Biosynthetic labeling of beta-hexosaminidase B: inhibition of the cellular uptake of lysosomal secretions containing [3H]hexosaminidase B by insulin-like growth factor-II in rat C6 glial cells.
The insulin-like growth factor-II/mannose-6-phosphate receptor binds two classes of ligands, IGF-II and lysosomal enzymes containing the mannose-6-phosphate recognition marker. To study the interaction of the two classes of ligands at the receptor level, we have isolated 'high uptake' forms of lysosomal enzymes containing mannose-6-phosphate that had been radiolabeled biosynthetically using a tissue culture model: Tay-Sachs disease fibroblasts were incubated in medium containing [3H]mannose, ammonium chloride and mannose-6-phosphate. Under the conditions of these experiments, the Tay-Sachs disease fibroblasts synthesized and secreted radiolabeled hexosaminidase B, as confirmed by measuring enzymatic activity of cell-conditioned medium. The enzyme secreted was recognized by antibodies raised against purified hexosaminidase A and B but not by nonimmune control sera in Western blotting and immunoprecipitation experiments. The radiolabeled cell-conditioned medium was partially purified by ion-exchange chromatography on a DEAE-Sephadex column. When partially purified [3H]hexosaminidase B was incubated with rat C6 glial cells which express large numbers of IGF-II/mannose-6-phosphate receptors, the enzyme was taken up specifically via the IGF-II/mannose-6-phosphate receptor as evidenced by carbohydrate competition experiments. The specific uptake of the radiolabeled lysosomal enzyme was partially inhibited by IGF-II and an antibody against the IGF-II/mannose-6-phosphate receptor (No. 3637). We conclude that the cellular uptake of a biosynthetically labeled lysosomal enzyme, hexosaminidase B, is partially inhibited by IGF-II. We hypothesize that IGF-II might be capable of modulating lysosomal pathways in vivo.
Kessler
U
U
Children's Hospital, Department of Pediatric Endocrinology, Munich, Germany.
Aumeier
S
S
Funk
B
B
Kiess
W
W
D000818:Animals; D002241:Carbohydrates / Q000494:pharmacology; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000378:metabolism / Q000494:pharmacology*; D008024:Ligands; D008247:Lysosomes / Q000201:enzymology* / Q000378:metabolism; D008360:Mannosephosphates / Q000378:metabolism*; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D013661:Tay-Sachs Disease / Q000201:enzymology* / Q000473:pathology; D001619:beta-N-Acetylhexosaminidases / Q000096:biosynthesis* / Q000378:metabolism
D002241:Carbohydrates; D006023:Glycoproteins; D008024:Ligands; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; C027693:mannose-6-phosphate; D007335:Insulin-Like Growth Factor II; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases