Hyal2 is a glycosylphosphatidylinositol-anchored, lipid raft-associated hyaluronidase

• Published in: Biochemical and biophysical research communications 2011-07, Vol.411 (1), p.175-179
• Main Authors: Andre, Benedicte, Duterme, Cecile, Van Moer, Kris, Mertens-Strijthagen, Jeannine, Jadot, Michel, Flamion, Bruno
• Format: Article
• Language: English
• Subjects: Animals; Cell Adhesion Molecules - chemistry; Cell Adhesion Molecules - genetics; Cell Adhesion Molecules - metabolism; Cell Line, Tumor; Cercopithecus aethiops; COS Cells; Detergents - chemistry; Glycosylphosphatidylinositol; Glycosylphosphatidylinositols - chemistry; Glycosylphosphatidylinositols - metabolism; GPI-Linked Proteins - chemistry; GPI-Linked Proteins - genetics; GPI-Linked Proteins - metabolism; Humans; Hyaluronidase; Hyaluronoglucosaminidase - chemistry; Hyaluronoglucosaminidase - genetics; Hyaluronoglucosaminidase - metabolism; Lipid raft; Membrane Microdomains - chemistry; Membrane Microdomains - enzymology; Mice; Rats
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2011.06.125

► Hyal2 is one of two somatic hyaluronidases with demonstrated enzymatic activity. ► We used MDA-MB231 cancer cells with high expression of Hyal2 and transfected COS cells. ► We show that Hyal2 is strongly linked to cell membranes through a GPI anchor. ► Hyal2 belongs to lipid rafts, like CD44. 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.