Figure 4.

GNeo delivery of GUS is M6P-independent. (a) MPS VII fibroblasts were treated with the indicated concentration of bGUS or GNeo-bGUS for 2 hours. The cells were washed, trypsin treated, sedimented by centrifugation, washed three times and subsequently assayed for β-glucuronidase activity. (b) MPS VII cells were treated with 5 nmol/l of the indicated bovine enzyme preparations in growth medium in the absence (open bars) or in the presence of 5 mmol/l mannose-6-phosphate (filled bars) or glucose-6-phosphate (gray bars), and assayed for β-glucuronidase activity. Assays of normal human foreskin fibroblasts (HFF) and MPS VII cells are shown for comparison. The difference between bGUS uptake in the presence of M6P was significant, whereas the inhibition by G6P was not. Analysis of variance showed that these differences were significant (P = 0.0104). The differences within the other data sets were not significant (P = 0.2915 for HFF cells, P = 0.8595 for GNeo-bGUS, P = 0.4577 for AP-bGUS, and P = 0.4513 for GNeo-AP-bGUS). (c) MPS VII cells were treated with 1 nmol/l of the indicated human enzymes in the presence (filled bars) and absence (open bars) of 5 mmol/l mannose-6-phosphate and assayed for β-glucuronidase activity. Assays of normal human fibroblasts (HFF) and untreated MPS VII cells are shown for comparison. (d) MPS VII cells were treated with medium in the absence (open bars) or with a mixture of heparin lyases (filled bars) 15 minutes prior to addition of 5 nmol/l bGUS or GNeo-bGUS or 1 nmol/l of hGUS or GNeo-AP-hGUS. Error bars are the SEM of triplicate uptake experiments. The difference between GNeo-bGUS uptake after heparinase digestion and bGUS was significant (P = 0.009) and probably reflected incomplete digestion by heparinases. The difference in hGUS uptake after heparinase was not significant (P = 0.2). The data were compared by Student's t-test. G6P, glucose 6-phosphate; M6P, mannose-6-phosphate; MPS, mucopolysaccharidosis; RFU, relative fluorescence units.