Production and characterization of a soluble, active form of Tva, the subgroup A avian sarcoma and leukosis virus receptor

Abstract

The receptor for the subgroup A avian sarcoma and leukosis viruses [ASLV(A)] is the cellular glycoprotein Tva. A soluble form of Tva, sTva, was produced and purified with a baculovirus expression system. Using this system, 7 to 10 mg of purified sTva per liter of cultured Sf9 cells was obtained. Characterization of the carbohydrate modification of sTva revealed that the three N glycosylation sites in sTva were differentially utilized; however, the O glycosylation common to Tva produced in mammalian and avian cells was not observed. Purified sTva demonstrates significant biological activity, specifically blocking infection of avian cells by ASLV(A) with a 90% inhibitory concentration of approximately 25 pM. A quantitative enzyme-linked immunosorbent assay, developed to assess the binding of sTva to ASLV envelope glycoprotein, demonstrates that sTva has a high affinity for EnvA, with an apparent dissociation constant of approximately 0.3 nM. Once they are bound, a very stable complex is formed between EnvA and sTva, with an estimated complex half-life of 6 h. The soluble receptor protein described here represents a valuable tool for analysis of the receptor-envelope glycoprotein interaction and for structural analysis of Tva.

FIG. 1

Comparison of sTva and naturally occurring forms of quail Tva. (A) Diagram of sTva and quail Tva₉₅₀ and Tva₈₀₀. The arrow indicates the site of Tva leader peptide cleavage; the sTva melittin leader peptide is cross hatched, the LDL-A motif (residues 11 to 50) is hatched, transmembrane domain is solid, the glycosylphosphotidylinositiol linkage signal is vertically hatched, the cytoplasmic tail is shaded, and the six-histidine tail is stippled. The numbers on the right indicate the sizes (in amino acids) of the mature proteins. (B) Comparison of WT Tva and sTva amino and carboxy termini. The arrows indicate the signal peptide cleavage sites. The four additional N-terminal amino acids in sTva are marked with a line above. Potential N-linked glycosylation sites are underlined. MSD, start of the membrane-spanning domain.

FIG. 2

Comparison of Tvas from mammalian and insect cells and analysis of purified sTva. (A) Cell lysates from human 293T cells transiently expressing either Tva₉₅₀ or sTva and lysates from Sf9 cells infected with bVTTva were subjected to SDS-PAGE (12.5% acrylamide), and Western blotting was performed with a polyclonal α-Tva serum. (B) Analysis of purified sTva. To assess the purity of sTva, 0.5 (left) and 10 (right) μg of the same preparation were analyzed by SDS-PAGE (12.5% acrylamide) and Coomassie blue staining. Molecular mass standards (in kilodaltons) are indicated.

FIG. 3

sTva is modified by asparagine-linked glycosylation. (A) sTva binds to ConA. Lysates of Sf9 cells infected with bVTTva were incubated with ConA sepharose. After extensive washing, ConA-bound proteins were eluted and analyzed by Western blotting as described in the legend to Fig. 2. (B) Analysis of sTva glycosylation by glycosidase digestion. sTva was acid precipitated from the medium of bVTTva-infected Sf9 cells. Pelleted sTva was resuspended in the appropriate glycosidase buffer and digested with PNGase F (F; lanes 2 and 5), neuraminidase (N; lanes 3, 4, and 5), and O-glycosidase (lanes 4 and 5). Following glycosidase digestion the samples were analyzed by SDS-PAGE and Western blotting as described in Fig. 2. Molecular mass standards (in kilodaltons) are indicated on the left.

FIG. 4

ELISA analysis of sTva-ASLV(A) envelope binding. (A) Titration of sTva. Increasing concentrations of purified sTva were incubated with captured gD-EnvA at the indicated temperature (○, 37°C; ◊, 25°C; □, 4°C) in an ELISA as described in Materials and Methods. The percent of sTva bound for each concentration is the average of three experiments. The error bars indicate standard deviations. The apparent K_ds for sTva-EnvA were estimated from the half-maximal binding and are 0.3, 0.2, and 0.5 nM at 37, 25, and 4°C, respectively. (B) Analysis of sTva-EnvA complex stability. The relative dissociation rate of the sTva-EnvA complex was determined by preincubating excess biotinylated sTva with gD-EnvA which was captured on an ELISA plate. After extensive washing, excess unlabeled sTva was incubated at 4°C for the indicated times. The percentage of labeled sTva bound is the average of three experiments. The error bars indicate standard deviations. The sTva-EnvA dissociation rates at 22 (○) and 4°C (●) are ∼5.5 and >6 h, respectively.