Characteristics of the Protein-Keratan Sulfate Core and of Keratan Sulfate Prepared from Bovine Nasal Cartilage Proteoglycan

Vincent C Hascall,Rick L Riolo

doi:10.1016/s0021-9258(19)45019-9

Vincent C Hascall, Rick L Riolo

Open Access

https://doi.org/10.1016/s0021-9258(19)45019-9

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Abstract

Abstract Protein-keratan sulfate core was isolated from bovine nasal cartilage proteoglycan after enzymatically removing chondroitin sulfate with chondroitinase AC from Flavobacterium heparinum. The core preparation exhibited a broad, approximately Gaussian distribution of buoyant densities in CsCl density gradients (between 1.43 and 1.58 g per ml with an average of 1.51 g per ml). Chemical and physical analyses of subfractions from the density gradient indicate that the core macromolecules have a wide polydispersity in molecular weights (350,000 to 550,000, with an average of 450,000). Molecules with larger molecular weights have greater buoyant densities and higher ratios of keratan sulfate to protein. The data suggest that the core molecules contain a protein (or combination of proteins) with a molecular weight of about 200,000 to which different amounts of keratan sulfate are attached. Differences in average keratan sulfate chain lengths and chain numbers appear to contribute to the polydispersity of the protein-keratan sulfate core. Keratan sulfate was isolated from the core preparation after proteolysis with papain and purification with DEAE-cellulose chromatography. Chemical and physical analyses of the keratan sulfate indicate that the polysaccharide exhibits a large polydispersity of molecular weights with a weight average molecular weight of about 8,500 and a z-average molecular weight of about 11,000, and that polysaccharide molecules with larger molecular weights have higher ratios of glucosamine to galactosamine. Alkali treatment of the keratan sulfate selectively destroys threonine and serine as it does for other skeletal keratan sulfates. After alkaline treatment the keratan sulfate chromatographs on Sephadex G-100 with a higher retention volume, Kd = 0.6, than that for the untreated preparation, Kd = 0.3, which indicates that the sizes of the molecules have been significantly reduced. The data are consistent with the hypothesis that the papain-prepared keratan sulfate consists of molecules which contain two polysaccharide chains, each with an average of about six repeat disaccharides, connected through a peptide bridge. Each chain probably contains a terminal sialic acid residue and is attached to the protein with a glycosidic bond between galactosamine and a threonine or serine residue. The high concentration of glutamic acid in the fraction suggests that the polysaccharide chains are also attached in some way to this amino acid. Such a model suggests that the intact proteoglycan macromolecules each contain about 60 keratan sulfate chains of variable lengths built onto the protein core structure.

Highlights

Isopycnic Centrifugation of Core in C.&l Gradients-Because proteins have lower buoyant densities than polysaccharides, the high proportion of protein to polysaccharide in the core preparation indicates that these molecules will have lower buoyant densities than the proteoglycans from which they were prepared
Any polydispersity in the ratio of polysaccharide to protein in the molecules would be reflected by differences in their buoyant densities. This suggests that isopycnic CsCl density gradients can be used to investigate the characteristics of the core preparation
Because the sulfated keratan sulfate portion of the core macromolecule has a higher buoyant density than the protein, the results suggest that the molecules vary in their relative contents of this polysaccharide and of protein

Summary

Introduction

CsCl density gradients (between 1.43 and 1.58 g per ml with an average of 1.51 g per ml). Equilibrium Centrifugation of Core Fractions-One model which is consistent with the physical and analytical data discussed above is that the molecules in the core preparation contain a protein structure of a certain molecular weight to which different amounts of keratan sulfate are attached.

Results

Conclusion