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Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
Published on: November 15, 2013
Binding modes of mammalian hepatic Gal/GalNAc receptors
1Department of Biology, Johns Hopkins University, Baltimore, MD 21218.
Abstract:
Mammalian Gal/GalNAc receptors show dramatic preference for three-branched oligosaccharide structures over two- or one-branched counterparts. The spatial arrangement of the Gal residues is extremely important for optimal binding. The three terminal Gal residues in the best triantennary ligand are about 15-30 A apart, and therefore the sugar-combining sites on the receptor may also have the same geometry. The results obtained with synthetic ligands containing Gal or GalNAc are in agreement with this concept. Photoaffinity labelling and GalNAc-lactoperoxidase catalysed iodolabelling of the receptors revealed that the minor subunits (52 and 60 kDa) were more readily labelled than the major subunit (43 kDa). The stoichiometry of binding was determined with synthetic ligands containing GalNAc. The results indicated that each subunit may have two sugar-combining sites. A model for subunit assembly is proposed on the basis of these results and the finding that coexpression of all subunits is necessary for the binding and processing of asialo-orosomucoid in transfected cells, whereas Gal-polylysine can be bound and processed by the cells expressing only the major subunit.
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