title: Annexin VI is a mannose-6-phosphate-independent endocytic receptor for bovine β-glucuronidase.
abstract: Endocytosis and transport of bovine liver β-glucuronidase to lysosomes in human fibroblasts are mediated by two receptors: the well-characterized cation-independent mannose 6-phosphate receptor (IGF-II/Man6PR) and an IGF-II/Man6PR-independent receptor, which recognizes a Ser-Trp*-Ser sequence present on the ligand. The latter receptor was detergent extracted from bovine liver membranes and purified. LC/ESI-MS/MS analysis revealed that this endocytic receptor was annexin VI (AnxA6). Several approaches were used to confirm this finding. First, the binding of bovine β-glucuronidase to the purified receptor from bovine liver membranes and His-tagged recombinant human AnxA6 protein was confirmed using ligand-blotting assays. Second, western blot analysis using antibodies raised against IGF-II/Man6PR-independent receptor as well as commercial antibodies against AnxA6 confirmed that the receptor and AnxA6 were indeed the same protein. Third, double immunofluorescence experiments in human fibroblasts confirmed a complete colocalization of the bovine β-glucuronidase and the AnxA6 receptor on the plasma membrane. Lastly, two cell lines were stably transfected with a plasmid containing the cDNA for human AnxA6. In both transfected cell lines, an increase in cell surface AnxA6 and in mannose 6-phosphate-independent endocytosis of bovine β-glucuronidase was detected. These results indicate that AnxA6 is a novel receptor that mediates the endocytosis of the bovine β-glucuronidase.
authors.affiliation: Department of Cell Biology and Physiology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico.
authors.forename: Alberto
authors.initials: A
authors.lastname: Ramírez-Mata
authors.forename: Colette
authors.initials: C
authors.lastname: Michalak
authors.forename: Guillermo
authors.initials: G
authors.lastname: Mendoza-Hernández
authors.forename: Alfonso
authors.initials: A
authors.lastname: León-Del-Río
authors.forename: Alfonso
authors.initials: A
authors.lastname: González-Noriega
mesh_terms: D000818:Animals; D017317:Annexin A6 / Q000032:analysis / Q000302:isolation & purification / Q000502:physiology*; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D002417:Cattle; D045744:Cell Line, Tumor; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004847:Epithelial Cells / Q000502:physiology; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D007739:L Cells; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D008360:Mannosephosphates / Q000494:pharmacology; D013058:Mass Spectrometry; D051379:Mice; D011485:Protein Binding / Q000502:physiology; D017527:Receptor, IGF Type 2 / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000032:analysis / Q000302:isolation & purification / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism
chemical_list: D017317:Annexin A6; D000906:Antibodies; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C027693:mannose-6-phosphate; D005966:Glucuronidase