title: Glycosylation of the Mr 46,000 mannose 6-phosphate receptor. Effect on ligand binding, stability, and conformation.
abstract: Using site-directed mutagenesis the N-glycosylation sites of the Mr 46,000 mannose 6-phosphate receptor (MPR 46) were identified as asparagine residues 57, 83, 107, and 113. The two outer asparagines carry high mannose-type and the two inner asparagines carry complex-type oligosaccharides. The glycosylation mutants were analyzed for stability, binding activity, and subcellular distribution. Replacing asparagine 57, 83, or 107 by threonine decreased only the stability of the receptor. Replacing asparagine 113 by threonine decreased the stability and binding activity. Deletion of three or all four N-glycosylation sites led in addition to an accumulation of the mutant receptors in endoplasmic reticulum-like structures. Nonglycosylated MPR 46 synthesized in the presence of tunicamycin, thus preserving the asparagine residues, had a normal stability and high affinity binding. The decreased stability and binding activity of the receptor mutants is therefore due to the exchange of asparagine residues rather than to the loss of N-linked oligosaccharides. The nonglycosylated receptor, however, displayed a decreased conformational stability after solubilization as a single cycle of freezing and thawing reduced the binding activity to one-third of the control. Simultaneously, the receptor lost its quaternary structure. It is concluded from these results that the N-glycosylation of the receptor is required for the stability of a high affinity conformation, but not for the binding itself or the intracellular stability.
authors.affiliation: Universität Göttingen, Abt. Biochemie II, Federal Republic of Germany.
authors.forename: M
authors.initials: M
authors.lastname: Wendland
authors.forename: A
authors.initials: A
authors.lastname: Waheed
authors.forename: B
authors.initials: B
authors.lastname: Schmidt
authors.forename: A
authors.initials: A
authors.lastname: Hille
authors.forename: G
authors.initials: G
authors.lastname: Nagel
authors.forename: K
authors.initials: K
authors.lastname: von Figura
authors.forename: R
authors.initials: R
authors.lastname: Pohlmann
mesh_terms: D002451:Cell Compartmentation; D002846:Chromatography, Affinity; D004252:DNA Mutational Analysis; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D008970:Molecular Weight; D011487:Protein Conformation; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D008024:Ligands; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins