The ligand-binding conformation of Mr 46,000 mannose 6-phosphate-specific receptor. Acquisition of binding activity during in vitro synthesis.
Purified Mr 46,000 mannose 6-phosphate-specific receptor (MPR 46) lost its ligand-binding activity after reductive alkylation and after enzymatic deglycosylation. Deglycosylated MPR 46 did not assemble to homodimers. Therefore, we investigated the role of N-glycosylation, intrasubunit disulfide bonds, and subunit assembly for the acquisition of ligand-binding activity during in vitro synthesis of MPR 46. Up to 21% of MPR 46 synthesized in a reticulocyte lysate supplemented with dog pancreas microsomes acquired ligand-binding activity provided that 1-5 mM glutathione was present during translation and during a chase following translation. Acquisition of ligand-binding activity after cotranslational membrane insertion and core glycosylation depended on formation of intrasubunit disulfide bonds and a conformational change. Formation of intrasubunit disulfide bonds was not sufficient for ligand-binding activity and is likely to precede the conformational change, which resulted in increased resistance toward trypsin, formation of highly antigenic epitopes, and association to dimers, concomitant with the acquisition of ligand-binding activity.
Hille
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A
Universität Göttingen, Biochemie II, Federal Republic of Germany.
Waheed
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A
von Figura
K
K
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005796:Genes; D005978:Glutathione / Q000494:pharmacology; D006600:Hexosephosphates / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008360:Mannosephosphates / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014357:Trypsin
D006600:Hexosephosphates; D008024:Ligands; D046911:Macromolecular Substances; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D004247:DNA; D014357:Trypsin; D005978:Glutathione