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, Stop155, and a truncated glycosylation-deficient form of the CD-MPR, Asn81/Stop155, 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 cross-linking experiments revealed that both Stop155 and Asn81/Stop155 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 Kd of Stop155 and Asn81/Stop155 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.
Marron-Terada
P G
PG
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
Bollinger
K E
KE
Dahms
N M
NM
D000143:Acids; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002412:Cations; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D019281:Dimerization; D005822:Genetic Vectors / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008360:Mannosephosphates / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D009844:Oligosaccharides; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D020033:Protein Isoforms / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017527:Receptor, IGF Type 2 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012685:Sepharose; D018411:Spodoptera / Q000235:genetics
D000143:Acids; D002412:Cations; D008360:Mannosephosphates; D009844:Oligosaccharides; D010446:Peptide Fragments; D020033:Protein Isoforms; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; C027693:mannose-6-phosphate; D001216:Asparagine; D012685:Sepharose