Structural analysis of chick-embryo cartilage proteoglycan by selective degradation with chondroitin lyases (chondroitinases) and endo-β-d-galactosidase (keratanase)

Abstract

Digestion of chick-embryo cartilage proteoglycan (type H) with chondroitin AC II lyase or keratanase, in the presence of EDTA, N-ethylmaleimide, phenylmethanesulphonyl fluoride and pepstatin, resulted in the removal of the bulk of the chondroitin sulphate or keratan sulphate chains respectively, without altering the protein portion of the macromolecule. An exhaustive treatment of the proteoglycan with chondroitin AC II lyase followed by digestion with keratanase yielded a core fraction having the enzymically modified linkage oligosaccharides. Zonal sedimentation of this core preparation on a sucrose gradient in 0.5% SDS resulted in a single narrow band with a sedimentation coefficient of 6S. In 4 M-guanidinium chloride, the core preparation showed a tendency to aggregate to multiple-molecular-weight forms which could dissociate in the presence of Triton X-100. The results indicate that the preponderance of glycosaminoglycans in the proteoglycan molecule is a main reason for both polydispersity and hydrophilicity of the proteoglycan preparation, and further suggest that the enzymic procedures could prove useful as a method to obtain new information about the structure and properties of proteoglycan core molecules.

The Uniform Galactose 4‐Sulfate Structure in the Carbohydrate‐Protein Linkage Region of Human Urinary Trypsin Inhibitor

“…Chondroitinase AC-I1 degrades a linkage hexasaccharide into a disaccharide unit and a tetrasaccharide core (Hascall et al, 1972;Oike et al, 1980;Sugahara et al, 1991). HPLC analysis of the chondroitinase AC-I1 digest gave rise to a single radiolabeled component emerging at the position of authentic dHexAa1-3GaI(4S)/31-3-Galpl -4Xyl-ol (Fig.…”

“…The purified UTI preparation, which has been demonstrated to consist of four isomers with different electric charges (Yuki et al, 1993), was treated with NaB'H, to release the chondroitin sulfate chain from the peptide and to prepare tracers by radiolabeling Xyl simultaneously at the reducing end of the linkage region. The resultant 'H-labeled chondroitin sulfate was mixed with a carrier prepared by alkaline NaBH, treatment of whale cartilage chondroitin 4-sulfate, isolated by gel filtration on Sephadex G-50 (data not shown) and digested with chondroitinase ABC to obtain the linkage hexasaccharide fraction (Hascall et al, 1972;Oike et al, 1980;Sugahara et al, 1991). Upon gel filtration of the digest on a Sephadex G-50 column two radioactive peaks were observed: a minor peak in the void volume and a major peak in the included volume (data not shown).…”

Basement membrane heparan sulfate proteoglycans are concentrated in the laminae rarae and in podocytes of the rat renal glomerulus.

“…Previous studies have shown that proteoglycans extracted from rat glomeruli are eluted from DEAESephacel at >0.45 M NaCl (17 NaDodSO4/PAGE. Electrophoresis of aliquots (=20,000 cpm) of the glomerular extracts, peaks 1 and 2 eluted from DEAE-Sephacel columns, and the immunoprecipitates was carried out on 5-10% gradient slab gels following a modification (19) (20) or with papain (30 pg/ml, 650C, 4 hr).…”

Purification and N-terminal amino acid sequence of a chondroitin sulphate/dermatan sulphate proteoglycan isolated from intima/media preparations of human aorta

“…Briefly, NHS-LC-biotin was used for labelling. For the specific degradation of the glycan chains, chondroitin sulphate lyases AC and ABC, heparinase/ heparitinase and keratanase were used in the presence of proteinase inhibitors (Oike et al, 1980;Kato et al, 1985). Electrophoresis of enzyme-treated and non-treated samples was performed as described above.…”