Production and characterization of recombinant human CLN2 protein for enzyme-replacement therapy in late infantile neuronal ceroid lipofuscinosis

L. Lin, Peter Lobel

Research output: Contribution to journalArticle

65 Scopus citations

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 ≈ 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 (EC50≈ 2 nM), where it remains active for long periods of time (t1/2≈ 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.

Original languageEnglish (US)
Pages (from-to)49-55
Number of pages7
JournalBiochemical Journal
Volume357
Issue number1
DOIs
StatePublished - Jul 1 2001

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Keywords

  • Endocytosis
  • Lysosomal storage
  • Overexpression
  • Tripeptidyl peptidase I

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