A proteoglycan consists of a core portein to which one or more glycosaminoglycans are covalently attached. In cartilage, proteoglycans are large, complex components of the extracellular matrix where they are found primarily organized into aggregates. It has been known for some time that proteoglycans isolated form immature and adult cartilages differ with respect to the size and composition of their glycosaminoglycan and protein moieties. More recent studies have brought forth evidence that there are different species of proteoglycans in cartilage. The purpose of this research is to re-evaluate the biochemical basis of the age-related diferences in light of this new evidence. More specifically, we intend to establish whether the age-related differences reflect differences in the composition of one or more of the proteoglycan species or whether they reflect differences in the relative proportions of the different species present at any one age. At first, we will restrict ourselves to the study of aggregating proteoglycans because the great majority (Greater than 80%) of articular cartilage proteoglycans are of the aggregating type and because it is difficult to rule out the possibility that some of the non-aggregating proteoglycans represent degradation products of the aggregates. Aggregating proteoglycans will be purified from the articular cartilage of immature calves (1-2 month old) or older steers (About 18 months old) and they will subjected to a number of prepartive procedures and analytical assays to characterize quantitatively and qualitatively each of the aggregating species at the two ages studied. We have obtained preliminary evidence that suggests that in steer articular cartilage there are two major species of aggregating proteoglycans whereas in the calf only one is apparent. In addition to studying the proteoglycans as they exists in the parent cartilages, we intend to examine the nature of the age-related differences at the level of biosynthesis. For this purpose, we will use a recently developed chondrocyte culture system in which calf and steer chondrocytes grown as monolayers continue to synthesize """"""""calf-like"""""""" and """"""""steer-like"""""""" proteoglycans for as long as three weeks following plating. We will use the system to ascertain that proteoglycan heterogeniety (i.e. more than one species) is not the result of partial degradation of a single proteoglycan species but rather than proteoglycans are synthesized as multiple species. The ability to manufacture typical immature-like or adult-like proteoglycans in vitro will be used to study the age-related differences at the level of biosynthesis and to examine whether heterogeneity can be detected prior to the addition of the glycosaminoglycans to the core protein(s) in the Golgi apparatus.
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