ABSTRACT
Neutrophils infiltrate tissues during inflammation, and when activated, they release ß-glucuronidase. Since inflammation is associated with carcinogenesis, we investigated how extracellular ß-glucuronidase changed the in vitro cellular response to the chemical carcinogen benzo(a)pyrene (B[a]P). For this we exposed human liver (HepG2) and lung (A549) cells to B[a]P in the presence or absence of ß-glucuronidase. ß-Glucuronidase reduced B[a]P-induced expression of CYP1A1 and CYP1B1 at 6 h after exposure, which did not depend on ß-glucuronidase activity, because the inhibitor D-saccharic acid 1,4-lactone monohydrate did not antagonize the effect of ß-glucuronidase. On the other hand, the inhibitory effect of ß-glucuronidase on CYP expression was dependent on signalling via the insulin-like growth factor receptor (IGF2R, a known receptor for ß-glucuronidase), because co-incubation with the IGF2R inhibitor mannose-6-phosphate completely abolished the effect of ß-glucuronidase. Extracellular ß-glucuronidase also reduced the formation of several B[a]P metabolites and B[a]P-DNA adducts. Interestingly, at 24 h of exposure, ß-glucuronidase significantly enhanced CYP expression, probably because ß-glucuronidase de-glucuronidated B[a]P metabolites, which continued to trigger the aryl hydrocarbon receptor (Ah receptor) and induced expression of CYP1A1 (in both cell lines) and CYP1B1 (in A549 only). Consequently, significantly higher concentrations of B[a]P metabolites and DNA adducts were found in ß-glucuronidase-treated cells at 24 h. DNA adduct levels peaked at 48 h in cells that were exposed to B[a]P and treated with ß-glucuronidase. Overall, these data show that ß-glucuronidase alters the cellular response to B[a]P and ultimately enhances B[a]P-induced DNA adduct levels.
Subject(s)
Benzo(a)pyrene/toxicity , Carcinogens/toxicity , Glucuronidase/pharmacology , Hepatocytes/drug effects , Lung/drug effects , Pneumonia/enzymology , Animals , Basic Helix-Loop-Helix Transcription Factors/agonists , Basic Helix-Loop-Helix Transcription Factors/metabolism , Benzo(a)pyrene/metabolism , Biotransformation , Carcinogens/metabolism , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1B1/genetics , Cytochrome P-450 CYP1B1/metabolism , DNA Adducts/metabolism , Disease Models, Animal , Hep G2 Cells , Hepatocytes/enzymology , Hepatocytes/pathology , Humans , Lipopolysaccharides , Lung/enzymology , Lung/pathology , Pneumonia/chemically induced , Pneumonia/genetics , Pneumonia/pathology , Receptor, IGF Type 2/agonists , Receptor, IGF Type 2/metabolism , Receptors, Aryl Hydrocarbon/agonists , Receptors, Aryl Hydrocarbon/metabolism , Signal Transduction/drug effects , Time FactorsABSTRACT
We have used a peptide-based targeting system to improve lysosomal delivery of acid α-glucosidase (GAA), the enzyme deficient in patients with Pompe disease. Human GAA was fused to the glycosylation-independent lysosomal targeting (GILT) tag, which contains a portion of insulin-like growth factor II, to create an active, chimeric enzyme with high affinity for the cation-independent mannose 6-phosphate receptor. GILT-tagged GAA was taken up by L6 myoblasts about 25-fold more efficiently than was recombinant human GAA (rhGAA). Once delivered to the lysosome, the mature form of GILT-tagged GAA was indistinguishable from rhGAA and persisted with a half-life indistinguishable from rhGAA. GILT-tagged GAA was significantly more effective than rhGAA in clearing glycogen from numerous skeletal muscle tissues in the Pompe mouse model. The GILT-tagged GAA enzyme may provide an improved enzyme replacement therapy for Pompe disease patients.
Subject(s)
Enzyme Replacement Therapy/methods , Glucan 1,4-alpha-Glucosidase/metabolism , Glycogen Storage Disease Type II/drug therapy , Glycogen Storage Disease Type II/enzymology , Glycogen/metabolism , Lysosomes/drug effects , Animals , Biological Transport/drug effects , Disease Models, Animal , Drug Delivery Systems/methods , Glucan 1,4-alpha-Glucosidase/genetics , Glycogen Storage Disease Type II/genetics , Glycosylation , HEK293 Cells , Half-Life , Humans , Insulin-Like Growth Factor II/genetics , Insulin-Like Growth Factor II/metabolism , Kinetics , Lysosomes/enzymology , Mice , Muscle, Skeletal/drug effects , Muscle, Skeletal/enzymology , Muscle, Skeletal/pathology , Mutant Chimeric Proteins/genetics , Mutant Chimeric Proteins/metabolism , Myoblasts/drug effects , Myoblasts/enzymology , Myoblasts/pathology , Plasmids , Receptor, IGF Type 2/agonists , Receptor, IGF Type 2/metabolism , TransfectionABSTRACT
The cation-independent mannose 6-phosphate receptor is a multifunctional protein which binds at the cell surface to two distinct classes of ligands, the mannose 6-phosphate (M6P) bearing proteins and IGF-II. Its major function is to bind and transport M6P-enzymes to lysosomes, but it can also modulate the activity of a variety of extracellular M6P-glycoproteins (i.e., latent TGFbeta precursor, urokinase-type plasminogen activator receptor, Granzyme B, growth factors, Herpes virus). The purpose of this review is to highlight the synthesis and potential use of high affinity M6P analogues able to target this receptor. Several M6P analogues with phosphonate, carboxylate or malonate groups display a higher affinity and a stronger stability in human serum than M6P itself. These derivatives could be used to favour the delivery of specific therapeutic compounds to lysosomes, notably in enzyme replacement therapies of lysosomal diseases or in neoplastic drug targeting. In addition, their potential applications in preventing clinical disorders, which are associated with the activities of other M6P-proteins involved in wound healing, cell growth or viral infection, will be discussed.
