title: Hyal2 is a glycosylphosphatidylinositol-anchored, lipid raft-associated hyaluronidase.
abstract: The rapid turnover rate of hyaluronan (HA), the major unbranched glycosaminoglycan of the extracellular matrix, is dependent on hyaluronidases. One of them, hyaluronidase-2 (Hyal2), degrades HA into smaller fragments endowed with specific biological activities such as inflammation and angiogenesis. Yet the cellular environment of Hyal2, a purported glycosylphosphatidylinositol (GPI)-anchored protein, remains uncertain. We have examined the membrane association of Hyal2 in MDA-MB231 cancer cells where it is highly expressed and in COS-7 cells transfected with native or fluorescent Hyal2 constructs. In both cell types, Hyal2 was strongly associated with cell membrane fractions from which it could be extracted using a Triton X-114 treatment (hydrophobic phase) but not an osmotic shock or an alkaline carbonate solution. Treatment of membrane preparations with phosphatidylinositol-specific phospholipase C released immunoreactive Hyal2 into the aqueous phase, confirming the protein is attached to the membrane through a functional GPI anchor. Hyal2 transfected in COS-7 cells was associated with detergent-resistant, cholesterol-rich membranes known as lipid rafts. The cellular immunofluorescent pattern of Hyal2 was conditioned by the presence of a GPI anchor. In summary, the strong membrane association of Hyal2 through its GPI anchor demonstrated in this study using biochemical methods suggests that the main activity of this enzyme is located at the level of the plasma membrane in close contact with the pericellular HA-rich glycocalyx, the extracellular matrix, or possibly endocytic vesicles.
authors: Molecular Physiology Research Unit, NARILIS, University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium. andreb@fundp.ac.be
authors: Benedicte
authors: B
authors: Andre
authors: Cecile
authors: C
authors: Duterme
authors: Kris
authors: K
authors: Van Moer
authors: Jeannine
authors: J
authors: Mertens-Strijthagen
authors: Michel
authors: M
authors: Jadot
authors: Bruno
authors: B
authors: Flamion
mesh_terms: D000818:Animals; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D003902:Detergents / Q000737:chemistry; D058851:GPI-Linked Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry / Q000201:enzymology*; D051379:Mice; D051381:Rats
chemical_list: D015815:Cell Adhesion Molecules; D003902:Detergents; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; C473096:Hyal2 protein, human; C469221:Hyal2 protein, mouse; D006821:Hyaluronoglucosaminidase