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.

Abstract

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.

• Authors: U Kessler, S Aumeier, B Funk, W Kiess
• Journal: Molecular and cellular endocrinology (Mol Cell Endocrinol) Vol. 90 Issue 1 Pg. 147-53 (Dec 1992) ISSN: 0303-7207 [Print] Ireland
• PMID: 1301395 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Carbohydrates
• Glycoproteins
• Ligands
• Mannosephosphates
• Receptor, IGF Type 2
• mannose-6-phosphate
• Insulin-Like Growth Factor II
• Hexosaminidase A
• Hexosaminidase B
• beta-N-Acetylhexosaminidases

Topics

• Animals
• Carbohydrates (pharmacology)
• Cells, Cultured
• Fibroblasts (drug effects, enzymology, metabolism)
• Glycoproteins (metabolism)
• Hexosaminidase A
• Hexosaminidase B
• Humans
• Insulin-Like Growth Factor II (metabolism, pharmacology)
• Ligands
• Lysosomes (enzymology, metabolism)
• Mannosephosphates (metabolism)
• Neuroglia (drug effects, metabolism)
• Rats
• Receptor, IGF Type 2 (metabolism)
• Tay-Sachs Disease (enzymology, pathology)
• beta-N-Acetylhexosaminidases (biosynthesis, metabolism)