Mannose 6-phosphate-independent endocytosis of beta-glucuronidase II. Purification of a cation-dependsnt receptor from bovine liver

A new binding protein, which recognizes a specific peptide sequence from pr onase digested bovine beta -glucuronidase, has been isolated from bovine li ver membranes, Prior work has shown that this peptide (IIIb2) contains a Se r-X-Ser sequence where X might be a posttranslational modified Trp, This re ceptor was detergent-extracted from total bovine liver membranes and purifi ed by affinity chromatography on a bovine beta -glucuronidase-Sepharose and a IIlb2 peptide-Sepharose column. Binding of bovine beta -glucuronidase to thr isolated receptor requires divalent cations, and their presence was ne cessary to maintain the receptor-ligand complex, Only the peptide sequence containing the fraction IIlb2 was able to impair the binding of the bovine enzyme to the receptor. Ilo other peptide from bovine beta -glucuronidase h ad an effect on binding. When analyzed by SDS-PAGE under reducing condition s? two bands were observed, a major band of 78 kDa and a faint band of 72 k Da. Rabbit antibodies against this binding protein revealed the presence of the 78 kDa protein ill membranes from bovine liver, human and bovine fibro blasts. These antibodies impaired human fibroblasts endocytosis of the bovi ne but not of the human beta -glucuronidase, which is taken up by a 300 kDa receptor that recognizes phosphomannosyl moieties in the enzyme, (C) 2001 Elsevier Science B.V. All rights reserved.