title: Expression and characterization of a soluble, active form of the jaagsiekte sheep retrovirus receptor, Hyal2.
abstract: Retrovirus entry into cells is mediated by specific interactions between virus envelope glycoproteins and cell surface receptors. Many of these receptors contain multiple membrane-spanning regions, making their purification and study difficult. The jaagsiekte sheep retrovirus (JSRV) receptor, hyaluronidase 2 (Hyal2), is a glycosylphosphatidylinositol (GPI)-anchored molecule containing no peptide transmembrane regions, making it an attractive candidate for study of retrovirus entry. Further, the hyaluronidase activity reported for human Hyal2, combined with its broad expression pattern, may point to a critical function of Hyal2 in the turnover of hyaluronan, a major extracellular matrix component. Here we describe the properties of a soluble form of human Hyal2 (sHyal2) purified from a baculoviral expression system. sHyal2 is a 54-kDa monomer with weak hyaluronidase activity compared to that of the known hyaluronidase Spam1. In contrast to a previous report indicating that Hyal2 cleaved hyaluronan to a limit product of 20 kDa and was active only at acidic pH, we find that sHyal2 is capable of further degradation of hyaluronan and is active over a broad pH range, consistent with Hyal2 being active at the cell surface where it is normally localized. Interaction of sHyal2 with the JSRV envelope glycoprotein was analyzed by viral inhibition assays, showing >90% inhibition of transduction at 28 nM sHyal2, and by surface plasmon resonance, revealing a remarkably tight specific interaction with a dissociation constant (KD) of 32 +/- 1 pM. In contrast to results obtained with avian retroviruses, purified receptor was not capable of promoting transduction of cells that do not express the virus receptor.
authors.affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Vigdorovich
authors.forename: Roland K
authors.initials: RK
authors.lastname: Strong
authors.forename: A Dusty
authors.initials: AD
authors.lastname: Miller
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D058851:GPI-Linked Proteins; D015686:Gene Products, env / Q000378:metabolism*; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000737:chemistry / Q000378:metabolism*; D022141:Jaagsiekte sheep retrovirus / Q000378:metabolism* / Q000472:pathogenicity; D007700:Kinetics; D011991:Receptors, Virus / Q000737:chemistry / Q000378:metabolism*; D012756:Sheep; D012995:Solubility; D014161:Transduction, Genetic
chemical_list: D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D015686:Gene Products, env; D011991:Receptors, Virus; D006820:Hyaluronic Acid; C473096:Hyal2 protein, human; D006821:Hyaluronoglucosaminidase