Deglycosylation of chondroitin sulfate proteoglycan and derived peptides.
In order to define the domain structure of proteoglycans as well as identify primary amino acid sequences specific for attachment of the various carbohydrate substituents, reliable techniques for deglycosylating proteoglycans are required. In this study, deglycosylation of cartilage chondroitin sulfate proteoglycan (CSPG) with minimal core protein cleavage was accomplished by digestion with chondroitinase ABC and keratanase, followed by treatment with anhydrous HF in pyridine. Nearly complete deglycosylation of secreted proteoglycan was verified within 45 min of HF treatment by loss of incorporated [3H]glucosamine label from the proteoglycan as a function of time of treatment, as well as by direct analysis of carbohydrate content and xylosyltransferase acceptor activity of unlabeled core protein preparations. The deglycosylated CSPG preparations were homogeneous and of high molecular weight (approximately 370,000). Comparison of the intact deglycosylated core protein preparations with newly synthesized unprocessed precursors (apparent Mr approximately 360,000) suggested that extensive proteolytic cleavage of the core protein did not occur during normal intracellular processing. Furthermore, peptide patterns generated after clostripain digestion of core protein precursor and of deglycosylated secreted proteoglycan were comparable. With the use of the clostripain digestion procedure, peptides were produced from unlabeled proteoglycan, and two predominant peptides from the most highly glycosylated regions (the chondroitin sulfate rich regions of the proteoglycan) were isolated, characterized, and deglycosylated. These peptides were found to follow similar kinetics of deglycosylation and to acquire xylose acceptor activity comparable to the intact core protein.
Campbell
S C
SC
Department of Pediatrics, University of Chicago, Illinois.
Krueger
R C
RC
Schwartz
N B
NB
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002645:Chickens; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D006031:Glycosylation; D015394:Molecular Structure; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis; D010455:Peptides / Q000032:analysis / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism*; D051381:Rats
D000596:Amino Acids; D002241:Carbohydrates; D011508:Chondroitin Sulfate Proteoglycans; D010446:Peptide Fragments; D010455:Peptides; D011509:Proteoglycans