EXPRESSION AND CHARACTERIZATION OF FUNCTIONAL BOVINE CATION-DEPENDENTMANNOSE B-PHOSPHATE RECEPTORS IN BACULOVIRUS-INFECTED INSECT CELLS

A recombinant baculovirus containing the cDNA for the full-length bovi ne cation-dependent mannose 8-phosphate receptor (CD-MPR) was generate d by homologous recombination. Spodoptera frugiperda (Sf9) and Trichop lusia ni 5B1-4 (High Five) insect cells, which do not contain endogeno us MPRs as determined by Western blot analyses and pentamannosyl phosp hate-agarose affinity chromatography, were infected with recombinant b aculovirus. The infected cells expressed 26-, 29-, 32-, 35-, and 39-kD a species that were immunologically reactive with antisera raised agai nst the native bovine CD-MPR and quantitative Western analysis demonst rated 1-2 X 10(6) CD-MPR. molecules/cell, a level which is comparable to that expressed normally in a variety of cell lines and tissues. Dig estion with endo-beta-N-acetylglucosaminidase H indicated that these m ultiple species were due to the presence of either 0, 1, 2, 3, or 4 N- linked oligosaccharide chains, respectively, and that the majority (87 %) of these carbohydrates were of the high-mannose type. The receptor produced was biologically active since it bound specifically to a pent amannosyl phosphate-agarose column and exhibited acid-dependent ligand dissociation. In addition, studies using a homobifunctional cross-lin king agent on the purified CD-MPR suggested that, like the receptor ex pressed in mammalian cells, the insect-produced CD-MPR existed as a di mer in the membrane. (C) 1995 Academic Press, Inc.