Purification and properties of β-glucuronidase from human placenta

Fe, Brot; Ce, Bell; Sly, W S

doi:10.1021/bi00596a001

Cited by 89 publications

(46 citation statements)

References 46 publications

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“…The fusion protein monomers migrated with an apparent molecular weight of about 100 kDa (Figure 2). The apparent molecular mass under non-denaturing conditions as determined by size exclusion chromatography was approximately 403 kDa (data not shown), which is in agreement with GUSh being active as a tetramer (Brot et al, 1978).…”

Section: Experimental Therapeuticssupporting

confidence: 71%

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug

et al. 2002

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Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme b-glucuronidase. The sequences encoding C28 and human enzyme b-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGk signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-bglucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-

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Section: Experimental Therapeuticssupporting

confidence: 71%

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug

et al. 2002

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“…The carbohydrates on glycoproteins often confer enhanced thermal stability, and removal of oligosaccharide chains often destabilizes glycoproteins (21). Human GUS has been shown to be relatively stable to thermal inactivation at 65°C (22)(23)(24)(25)(26). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Chemically modified β-glucuronidase crosses blood–brain barrier and clears neuronal storage in murine mucopolysaccharidosis VII

Grubb¹,

Vogler

Levy

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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113

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Enzyme replacement therapy has been used successfully in many lysosomal storage diseases. However, correction of brain storage has been limited by the inability of infused enzyme to cross the blood-brain barrier. The newborn mouse is an exception because recombinant enzyme is delivered to neonatal brain after mannose 6-phosphate receptor-mediated transcytosis. Access to this route is very limited after 2 weeks of age. Recently, several studies showed that multiple infusions of high doses of enzyme partially cleared storage in adult brain. These results raised the question of whether correction of brain storage by repeated high doses of enzyme depends on mannose 6-phosphate receptor-mediated uptake or whether enzyme gains access to brain storage by another route when brain capillaries are exposed to prolonged, high levels of circulating enzyme. To address this question, we used an enzyme whose carbohydrate-dependent receptor-mediated uptake was inactivated by chemical modification. Treatment of human ␤-glucuronidase (GUS) with sodium metaperiodate followed by sodium borohydride reduction (PerT-GUS) eliminated uptake by mannose 6-phosphate and mannose receptors in cultured cells and dramatically slowed its plasma clearance from a t1 ͞2 of <10 min to 18 h. Surprisingly, PerT-GUS infused weekly for 12 weeks was more effective in clearing central nervous system storage than native GUS at the same dose. In fact, PerT-GUS resulted in almost complete reversal of storage in neocortical and hippocampal neurons. This enhanced correction of neuronal storage by long-circulating enzyme, which targets no known receptor, suggests a delivery system across the blood-brain barrier that might be exploited therapeutically.deglycosylation ͉ enzyme replacement therapy ͉ hippocampal neurons ͉ lysosomal storage disease ͉ receptor-mediated endocytosis T he mucopolysaccharidosis (MPS) diseases, including MPS VII, are characterized by widespread storage of glycosaminoglycans in lysosomes in brain and viscera (1). Enzyme replacement therapy (ERT) has resulted in clearance of stored material from visceral organs and in clinical improvement in many patients with lysosomal storage diseases (LSDs) (2-5). Unfortunately, little infused enzyme crosses the blood-brain barrier (BBB), so limited improvement has been achieved in the central nervous system (CNS) (6).We have focused on improving delivery of enzyme to the brain by using ␤-glucuronidase (GUS) in the mouse model of MPS VII (Sly syndrome). When enzyme was infused into newborn mice, considerable enzyme was delivered to brain, and CNS storage was reduced (7-9). However, brain storage was resistant to clearance if ERT was begun after 2 weeks of age. Recent studies indicated that this enzyme delivery to the CNS in the newborn period was caused by mannose 6-phosphate receptor (M6PR)-mediated transcytosis (10). Down-regulation of this receptor by age 2 weeks appeared to explain the resistance of brain to ERT in the adult. More recently, however, we observed that multiple infusions of large doses ...

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“…dose of 131 I-P-GUS was taken up per gram of brain by 10 min in 2-day-old mice. This uptake rate is very high for a protein of this size (312 kDa) (24,25), exceeding that of morphine by Ͼ50-fold (26) and of interleukin-1 by at least 10-fold (27). The K in value of 131 I-P-GUS and the V i value of 131 I-P-GUS were both larger than those of 125 I-albumin, clearly demonstrating specific uptake by brain.…”

Section: Discussionmentioning

confidence: 99%

Developmentally regulated mannose 6-phosphate receptor-mediated transport of a lysosomal enzyme across the blood-brain barrier

Urayama

Grubb

Sly

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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151

138

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Mucopolysaccharidosis type VII is a lysosomal storage disorder resulting from inherited deficiency of ␤-glucuronidase (GUS). Mucopolysaccharidosis type VII is characterized by glycosaminoglycan storage in most tissues, including brain. In these disorders, enzyme delivery across the blood-brain barrier (BBB) is the main obstacle to correction of lysosomal storage in the CNS. Prior studies suggested mouse brain is accessible to GUS in the first 2 weeks of life but not later. To explore a possible role for the mannose 6-phosphate͞insulin-like growth factor II receptor in GUS transport across the BBB in neonatal mice, we compared brain uptake of phosphorylated GUS (P-GUS) and nonphosphorylated GUS (NP-GUS) in newborn and adult mice. 131 I-P-GUS was transported across the BBB after i.v. injection in 2-day-old mice. The brain influx rate (Kin) of 131 I-P-GUS in 2-day-old mice was 0.21 l͞g⅐min and decreased with age. By 7 weeks of age, transport of 131 I-P-GUS was not significant. Capillary depletion revealed that 62% of the 131 I-P-GUS in brain was in brain parenchyma in 2-day-old mice. In addition, uptake of 131 I-P-GUS into brain was significantly reduced by coinjection of unlabeled P-GUS or M6P in a dose-dependent manner. In contrast, the Kin of 131 I-NP-GUS (0.04 l͞g⅐min) was significantly lower than 131 I-P-GUS in 2-day-old mice. Transcardiac brain perfusion confirmed that neither 131 I-P-GUS nor 131 I-NP-GUS crossed the BBB in adult mice. These results indicate that 131 I-P-GUS transport into brain parenchyma in early postnatal life is mediated by the mannose 6-phosphate͞insulin-like growth factor II receptor. This receptor-mediated transport is not observed in adult mice.␤-glucuronidase ͉ mannose 6-phosphate͞insulin-like growth factor II receptor ͉ central nervous system ͉ lysosomal storage disease ͉ phosphorylated ␤-glucuronidase

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Purification and properties of β-glucuronidase from human placenta

Cited by 89 publications

References 46 publications

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug

Chemically modified β-glucuronidase crosses blood–brain barrier and clears neuronal storage in murine mucopolysaccharidosis VII

Developmentally regulated mannose 6-phosphate receptor-mediated transport of a lysosomal enzyme across the blood-brain barrier

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