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

Abstract

A proteoglycan (PG) was purified to homogeneity from intima/media preparations of human aorta specimens by the following chromatographic steps: Sepharose Q anion exchange, Sepharose CL-4B size exclusion, hydroxyapatite, MonoQ anion exchange and TSK G 4000 SW size exclusion. The purity of the preparation was established by SDS/PAGE using direct staining by silver or Dimethylmethylene Blue, as well as by Western blots of biotin-labelled samples. The electrophoretic mobility of the native PG was less than that of a 200000-Mr standard protein. After treatment with chondroitin sulphate lyase ABC, a core protein of Mr 15000 was revealed. The Mr of the glycosaminoglycan (GAG) peptides was less than 24000, by comparison with a keratan sulphate peptide. The composition of the GAG chains was determined by differential digestion of the PG by chondroitin sulphate lyases AC/ABC or chondroitin sulphate lyase AC alone followed by anion-exchange chromatography of the resulting disaccharides. The GAG chains are composed of approximately one-third of dermatan sulphate and two-thirds chondroitin sulphate disaccharide units. The sequence of the 20 N-terminal amino acids is identical with the sequence previously reported for PG I isolated from human developing bone [Fisher, Termine & Young (1989) J. Biol. Chem. 264, 4571-4576]. The assignment of glycosylation sites to the serine residues in positions 5 and 10 was confirmed. The findings indicate that the chondroitin sulphate/dermatan sulphate PG is a major PG in intima/media preparations of human aorta and represents a biglycan-type PG.

Proteoglycan synthesis in haematopoietic cells: isolation and characterization of heparan sulphate proteoglycans expressed by the bone-marrow stromal cell line MS-5

“…Proteoglycans were isolated as described elsewhere [26,27]. Briefly, proteoglycans were extracted from metabolically labelled cells using 4 M guanidinium chloride (pH 5.8) containing 50 mM sodium acetate, 50 mM EDTA, 0.1 M 6-aminohexanoic acid, 50 mM benzamidine hydrochloride, 5 mM PMSF, 10 mM Nethylmaleimide and 0.5 % (w\v) CHAPS.…”

Role of glycosaminoglycans of biglycan in BMP-2 signaling

“…Small leucine rich proteoglycans (SLRPs) are a large family of extracellular matrix proteins composed of a core protein that includes 10–20 leucine-rich repeats (LRRs) and covalently bound glycosaminoglycan chains (GAGs) [1, 2]. The latter are highly negative-charged polymers of repeating disaccharides.…”

Analysis of Intimal Proteoglycans in Atherosclerosis-prone and Atherosclerosis-resistant Human Arteries by Mass Spectrometry

“…Biglycan was definitively demonstrated in human intima by protein sequencing (19). In addition, immunologic and/or mRNA transcript analyses have provided evidence for additional proteoglycans in human intima, predominantly versican, decorin, perlecan, and lumican (20 -26).…”