Title

Aggrecan synthesis and secretion: A paradigm for molecular and cellular coordination of multiglobular protein folding and intracellular trafficking

Date of Completion

January 1998

Keywords

Biology, Molecular|Biology, Genetics

Degree

Ph.D.

Abstract

Aggrecan is the large aggregating proteoglycan of cartilage. The aggrecan protein core has an multi-domain structure which consists of three globular domains, G1, G2 and G3, and a long GAG domain between G2 and G3 on which keratan sulfate (KS) and chondroitin sulfate (CS) attachments occur. G1 and G2 reside at the amino terminus and G3 at the carboxyl terminus of aggrecan. The C-terminal globular G3 domain appears in a family of structurally related proteoglycans. As secreted proteins with multiple globular domains, members of the aggrecan family would be expected to undergo chaperone-mediated surveillance in the ER lumen.^ Recombinant DNA technologies have been used to create gene constructs and test the secretion behavior of selected aggrecan domains. Different constructs have been fabricated with a common N-terminal signal sequence, a GAG consensus region, and a histidine "tag" at the C-terminus. CHO cells were transfected with these different vector-containing constructs. The proteins were metabolically labeled, and then harvested from the cells and spent media. The protein secretion rate was measured. The N-terminal G1 domain was secreted at a rate much lower than the C-terminal G3 domain. A hybrid construct, (G1 domain-GAG consensus domain-G3 domain), or "mini-aggrecan", was secreted as a neoproteoglycan at a rate intermediate between G1 and G3; this result suggests that G3 facilitates secretion of G1 and, consequently, of aggrecan itself. Constructs were made with G1 or G3 on the contralateral flank of the GAG consensus domain, opposite their usual sites. Contralateral G1 protein was secreted at a lower rate than normal G1 protein whereas the contralateral G3 protein secretion rate is similar to that of normal G3 protein. In situ cross-linking results showed that the molecular chaperone Hsp25 became cross-links to G3 but did not cross-link to G1. Moreover, non-cross-linked Hsp25 is secreted in a complex with contralateral G3 protein but is not secreted with normal G3 protein. We suggest that Hsp25 interacts with nascent G3, assisting its folding into proper globular conformation, followed by G3 acting as an intramolecular chaperone, assisting G1 to become globular. ^