Characterization of truncated and glycosylation-deficient forms of the cation-dependent mannose 6-phosphate receptor expressed in baculovirus-infected insect cells

A soluble truncated form of the cation-dependent mannose 6-phosphate receptor (CD-MPR) encoding only the extracytoplasmic region, Stop(155), and a truncated glycosylation-deficient form of the CD-MPR, Asn(81)/Stop(155), which has been modified to contain only one N-linked glycosylation site at position 81 instead of five, were purified from baculovirus-infected High Five insect cells. The glycosylated recombinant proteins were functional in ligand binding and acid-dependent dissociation as assessed by pentamannosyl phosphate-agarose affinity chromatography. Gel filtration, sucrose gradients, and crosslinking experiments revealed that both Stop(155) and Asn(81)/Stop(155) are dimeric, demonstrating that the transmembrane and cytoplasmic region of the receptor as well as N-linked oligosaccharides at positions 31, 57, and 87 are not required for dimerization. The K-d Of Stop(155) and Asn(81)/Stop(155) for the lysosomal enzyme, beta-glucuronidase, was 0.2 and 0.3 nM, respectively. These values are very similar to those reported for the full-length CD-MPR, demonstrating that the extracellular region of the CD-MPR is sufficient for high-affinity binding and that oligosaccharides at positions 31, 57, and 87 do not influence ligand binding.