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Fig. 5. Histochemical evidence of GUS activity in MR^-/- (A-L) and MR^+/+ (M-X) mice 24 h after a single infusion of P-GUS at doses of 0.4 (A-D and M-P), 1.6 (E-H and Q-T) or 6.4 mg/kg (I-L and U-X). The meninges had stainable enzyme activity at both 1.6 (E) and 6.4 mg/kg (I) GUS in the MR^-/- mice, but MR^+/+ mice had enzyme in meninges only in those receiving the highest dose of the enzyme (U). The kidney in both MR^-/- (B, F, and J) and MR^+/+ (N, R, and V) mice showed a dose-dependent increase in histochemically demonstrable enzyme with the highest activity in the glomeruli. The liver also had a dose-based response to the increasing enzyme infusions, but the localization of activity depended on the genotype. Hepatocytes and Kupffer cells both contained enzyme in the MR^-/- (C, G, and K) mice, but the Kupffer cells preferentially took up enzyme in the MR^+/+ (O, S, and W) mice. The spleen also showed a dose-response and distinctive distribution of GUS activity. Enzyme was preferentially distributed to the perifollicular red pulp area in the MR^-/- mice (D, H, and L). The distribution in the MR^+/+ mice (P, T, and X) was more diffuse.

Supporting Text

The his-tagged placental alkaline phosphatase (AP) was purified from culture supernatant from overexpressing cells (GenHunter, Nashville, TN) on a Ni-NTA column. P-GUS was treated with 50 units/mg of purified AP overnight in a dialysis bag at 37°C with several changes of 25 mM Tris, pH 8.0/140 mM NaCl. The AP was removed from the enzyme by passing over the Ni-NTA column. The removal of phosphate from AP-GUS was verified by demonstrating that susceptibility to mannose 6-phosphate receptor (MPR)-dependent uptake by mucopolysaccharidosis (MPS) VII fibroblasts had been completely lost. Susceptibility to mannose receptor (MR)-dependent uptake was demonstrated with macrophage cell line J7774.