We have studied intracellular catabolism of heparan Sulfate proteoglycans (HSPGs) in Drosophila cell lines, Kc and S2, by a series of pulse-chase experiments Using [S-35]Sulfate as a precursor in metabolic labeling experiments. HSPGs in culture medium and cell layer were separately purified by serial chromatographic procedures using Q-Sepharose and Superose 6 for characterization. Analysis of intact HSPG on Superose 6 chromatography revealed that Kc and S2 cells synthesize one major molecular species with slightly differing in sizes (estimated to be 54 kDa in Ke and 78 kDa in S2 cells). Analysis of glycosaminoglycans for S-35-labeled macromolecules showed that the majority of S-35-labeled macromolecules in Kc and S2 cells are HSPGs (similar to60% and similar to80%, respectively). Results from continuous labeling and 2 h pulse labeling-chase experiments revealed that, in both cell lines, the intact HSPGs were degraded in multiple phases; the degradation of HSPG was rapid in the early phase (with half-lives of similar to6 h in Kc and similar to3 h in S2 cells) and slow in the later phase (with half-lives >80 h in both Kc and S2 cells). The rapid degradation appeared similar to that observed for glycosylphosphatidylinositol-anchored HSPGs (glypicans) in mammalian cell cultures. While the slow degradation appeared similar to that observed for transmembrane HSPGs (syndecans) in mammalian cell cultures. These experiments suggested that vertebrates and invertebrates shared common mechanisms for intracellular HSPG catabolism. (C) 2004 Elsevier Inc. All rights reserved.