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.

FIG. 1.

Structure and properties of purified sHyal2. (A) The structures of native Hyal2 and sHyal2 are shown (both drawn to scale). Amino acids 1 to 21 constitute the endoplasmic reticulum signal peptide for both proteins. A GPI anchor is predicted to replace all residues following amino acid 447 in Hyal2, which also contains a C-terminal hydrophobic tail at residues 456 to 473 that localizes the protein in the membrane prior to GPI anchor addition. In sHyal2, a C-terminal His₆ tag (attached with an Ala₃ linker) replaces the GPI anchor attachment site and the hydrophobic tail of the native sequence. (B) Size exclusion chromatography using a Superdex 200 HR 10/30 column shows that sHyal2 is monomeric in solution and migrates with an apparent molecular mass of ∼40 kDa. Overlaid onto the trace of sHyal2 is a trace of Bio-Rad gel filtration chromatography standards with sizes in kilodaltons indicated. (C) SDS-PAGE of sHyal2 and protein standards under reducing conditions indicates a molecular mass for sHyal2 of ∼50 kDa.

FIG. 2.

sHyal2 is a weak neutral-pH-active hyaluronidase. (A) Fifty-microgram samples of HA were incubated at 37°C and pH 3.8 for 14 h in a total volume of 40 μl with no added protein, with various amounts of sperm hyaluronidase Spam1, or with 1.6 μg of sHyal2. The samples were separated in 0.5% agarose and visualized with Stains-All. (B) Fifty-microgram samples of HA were incubated at 37°C for 14 h in a total volume of 40 μl with no added protein at pH 3.8 or with sHyal2 (1.0 μg per sample) at the indicated pH values. Samples were analyzed as in panel A.

FIG. 3.

sHyal2 is capable of complete digestion of hyaluronan. (Left panel) Fifty-microgram samples of HA were incubated with no added protein or with various amounts of sHyal2 at 37°C and pH 5.5 for 14 h in a total volume of 40 μl. Samples were analyzed by agarose gel electrophoresis as in Fig. 2. (Right panel) To examine the effect of repeated sHyal2 treatment on HA degradation, a 50-μg sample of HA was incubated with 1.6 μg of sHyal2 for 12 h, 1.6 μg of sHyal2 in PNEA buffer was added to the sample, and the sample was incubated for an additional 12 h (lane labeled “1.6 μg sHYAL2 × 2”). In parallel, 50-μg samples of HA were incubated with or without 1.6 μg of sHYAL2 for 12 h, PNEA buffer without sHyal2 was added, and the samples were incubated for an additional 12 h (lanes labeled “1.6 μg sHyal2” and “No added protein”, respectively). Samples were analyzed by agarose gel electrophoresis as in Fig. 2.

FIG. 4.

sHyal2 specifically inhibits transduction by a JSRV-pseudotype retroviral vector. PJ4/LAPSN (similar to PJ14/LAPSN) (46) and PA317/LAPSN (39) vector-producing cell lines were used to generate JSRV-pseudotype and amphotropic MLV-pseudotype vectors. The LAPSN vector encodes human placental alkaline phosphatase (AP) and bacterial neomycin phosphotransferase. These vector stocks were used to transduce NIH 3T3 TK⁻ cells expressing human Hyal2 and that also express the endogenous mouse amphotropic retrovirus receptor Pit2. Experiments were carried out in the presence of various concentrations of purified sHyal2 that was added to the cells just prior to virus addition. Two days after virus exposure, the cells were stained for AP and AP⁺ foci were counted. Results are expressed as a percentage of the transduction rate observed without sHyal2 addition for each vector. Each data point represents the average of three experiments, and standard deviations are shown.

FIG. 5.

Measurement of the kinetics of the interaction between sHyal2 and JSU-IgG. (A) Reference-subtracted data (gray) illustrating the interaction between the immobilized JSU-IgG and sHyal2 are shown. Following the initial phase of buffer flow, solutions of indicated concentrations of sHyal2 in buffer were injected during the time interval of 0 to 200 s at a rate of 20 μl/min. This was followed by a return to initial buffer flow to allow for measurements of dissociation of the complexes formed. BIAevaluation software was used to build a curve fit (black line) to the data. (B) Deviations of data from the fit in panel A are shown.