Extracellular Processing of the Cartilage Proteoglycan Aggregate and Its Effect on CD44-mediated Internalization of Hyaluronan

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In many cells hyaluronan receptor CD44 mediates the endocytosis of hyaluronan and its delivery to endosomes/lysosomes. The regulation of this process remains largely unknown. In most extracellular matrices hyaluronan is not present as a free polysaccharide but often is found in complex with other small proteins and macromolecules such as proteoglycans. This is especially true in cartilage, where hyaluronan assembles into an aggregate structure with the large proteoglycan termed aggrecan. In this study when purified aggrecan was added to FITC-conjugated hyaluronan, no internalization of hyaluronan was detected. This suggested that the overall size of the aggregate prevented hyaluronan endocytosis and furthermore that proteolysis of the aggrecan was a required prerequisite for local, cell-based turnover of hyaluronan. To test this hypothesis, limited C-terminal digestion of aggrecan was performed to determine whether a size range of aggrecan exists that permits hyaluronan endocytosis. Our data demonstrate that only limited degradation of the aggrecan monomer was required to allow for hyaluronan internalization. When hyaluronan was combined with partially degraded, dansyl chloride-labeled aggrecan, blue fluorescent aggrecan was also visualized within intracellular vesicles. It was also determined that sonicated hyaluronan of smaller molecular size was internalized more readily than high molecular mass hyaluronan. However, the addition of intact aggrecan to hyaluronan chains sonicated for 5 and 10 s reblocked their endocytosis, whereas aggregates containing 15-s sonicated hyaluronan were internalized. These data suggest that hyaluronan endocytosis is regulated in large part by the extracellular proteolytic processing of hyaluronan-bound proteoglycan.