Expression and characterization of functional bovine cation-dependent mannose 6-phosphate receptors in baculovirus-infected insect cells.
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.
Dahms
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Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Brzycki-Wessell
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D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002236:Carbohydrate Conformation; D002412:Cations; D002417:Cattle; D002846:Chromatography, Affinity; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D046911:Macromolecular Substances; D009844:Oligosaccharides / Q000032:analysis; D017527:Receptor, IGF Type 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D018411:Spodoptera / Q000378:metabolism*; D014162:Transfection
D002412:Cations; D046911:Macromolecular Substances; D009844:Oligosaccharides; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; D000118:Acetylglucosaminidase