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The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan heterogeneity and the pathway of proteolytic degradation.

ABSTRACT: 1. CaCl2-extracted proteoglycan from bovine nasal cartilage was degraded by four tissue proteinases till no further decrease in hydroynamic size was obtained. The proteoglycan and its final degradation products were then fractionated by Sepharose 2B chromatography. 2. The average size of the degradation products was least for cathepsin B and lysosomal elastase, and greatest for cathepsin D and cathepsin G. The latter two proteinases also produced degradation products that showed the widest range of sizes. 3. The structure of the degradation products ranged from peptides containing a single glycosaminoglycan chain to those containing twelve or more chains. Of the four proteinases, only cathepsin B produced peptides that contained a single chondroitin sulphate chain. 4. The proteoglycan was very heterogeneous with respect to size and chemical composition. Its behaviour on electrophoresis suggested that at least two genetically distinct core proteins might exist. 5. Irrespective of their structural variations, all proteoglycan molecules were able to interact with hyaluronic acid. In contrast, none of the degradation products were capable of this type of interaction. 6. A pathway for the proteolytic degradation of proteoglycans is postulated in which the sites of initial cleavage may be common to the majority of proteinases, whereas the production of the final clusters is dependent on the specificity of the proteinase. Only those proteinases of broadest specificity can produce single-chain chondroitin sulphate-peptides.

SUBMITTER: Roughley PJ

PROVIDER: S-EPMC1183710 | biostudies-other | 1977 Dec

REPOSITORIES: biostudies-other

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Project description:1. Proteoglycans were extracted from bovine nasal cartilage with 2.0M-CaC2 or with 0.15M-KCl followed by 2.0M-CaC2.. Proteoglycan fractions were prepared from the extracts by density-gradient centrifugation in CsCl under 'associative' and 'dissociative' conditions. 2. The heterogeneity of the proteoglycan fractions was investigated by large-pore-gel electrophoresis. It was concluded that extracts made with 2.0M-CaCl2 or sequential 2.0M-CaCl2 contain two major species of proteoglycan 'subunit' of different hydrodynamic size, together with proteoglycan aggregates. Both 'subunits' have mobilities that are greater than those of proteoglycans obtained from pig articular cartilage McDevitt & Muir (1971) Anal. Biochem. 44, 612-622] and are therefore probably smaller in size than the latter. 3. Proteoglycan fractions isolated from cartilage extracted lith 0.15M-KCl separated into two main components on large-pore-gel electrophoresis with mobilities greater than those of proteoglycans extracted with 2.0M-CaCl2. Proteoglycans extracted at low ionic strength from bovine nasal cartilage are of similar hydrodynamic size to those extracted from pig articular cartilage under the same conditions [McDevitt & Muir (1971) Anal. Biochem. 44, 612-622]. 4. The role of endogenous proteolytic enzymes in producing proteoglycan heterogeneity, particularly in low-ionic-strength cartilage extracts is discussed. 5. Hyaluronic acid and 'link proteins' were present in the proteoglycan fraction separated from KCl extracts as well as in the fraction separated from CaCl2 extracts. Hyaluronic acid can only be identified in proteoglycan fractions by large-pore-gel electrophoresis after proteolysis and further purification of the fraction. 6. Collagen was extracted by both salt solutions and was tentatively identified as type II. Small amounts of collagen appear to be associated with the proteoglycan-aggregate fraction from the high-ionic-strength extract but not with the corresponding fraction from the KCl extract.

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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The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan heterogeneity and the pathway of proteolytic degradation.

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