CATABOLISM OF AGGRECAN BY EXPLANT CULTURES OF HUMAN ARTICULAR-CARTILAGE IN THE PRESENCE OF RETINOIC ACID

The N-terminal amino acid sequence of human aggrecan was determined an d it was shown that two sequences were present. The major sequence, AV TVE-, accounted for 60% of the aggrecan and started at alanine residue 17 of the human aggrecan core protein cDNA sequence (K. Doege et al. (1991) J. Biol. Chem. 266, 894-920). The other N-terminal sequence, VE TX-, started at valine residue 20. Characterization of aggrecan core p rotein peptides present in the matrix of adult human articular cartila ge showed that at least 11 aggrecan core proteins were present with ap proximate M(r) between 300,000 and 43,000. All these core proteins wer e found to have the same N-terminal sequences as that observed in huma n aggrecan. When articular cartilage was placed in explant culture in medium containing 10(-6) M retinoic acid there was a 3.5-fold increase in the loss of aggrecan into the culture medium compared to tissue ma intained in medium alone or medium containing 20% (v/v) newborn calf s erum. Analysis of the aggrecan core protein fragments that were releas ed to the culture medium containing 10(-6) M retinoic acid showed the presence of 13 core protein peptides of M(r) between 300,000 and 43,00 0. The 11 smaller peptides of M 230,000 to 43,000 were shown to have t he N-terminal sequence ARGS-. This sequence which starts at residue 39 3 of the human aggrecan core protein is located within the interglobul ar region between the G1 and G2 domains and is the site of aggrecan ca tabolism by the putative protease aggrecanase. The presence of core pr oteins of varying sizes but with the same N-terminal sequence reflects proteolytic processing from the C-terminal end of the core protein th at was also observed in the aggrecan macromolecules extracted from the matrix of human articular cartilage. This proteolytic processing was also evident but to a lesser extent in newly synthesized S-35-labeled aggrecan macromolecules. (C) 1995 Academic Press, Inc.