Role of the C-terminal G3 domain in sorting and secretion of aggrecan coreprotein and ubiquitin-mediated degradation of accumulated mutant precursors

Aggrecan is a complex multidomain macromolecule that undergoes extensive pr ocessing and post-translational modification. A thorough understanding of t he events and signals that promote translocation of aggrecan through the se cretory pathway is lacking. To investigate which features of the C-terminal G3 region are necessary for:successful translocation of the core protein, a number of deletion constructs based on the chick aggrecan cDNA sequence w ere prepared and transiently expressed in COS-l cells and the natural host, embryonic chick chondrocytes; stable cell lines were established as well. The present results clearly establish a precise requirement for that portio n of the G3 C-lectin domain encoded by exon 15 for: (i) translocation from the endoplasmic reticulum (ER) to the Golgi, (ii) secretion from the cell, (iii) galactosylation of chondroitin sulfate (CS):chains, (iv) generation o f Ca+2-dependent galactose binding ability, Furthermore, in the absence of this subdomain there is excess accumulation in the ER of expression product s leading to a stress-related response involving the chaperones Grp78 and p rotein disulfide isomerase, followed by degradation via a ubiquitin-proteos ome pathway. All of these events in the model system faithfully mimic the n aturally occurring nanomelic mutant, which also elicits a ubiquitin-mediate d degradation response due to the accumulation of the truncated core protei n precursor. This study represents the first report of the mode of degradat ion of overexpressed or misfolded proteoglycans and suggests that, although proteoglycans follow different glycosylation pathways from other glycoprot eins, they are monitored by an ER surveillance system similar to that which detects other misfolded proteins.