Role of the C-terminal G3 domain in sorting and secretion of aggrecan core protein and ubiquitin-mediated degradation of accumulated mutant precursors.
Aggrecan is a complex multidomain macromolecule that undergoes extensive processing and post-translational modification. A thorough understanding of the events and signals that promote translocation of aggrecan through the secretory 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 were prepared and transiently expressed in COS-1 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 portion 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 of Ca(+2)-dependent galactose binding ability. Furthermore, in the absence of this subdomain there is excess accumulation in the ER of expression products leading to a stress-related response involving the chaperones Grp78 and protein disulfide isomerase, followed by degradation via a ubiquitin-proteosome pathway. All of these events in the model system faithfully mimic the naturally occurring nanomelic mutant, which also elicits a ubiquitin-mediated degradation response due to the accumulation of the truncated core protein precursor. This study represents the first report of the mode of degradation of overexpressed or misfolded proteoglycans and suggests that, although proteoglycans follow different glycosylation pathways from other glycoproteins, they are monitored by an ER surveillance system similar to that which detects other misfolded proteins.
Domowicz
M S
MS
Departments of Pediatrics and Biochemistry & Molecular Biology, Committee on Developmental Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Pirok
E W
EW
Novak
T E
TE
Schwartz
N B
NB
D053674:Aggrecans; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D002809:Chondroitin Sulfates / Q000378:metabolism; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D005091:Exons; D016326:Extracellular Matrix Proteins; D005690:Galactose / Q000378:metabolism; D017353:Gene Deletion; D006056:Golgi Apparatus / Q000378:metabolism; D006360:Heat-Shock Proteins; D007150:Immunohistochemistry; D037102:Lectins / Q000737:chemistry; D037181:Lectins, C-Type; D018832:Molecular Chaperones / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection; D014178:Translocation, Genetic; D014452:Ubiquitins / Q000378:metabolism*
D053674:Aggrecans; D002352:Carrier Proteins; D018076:DNA, Complementary; D000091342:Endoplasmic Reticulum Chaperone BiP; D016326:Extracellular Matrix Proteins; D006360:Heat-Shock Proteins; D037102:Lectins; D037181:Lectins, C-Type; D018832:Molecular Chaperones; D009097:Multienzyme Complexes; D011509:Proteoglycans; D014452:Ubiquitins; D002809:Chondroitin Sulfates; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D019704:Protein Disulfide-Isomerases; D002118:Calcium; D005690:Galactose