Characterization of the role of domains and subdomains of aggrecan in post-translational processing and secretion

Aggrecan is the major proteoglycan in the articular cartilage. This molecule is crucial to the chondroskeletal morphogenesis during development and it is also important in the proper functioning of articular cartilage by providing hydrated gel structures (via its interaction with hyaluronic acid and link protein) which endow the cartilage with load-bearing properties. Aggrecan is a multimodular molecule expressed by chondrocytes. To determine the role of each aggrecan domain in its post-translational modification and secretion, we generated a number of recombinant DNA constructs bearing various aggrecan domains individually or in combinations with other domains. We observed that G1 and G2 domains inhibited product secretion and GAG chain attachment whereas, G3 domain promoted secretion and GAG chain attachment. Knowing that G1 and G2 inhibited secretion, we set to identify which region of G1 and G2 shared the inhibition of secretion properties. Utilizing recombinant genes, we demonstrated that the first tandem repeats of G1 and G2 inhibited product secretion if expressed alone or in combinations with other domains and subdomains. We next tried to isolate the amino acid residues in the first tandem repeat of G1 involved in the inhibition of secretion by mutagenesis experiments. C-terminal mutagenesis of the first tandem repeat of G1 did not promote the secretion, however, N-terminal mutagenesis (point mutations and deletions) gave rise to enhancement of secretion. We noticed that there were four N-terminal residues, FHYR, which seemed to be important in inhibition of secretion, however, we next realized that these four residues were part of a stretch of 55 amino acids required for the inhibitory property. Yeast two-hybrid analyses also indicated that TR1 of G1 is interacting with ER-bound chaperones.