Expression and characterization of functional bovine cation-dependent mannose 6-phosphate receptors in...

A recombinant baculovirus containing the cDNA for the full-length bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) was generated by homologous recombination. Spodoptera frugiperda (Sf9) and Trichoplusia ni 5B1-4 (High Five) insect cells, which do not contain endogenous MPRs as determined by Western blot analyses and pentamannosyl phosphate-agarose affinity chromatography, were infected with recombinant baculovirus. The infected cells expressed 26-, 29-, 32-, 35-, and 39-kDa species that were immunologically reactive with antisera raised against the native bovine CD-MPR and quantitative Western analysis demonstrated 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. Digestion with endo-beta-N-acetylglucosaminidase H indicated that these multiple 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 pentamannosyl phosphate-agarose column and exhibited acid-dependent ligand dissociation. In addition, studies using a homobifunctional cross-linking agent on the purified CD-MPR suggested that, like the receptor expressed in mammalian cells, the insect-produced CD-MPR existed as a dimer in the membrane.