BIOLOGY OF HYALURONIDASES

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Hyaluronidases are a group of neutral- and acid- active enzymes found throughout the animal kingdom, critical to such fundamental process as cell motility, embryological development, wound healing, angiogenesis and tumorigenesis. Despite their obvious impotence, these enzymes are not well understood because of the lack of a rapid, sensitive assay. This problem has now been overcome with our development of an ELISA-like assay sufficiently sensitive so that kinetics can be performed, and activity in small samples can be measured. In an organ survey, high levels of activity were found in liver, lung, kidney and spleen. The liver enzyme as well as the serum enzyme have now been purified to homogeneity an dare indeed different proteins. Hyaluronidases appear to be a broad family of enzyme proteins. We propose to isolate other enzymes of this family, and using a coordinated biochemical, immunological, and molecular biology approach, examine the biology of each, identify precursors forms, and establish precise histolocalization, both in cultured cells and in tissue sections. The biotinylated HA-binding protein probe will also permit simultaneous visualization of substrate. We predict that the lymphatic system, as exemplified by the spleen enzyme, is the major catabolic route for HA. The liver and the kidneys are responsible for additional catabolic pathways, of free HA in the circulation in what may be a hierarchy dependent on HA polymer size. The hyaluronidase in fibroblasts has also been detected now, by direct assay and by Western blot utilizing the polyclonal antibodies directed against the purified liver enzyme. detection of this neutral enzyme activity, not observed previously in normal mammalian fibroblasts, was facilitated by extracting in the presence of high salt, which appears to disaggregate an enzyme-inhibitor complex. We will use appropriate cDNA libraries to sequence hyaluronidase enzymes. Consensus sequences, as well as modular domain patterns will be identified in this new enzyme family. Why is there such a wide repertoir of hyaluronidase in vertebrate tissue? And why does the HA substrate itself exist in such a wide variety of states: free in the circulation, cell-bound, fully hydrated or in association with a spectrum of HA-binding proteins. Toward an answer to such questions, we will attempt to correlate enzyme kinetics and patterns of hyaluronidase protein structure in various tissues with the molecular state an location of the attendant HA substrate and the requirements for tissue-specific functions.