Abstract
The epiphysis of developing bones is a cartilaginous structure that is eventually replaced by bone during skeletal maturation. We have separated a dermatan sulfate proteoglycan, epiphycan, from decorin and biglycan by using dissociative extraction of bovine fetal epiphyseal cartilage, followed by sequential ion-exchange, gel permeation, hydrophobic, and Zn2+ chelate chromatographic steps. Epiphycan is a member of the small leucine-rich proteoglycan family, contains seven leucine-rich repeats (LRRs), is related to osteoglycin (osteoinductive factor) (Bentz, H., Nathan, R. M., Rosen, D. M., Armstrong, R. M., Thompson, A. Y., Segarini, P. R., Mathews, M. C., Dasch, J., Piez, K. A., and Seyedin, S. M. (1989) J. Biol. Chem. 264, 20805-20810), and appears to be the bovine equivalent of the chick proteoglycan PG-Lb (Shinomura, T., and Kimata, K. (1992) J. Biol. Chem. 267, 1265-1270). The intact proteoglycan had a median size of approximately 133 kDa. The core protein was 46 kDa by electrophoretic analysis, had a calculated size of 34,271 Da, and had two approximately equimolar N termini (APTLES ... and ETYDAT ... ) separated by 11 amino acids. There were at least three O-linked oligosaccharides in the N-terminal region of the protein, based on blank cycles in Edman degradation and corresponding serine or threonine residues in the translated cDNA sequence. The glycosaminoglycans ranged in size from 23 to 34 kDa were more heterogeneous than those in other dermatan sulfate small leucine-rich proteoglycans and were found in the acidic N-terminal region of the protein core, N-terminal to the LRRs. A four-cysteine cluster was present at the N terminus of the LRRs, and a disulfide-bonded cysteine pair was present at the C terminus of the protein core. The seventh LRR and an N-linked oligosaccharide were between the two C-terminal cysteines. An additional potential N-glycosylation site near the C terminus did not appear to be substituted at a significant level.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U77127
The exact roles of the SLRPs are unclear at present, but it is thought that fibromodulin and decorin are involved in the process of collagen fibrillogenesis [2, 3] and may play a crucial role in optimizing the diameter of collagen fibrils that will eventually be replaced during remodeling of the cartilage during calcification
The results indicated that the first peak contained biglycan, and the second peak contained decorin and a second somewhat larger proteoglycan
Summary
Materials—Guanidine hydrochloride (GdnHCl) was from Research Plus Laboratories. Phenylmethylsulfonyl fluoride, iodoacetamide, sodium citrate, and sodium chloride were from Sigma. Octyl-Sepharose Chromatography—The DEAE-bound and eluted proteoglycan-containing fractions from the gel permeation chromatography were applied to an octyl-Sepharose column that had been equilibrated with 2 M GdnHCl and 0.15 M sodium acetate, pH 6.3, at 25 °C and allowed to bind for 2 h. The molecular masses of the intact proteoglycans were estimated by gel permeation chromatography on a Superose 6 10/30 column that was eluted at a flow rate of 0.4 ml/min in 4 M GdnHCl, 50 mM sodium acetate, pH 5.8, and 0.05% CHAPS and that had been calibrated with 14C-labeled molecular mass markers. Isolation and Analysis of Core Proteins—The 125I-labeled SLRPs epiphycan, decorin, and biglycan (400,000 cpm each) were digested with chondroitinase ABC (10 units/ml) for 24 h at 37 °C in 0.1 M Tris, 30 mM sodium acetate, 0.2% bovine serum albumin, 10 mM EDTA, 10 mM N-ethylmaleimide, 5 mM phenylmethylsulfonyl fluoride, and 1 mg/ml pepstatin A. Molecular size estimates for GAG chains are based on the data of Wasteson [17]
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