2). The N-terminal amino acid sequence of four of these protein-core bands (M(r) 144,000, 173,000, 214,000 and 266,000) yielded sequences LGQRPPV-Y-PQLF(E), AGEGP(S)GILEL-GAP(S)-AP(D)M, GLG-VEL-LPGE and (A)RGSVIL-AKPDFEV-P-A. A comparison of these N-terminal amino acid sequences with the published proteoglycan sequence for bovine nasal cartilage [Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259], rat chondrosarcoma [Doege, Sasaki, Horigan, Hassell & Yamada (1987) J. Biol. Chem. 262, 17757-17769] and human articular cartilage [Doege, Sasaki, Kimura & Yamada (1991) J. Biol. Chem. 266, 894-902] permitted assignment of their relative positions on the core protein. Furthermore, on the basis of this similarity to published sequence, putative sites of enzymic cleavage were constructed. These theoretical cleavage sites revealed a glutamic acid residue in the P1 position and an uncharged polar or non-polar residue in the P1' position.
Loulakis
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P
Institute for Arthritis and Autoimmunity, Miles Inc., West Haven, CT 06516.
Shrikhande
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A
Davis
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Maniglia
C A
CA
D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D006868:Hydrolysis; D007375:Interleukin-1 / Q000494:pharmacology*; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism*; D016622:Silver Staining
C508321:Acan protein, rat; D053674:Aggrecans; D004798:Enzymes; D016326:Extracellular Matrix Proteins; D007375:Interleukin-1; D037181:Lectins, C-Type; D010446:Peptide Fragments; D011509:Proteoglycans