title: Production and characterization of recombinant human CLN2 protein for enzyme-replacement therapy in late infantile neuronal ceroid lipofuscinosis.
abstract: Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal recessive childhood disease caused by mutations in the CLN2 gene, which encodes the lysosomal enzyme tripeptidyl peptidase I. As a step towards understanding the protein and developing therapeutics for the disease, we have produced and characterized recombinant human CLN2 (ceroid lipofuscinosis, neuronal 2) protein from Chinese-hamster ovary cells engineered to secrete high levels of the enzyme. The protein was secreted as an inactive soluble proenzyme of approximately 65 kDa that appears as a monomer by gel filtration. Upon acidification, the protein is processed to mature form and acquires activity. The enzyme is efficiently delivered to the lysosomes of LINCL fibroblasts by mannose 6-phosphate-receptor-mediated endocytosis (EC(50) approximately 2 nM), where it remains active for long periods of time (t(1/2) approximately 12 days). In addition, the enzyme is taken up by rat cerebellar granule neurons by mannose 6-phosphate-dependent and -independent mechanisms. Treatment of LINCL fibroblasts with recombinant CLN2 protein restores normal enzyme activity and ameliorates accumulation of the major storage protein, mitochondrial ATP synthase subunit c.
authors: Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854, USA.
authors: L
authors: L
authors: Lin
authors: P
authors: P
authors: Lobel
mesh_terms: D000626:Aminopeptidases; D000818:Animals; D001120:Arginine; D001692:Biological Transport; D016466:CHO Cells; D002460:Cell Line; D018388:Codon, Terminator; D006224:Cricetinae; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D004705:Endocytosis; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology; D006579:Heterozygote; D006801:Humans; D007223:Infant; D007700:Kinetics; D008969:Molecular Sequence Data; D009472:Neuronal Ceroid-Lipofuscinoses / Q000188:drug therapy*; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism* / Q000627:therapeutic use; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000627:therapeutic use; D057057:Serine Proteases; D014162:Transfection; D000091346:Tripeptidyl-Peptidase 1
chemical_list: D018388:Codon, Terminator; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; C000717269:Tpp1 protein, rat; D000091346:Tripeptidyl-Peptidase 1; D001120:Arginine; D010450:Endopeptidases; D010447:Peptide Hydrolases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C000717267:TPP1 protein, human