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Structural analysis of chick-embryo cartilage proteoglycan by selective degradation with chondroitin lyases (chondroitinases) and endo-beta-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.

SUBMITTER: Oike Y

PROVIDER: S-EPMC1162197 | biostudies-other | 1980 Oct

REPOSITORIES: biostudies-other

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Project description:1. Chondroitin sulphate was isolated from different regions of rat costal cartilage after extensive proteolysis of the tissues. The molecular weight, determined by gel chromatography, of the polysaccharide obtained from an actively growing region (lateral zone) near the osteochondral junction was higher than that of the polysaccharide isolated from the remaining portion of the costal cartilage (medial zone). 2. In both types of cartilage the molecular weight of chondroitin sulphate, labelled with [(35)S]sulphate, remained unchanged in vivo over a period of 10 days, approximately corresponding to the half-life of the chondroitin sulphate proteoglycan. The molecular-weight distribution of chondroitin [(35)S]sulphate, labelled in vivo or in vitro, was invariably identical with that of the bulk polysaccharide from the same tissue. It is concluded that the observed regional variations in molecular-weight distribution were established at the time of polysaccharide biosynthesis. 3. In tissue culture more than half of the (35)S-labelled polysaccharide-proteins of the two tissues was released into the medium within 10 days of incubation. The released materials were of smaller molecular size than were the corresponding native proteoglycans. In contrast, the molecular-weight distribution of the chondroitin [(35)S]sulphate (single polysaccharide chains) remained constant throughout the incubation period. 4. A portion (about 20%) of the total radioactive material released from (35)S-labelled cartilage in tissue culture was identified as inorganic [(35)S]sulphate. No corresponding decrease in the degree of sulphation of the labelled polysaccharide could be detected. These findings suggest that a limited fraction of the proteoglycan molecules had been extensively desulphated. 5. It is suggested that the initial phase of degradation involves proteolytic cleavage of the proteoglycan, but the constituent polysaccharide chains remain intact. The partially degraded proteoglycan may be eliminated from the cartilage by diffusion into the circulatory system. An additional degradative process, which may occur intracellularly, includes desulphation of the polysaccharide, probably in conjunction with a more extensive breakdown of the polymer.

Project description:Structural and associated biomechanical gradients within biological tissues are important for tissue functionality and preventing damaging interfacial stress concentrations. Articular cartilage possesses an inhomogeneous structure throughout its thickness, driving the associated variation in the biomechanical strain profile within the tissue under physiological compressive loading. However, little is known experimentally about the nanostructural mechanical role of the collagen fibrils and how this varies with depth. Utilising a high-brilliance synchrotron X-ray source, we have measured the depth-wise nanostructural parameters of the collagen network in terms of the periodic fibrillar banding (D-period) and associated parameters. We show that there is a depth dependent variation in D-period reflecting the pre-strain and concurrent with changes in the level of intrafibrillar order. Further, prolonged static compression leads to fibrillar changes mirroring those caused by removal of extrafibrillar proteoglycans (as may occur in aging or disease). We suggest that fibrillar D-period is a sensitive indicator of localised changes to the mechanical environment at the nanoscale in soft connective tissues. STATEMENT OF SIGNIFICANCE: Collagen plays a significant role in both the structural and mechanical integrity of articular cartilage, allowing the tissue to withstand highly repetitive loading. However, the fibrillar mechanics of the collagen network in cartilage are not clear. Here we find that cartilage has a spatial gradient in the nanostructural collagen fibril pre-strain, with an increase in the fibrillar pre-strain with depth. Further, the fibrillar gradient changes similarly under compression when compared to an enzymatically degraded tissue which mimics age-related changes. Given that the fibrils potentially have a finite capacity to mechanically respond and alter their configuration, these findings are significant in understanding how collagen may alter in structure and gradient in diseased cartilage, and in informing the design of cartilage replacements.

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

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