The extracellular processing of aggrecan aggregate and its effect on CD44 mediated internalization of hyaluronan.

Abstract

In many cells the 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 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 if a size range of aggrecan exists that was permissive of hyaluronan endocytosis. The 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 that is of smaller molecular size was internalized more readily than high molecular mass hyaluronan. However, the addition of intact aggrecan to 5 and 10 second sonicated hyaluronan chains re-blocked their endocytosis while aggregates containing 15 second sonicated hyaluronan were internalized. These data suggest that hyaluronan endocytosis is regulated in large part by the extracellular proteolytic processing of hyaluronan-bound proteoglycan.