Receptor-mediated uptake of beta-glucuronidase into primary astrocytes and C6 glioma cells from rat brain

Abstract

The ability of both primary astrocytes from rat cerebrum and a rat C6 glioma cell line to take up lysosomal enzymes by receptor-mediated endocytosis was compared. The beta-glucuronidase secreted by 3T3 fibroblasts was purified to homogeneity by antibody affinity chromatography, iodinated and used as a typical enzyme to determine the nature of receptors involved in its uptake into glial cells. Both primary astrocytes and C6 glioma cells took up 125I-labelled enzyme in a rapid and saturable manner indicative of specific receptors, while immunostaining with an anti-mouse beta-glucuronidase antibody showed that the enzyme was distributed in a mainly punctate pattern after uptake, characteristic of that of lysosomes. Subcellular fractionation of C6 glioma cells following endocytosis revealed that the enzyme became localised in lysosomes, after first passing through an endosomal compartment. Uptake of enzyme was reduced markedly after its sugar side chains had been removed with N-glycanase, indicating that endocytosis was mediated via a carbohydrate-recognising receptor. A range of carbohydrates and glycoproteins were tested for their ability to inhibit receptor-mediated endocytosis but of these only sialic acid had a notable effect. Further evidence that endocytosis of beta-glucuronidase into primary astrocytes and C6 gliomas may be mediated via sialic acid receptors was provided by the large reduction in rate of uptake observed following removal of this sugar from the enzyme with sialidase.