ABSTRACT
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
PURPOSE: To determine matrix metalloproteinase (MMP) 2 and MMP 9 expression in acute and chronic experimentally wounded canine corneas and keratectomy samples from canine patients with spontaneous chronic corneal epithelial defects (SCCEDs). METHODS: Mechanical debridement was performed unilaterally in 25 healthy dogs for the acute wound study. Twenty-four hours (n = 8), 48 hours (n = 5), 72 hours (n = 3), or 1 week (n = 9) after wounding, the dogs were euthanized. Debridement was performed once weekly for 8 weeks for the chronic study (n = 8). Therapeutic superficial keratectomies (n = 16) were performed on SCCED patients. Gelatin zymography and immunohistochemistry were performed. RESULTS: Acute wounds showed upregulation of MMP 9 at all time points. At 7 days after wounding, values diminished markedly but remained elevated above those of unwounded controls. SCCED and chronic wound samples showed a significant increase in MMP 9 compared with controls but were less than that of acute wounds. There was no significant difference between chronic wounds versus SCCED samples. Fellow control eyes showed significant upregulation of MMP 9 in tandem with wounded eyes. There was no significant difference in values for MMP 2 in wounded corneas or SCCED compared with those of controls. Immunhistochemistry localized MMP 9 to predominantly the epithelium with some staining of keratinocytes and stroma. CONCLUSIONS: The dog exhibits similar MMP expression during corneal wound healing to that of other species. The lack of significant difference in MMP expression between SCCED and chronic wounds suggest that MMP 2 and 9 are not involved in the pathophysiology of SCCED and are more likely altered secondary to a chronic epithelial defect.
Subject(s)
Corneal Diseases/enzymology , Corneal Injuries , Eye Injuries/enzymology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Acute Disease , Animals , Chronic Disease , Debridement , Disease Models, Animal , Dogs , Epithelium, Corneal/pathology , Immunoenzyme Techniques , Up-Regulation , Wound HealingABSTRACT
Thionucleosides are uniquely present in tRNA. In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34. The s(2) group of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance. Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli. We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E. coli. We also cloned mnmA from E. coli, and overproduced and purified the protein. Using a gel mobility shift assay, we show that MnmA binds to unmodified E. coli tRNA(Lys) with affinity in the low micromolar range. MnmA does not bind observably to the nonsubstrate E. coli tRNA(Phe). Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion. ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence. We have reconstituted the in vitro biosynthesis of s(2)U using unmodified E. coli tRNA(Glu) as a substrate. The activity requires MnmA, Mg-ATP, l-cysteine, and the cysteine desulfurase IscS. HPLC analysis of thiolated tRNA digests using [(35)S]cysteine confirms that the product of the in vitro reaction is s(2)U. As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine. Small RNAs that represent the anticodon stem loops for tRNA(Glu) and tRNA(Lys) are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region.
