Biosynthesis of small proteoglycan II (decorin) by chondrocytes and evidence for a procore protein.
We have studied the biosynthesis of cartilage dermatan sulfate proteoglycan II (DS-PGII) (decorin) using in vitro translation of mRNA to determine the size of the primary gene product and by radiolabeling the protein in the presence of tunicamycin to inhibit the addition of Asn-linked oligosaccharides. Pulse-chase experiments were performed to examine post-translational processing and secretion. Inhibitors of oligosaccharide processing were used to determine whether DS-PGII molecules containing partially processed oligosaccharides could become proteoglycans and be secreted. Cell-free translation of sucrose gradient-fractionated RNA and subsequent immunoprecipitation of the core protein confirmed that the functional translated mRNA is in the size range of the two mRNA species observed by hybridization of chondrocyte RNA with a bone PGII cloned probe and that the translation product is a single protein with an apparent molecular mass of 42 kDa. Digestion of the intact proteoglycan (average molecular mass = 103 kDa) with chondroitinase ABC or AC results in an approximately 48-49-kDa product. Chondrocytes treated with tunicamycin to inhibit Asn-linked oligosaccharide addition synthesize and secrete a glycosaminoglycan (GAG)-substituted proteoglycan (average molecular mass = 86 kDa), yielding a 42-kDa core protein after chondroitinase ABC digestion, showing that Asn-linked oligosaccharides are not required for the addition of GAG chains or secretion. Following a short pulse (10 min) of [3H]leucine, three glycosylated forms of the DS-PGII core protein were observed, one of which is likely to be the precursor form of PGII predicted by the implied protein sequence of both bovine and human cDNA clones. Following the apparent cleavage of the propeptide, GAG-substituted intracellular core protein is detectable. Susceptibility to endoglycosidase H indicates that approximately one-third of the secreted core protein contains exclusively complex-type Asn-linked oligosaccharides and approximately two-thirds contain high mannose as well as complex-type oligosaccharides. Secreted DS-PGII appears to be fully substituted with three Asn-linked oligosaccharide chains. Inhibitors of oligosaccharide processing, however, permitted secretion of GAG-substituted DS-PGII that was fully (three chains) or incompletely (one or two chains) substituted with partially processed Asn-linked carbohydrate chains. By comparison of chondrocyte DS-PGII with fibroblast DS-PGII, we conclude that the addition and processing of Asn-linked carbohydrate chains are directed by the amino acid sequence of the core protein. The results reported here also suggest that the addition of xylose, the initial step in GAG chain synthesis, occurs early in biosynthesis and is determined by the primary amino acid sequence of the core protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Sawhney
R S
RS
Department of Medicine, Rush Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612.
Hering
T M
TM
Sandell
L J
LJ
D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002474:Cell-Free System; D002478:Cells, Cultured; D058575:Decorin; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006596:Hexosaminidases / Q000494:pharmacology; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011509:Proteoglycans / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D053674:Aggrecans; C546878:DCN protein, human; D058575:Decorin; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D037181:Lectins, C-Type; D009844:Oligosaccharides; D011509:Proteoglycans; D012333:RNA, Messenger; D014415:Tunicamycin; D006596:Hexosaminidases