EFFECT OF LINK PROTEIN-CONCENTRATION ON ARTICULAR-CARTILAGE PROTEOGLYCAN AGGREGATION

Previous work has shown that alterations in proteoglycan aggregates ar e among the first changes detected with aging, disuse, and degeneratio n of articular cartilage, yet the cause or causes of these alterations remain unknown. To determine if differences in link protein concentra tion can explain alterations in the assembly size, and stability of ar ticular cartilage proteoglycan aggregates, we isolated proteoglycan mo nomer (aggrecan) and link protein from adult bovine articular cartilag e and then assembled proteoglycan aggregates from aggrecan and 0.8% hy aluronan relative to aggrecan weight, in the presence of 0, 2, 4, 6, 8 , 10, 15, and 20% concentrations of link protein relative to aggrecan weight. We determined the amount, sedimentation coefficient, and stabi lity of the aggregates by analytical ultracentrifugation and measured their dimensions by electron microscopy with use of the monolayer tech nique. Increased aggregate size, as determined by ultracentrifugation, was directly correlated with an increased number of aggrecans per agg regate and with increased hyaluronan length, as determined by electron microscopy. The concentration of link protein significantly influence d aggregation: concentrations of 6-8% produced maximum aggregation, ag gregate stability, and uniformity of aggrecan spacing; concentrations greater than 10% led to the formation of superaggregates (aggregates w ith sedimentation velocities greater than 100 S that may result from l inking two or more hyaluronan filaments) but decreased aggregate stabi lity; and concentrations of less than 4% link protein significantly de creased aggregation, the size and stability of aggregates, and the reg ularity of aggrecan spacing. The latter observations suggest that a de cline in the concentration of link protein could decrease the organiza tion and stability of the articular cartilage matrix.