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Incorporation of mannose 6-phosphate receptors into liposomes. Receptor topography and binding of alpha-mannosidase.

ABSTRACT: A receptor that binds the lysosomal enzyme alpha-mannosidase via mannose 6-phosphate moieties (mannose 6-phosphate receptor) was purified from Swarm-rat chondrosarcoma and bovine liver microsomal membranes. Receptor-reconstituted liposomes were prepared by dialysis of taurodeoxycholate-dispersed lipids with purified mannose 6-phosphate receptor. Liposomes appeared by electron microscopy as 60-120 nm unilamellar vesicles. Receptor-reconstituted liposomes retained the ability to bind alpha-mannosidase specifically. Binding was saturable with an apparent Kd of 1 nM and was competitively inhibited by mannose 6-phosphate (Ki 2mM). Liposomes containing entrapped 125I-bovine serum albumin were used to demonstrate that treatment with 0.045% taurodeoxycholate rendered liposomes permeable to macromolecules without solubilizing the membrane. Receptor orientation in the liposome membrane was established by measuring binding of ligand to intact and detergent-treated liposomes. Unlike coated vesicles, which contain cryptic mannose 6-phosphate receptors [Campbell, Fine, Squicciarini & Rome (1983) J. Biol. Chem. 258, 2526-2533], treatment of liposomes with detergent revealed no additional cryptic binding sites. In addition, treatment of liposomes with 0.75% trypsin abolished total receptor binding activity. The results suggest that the receptor is inserted with its binding site facing the outside of the liposome.

SUBMITTER: Campbell CH

PROVIDER: S-EPMC1152262 | biostudies-other | 1983 Aug

REPOSITORIES: biostudies-other

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Identification of a fourth mannose 6-phosphate binding site in the cation-independent mannose 6-phosphate receptor.

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Project description:The 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) plays a key role in the delivery of lysosomal enzymes to the lysosome by binding newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and diverting them from the secretory pathway. Previous studies on a truncated form of the receptor comprised of only the soluble extracellular region (sCD-MPR, residues 1-154) have shown that the CD-MPR exists as a homodimer and exhibits two distinct conformations in the ligand-bound versus ligand-unbound states, involving changes in quaternary structure and positioning of loop D, the residues of which form a side of the binding pocket in the presence of ligand. To determine the role of intermonomer contacts in the functioning of the sCD-MPR, site-directed mutagenesis was used to generate a construct lacking a salt bridge (Glu19-Lys137) that tethers the N-terminal alpha-helix of one subunit to loop D of the other subunit in the ligand-bound form. Here we show by surface plasmon resonance analyses and NMR spectroscopy that the elimination of this intermonomer salt bridge significantly decreases the binding affinity of the mutant receptor (E19Q/K137M) toward lysosomal enzymes and Man-6-P. Analyses of the E19Q/K137M mutant receptor crystallized under various conditions revealed an altered quaternary structure that is intermediate between those observed in the ligand-bound and ligand-unbound states. Taken together, the results demonstrate a key role for intermonomer interactions in the structure and functioning of the CD-MPR.

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P-Type Lectins: Cation-Dependent Mannose-6-Phosphate Receptor

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