Is HYAL-1 an authentic lysosomal enzyme?

Introduction: Hyaluronan is a glucosaminoglycan (GAG) found in the extracellular matrix (ECM) of many tissues where it influences many biological processes such as cell migration and proliferation. Its levels under normal cellular conditions are held in check by the balance between its synthesis by HA synthases and its catabolism by a family of depolymerising enzymes, Hyaluronidases, six of which are already known in mammals (Csoka et al., 2001). The most predominant and active of these are Hyal-1 and Hyal-2. They degrade the polymer to progressively smaller and different sizes of fragments which exert a wide and occasionally opposing spectrum of biological activities (Stern, 2008). Hyaluronidase-1 is the most ubiquitously distributed hyaluronidase, by virtue of its mRNA expression, in all mammalian tissues with exception of adult brain. The highest specific activity of the protein is found in serum, from which it derives the name, "plasma Hyal-1" (Frost et al., 1997), but it is tightly regulated by association with its inhibitors (Stern, 2005). At the same time, Hyal-1 is the most abundant hyaluronidase in somatic tissues particularly those with the highest turnover rate for HA, its substrate, namely the liver, kidney and the spleen (Csoka et al., 2001; Laurent and Fraser, 1992). Apart from its acidic optimal pH, no other evidence has ever been experimentally established in support of the lysosomal localization that is attributed to the protein. We investigated the subcellular localization of Hyal-1 by classical centrifugation methods, based on comparing the behavior of an intracellular compound under different centrifugation systems with that of known reference marker enzymes under the same conditions. Methods: Experiments were carried out on perfused rodent liver and kidney as well as on human hepatoma cells, HepG2. Detection of the protein was by immunodetection (Western blotting) and zymography activity. These assays were carried out on perfused tissue fraction
(DOCMED00)--FUNDP, 2009