Mannose 6-phosphate-independent endocytosis of beta-glucuronidase by human fibroblasts. I. Evidence for the existence of a membrane-binding activity.
Prior work has shown that endocytosis of bovine beta-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 beta-glucuronidase that behaved as competitive inhibitor of the endocytosis of bovine beta-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 beta-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 beta-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 beta-glucuronidase-receptor complexes. All together, these data reinforce the presence of two endocytic systems for the recapture and targeting of beta-glucuronidase in human fibroblasts.
González-Noriega
A
A
Departmento de Biología Celular, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico. gonor@servidor.unam.mx
Michalak
C
C
Cruz-Perez
J R
JR
Masso
F
F
D001667:Binding, Competitive; D001692:Biological Transport; D002413:Cations, Divalent; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D005347:Fibroblasts / Q000378:metabolism*; D005966:Glucuronidase / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008360:Mannosephosphates / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification / Q000494:pharmacology; D011402:Pronase; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature
D002413:Cations, Divalent; D008024:Ligands; D008360:Mannosephosphates; D008565:Membrane Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C027693:mannose-6-phosphate; D005966:Glucuronidase; D011402:Pronase