Mannose 6-phosphate-independent endocytosis of β-glucuronidase by human fibroblasts: I. evidence for the existence of a membrane-binding activity

Abstract

Prior work has shown that endocytosis of bovine β-glucuronidase by human fibroblasts can be mediated by the existence of a Man6P-independent receptor for the recapture and targeting to lysosomes. In this study, we have isolated a peptide (IIIb2) from pronase digested bovine β-glucuronidase that behaved as competitive inhibitor of the endocytosis of bovine β-glucuronidase by human fibroblasts. This peptide contained a Ser–X–Ser sequence, where X is probably a posttranslational modified Trp. Antibodies raised against this peptide impaired the endocytosis of the bovine but not the human β-glucuronidase, implying that the new recognition marker for the endocytosis of acid hydrolases might reside in a single discrete stretch of amino acid sequence. On the other hand, bovine β-glucuronidase has been shown to bind specifically to receptors of human fibroblast membranes. The binding was saturable, divalent cation-dependent and was competitively inhibited by the IIIb2 peptide, but not by mannose 6-phosphate. Results presented suggested an interplay between manganese concentrations, temperature and pH on the dissociation of the β-glucuronidase-receptor complexes. All together, these data reinforce the presence of two endocytic systems for the recapture and targeting of β-glucuronidase in human fibroblasts.